Article

The human c-fos serum response factor and the yeast factors GRM/PRTF have related DNA-binding specificities

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Abstract

One of the elements that mediates growth factor and serum inducibility of the human c-fos gene is a region of dyad symmetry that lies between nucleotides -320 and -299 of the human gene. A mammalian protein specifically binds to this sequence element and has been termed the serum response factor (SRF). Gel-shift analysis and competition experiments demonstrate that there is a factor in the yeast Saccharomyces cerevisiae that binds specifically to the human c-fos SRE. The methylation interference pattern of the yeast factor is identical to that of the mammalian SRF. Regulatory elements of cell-type-specific genes in yeast have homologies to the c-fos SRE and complete for binding of both the mammalian and yeast factors to the SRE. Antisera to the gene product of the MCM1 locus react with the yeast SRE-binding factor. These data suggest that this yeast protein is closely related or identical to the factors [general regulator of mating type (GRM) and pheromone/receptor transcription factor (PRTF)] that are required for the regulation of cell-type-specific genes in yeast.

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... This region, which contains residues common to the entire group of proteins, has been termed the MADS (MCM1-Arg80--_agamous-deficiens-SRF) box ). Although the DNA-binding specificity of MCM1 is clearly related to that of SRF (Hayes et al. 1988;Passmore et al. 1989), the binding specificities of the other proteins are uncharacterized. ...
... within the basic region, also have overlapping but nonidentical sequence specificities (Hayes et al. 1988;Passmore et al. 1989; J. Wynne, S. Dalton, and R. Treisman, unpubl.). ...
Article
Serum response factor (SRF) is a transcription factor that binds the sequence CC(A/T)6GG found in a number of growth factor-inducible and muscle-specific promoters. We describe the isolation and characterization of cDNA clones encoding a family of three human SRF-related DNA-binding proteins. Each of these RSRF (related to SRF) proteins contains an 86-amino-acid amino-terminal region related to the SRF DNA-binding domain: In RSRFC4 and RSRFC9, this region is identical, whereas that present in RSRFR2 differs by seven conservative amino acid substitutions. The DNA-binding specificity of the RSRF proteins, which recognize the consensus sequence CTA(A/T)4TAG, is distinct from that of SRF. The entire RSRF common region is required for DNA binding, and the differential sequence specificity of the RSRFs and SRF is the result of differences in the basic amino-terminal part of this domain. The RSRF proteins bind DNA as dimers and can dimerize with one another but not with SRF. Although the RSRF mRNAs are expressed in many cell types, RSRFR2 mRNA is expressed at elevated levels in several B-cell lines. Consistent with this, extracts from many cell types form CTA(A/T)4TAG-binding complexes that contain RSRF proteins, and oligonucleotides containing RSRF-binding sites function as promoter elements in transfection assays. Like SRF-binding sites, RSRF-binding sites are found in the regulatory sequences of a number of growth factor-inducible and muscle-specific genes, and we show that RSRF polypeptides are components of previously characterized binding activities that interact with these elements. We discuss the potential role of RSRF proteins in the regulation of these genes.
... This is likely to be another important factor in enabling the transgenic tomato lines to develop the abscission zone in pedicel. It is known that MADS-box proteins can regulate gene expression by recognizing and binding to a DNA sequence CArG boxes, CC(A/T) 6 GG, that allows them to regulate downstream genes (Hayes et al., 1988;Riechmann et al., 1996). In this case, it is possible that some of them might be activated on transcriptional level as direct target of PsJOINTLESS. ...
Article
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MADS-box proteins have been implicated in many biological processes. However, plant MADS-box proteins functioning in floral organ abscission and the underlying physiological mechanisms remain poorly understood. Here, we report the identification and functional characterization of PsJOINTLESS isolated from 'Kuerlexiangli'. PsJOINTLESS had a complete open reading frame of 672bp, encoding a 224 amino acid peptide, and shared high sequence identities with MADS-box from other plants. PsJOINTLESS was subcellularly targeted to the nucleus, supporting its role as a transcription factor. Expression levels of PsJOINTLESS in the calyx tube were strongly induced by calyx abscission treatment at 6d after full bloom. Overexpression of PsJOINTLESS in tomato enhanced the rate of pedicel abscission rate. Of special note, the transgenic plants increased the abscission zone cell layer compared with wild type. Furthermore, the tomato transgenic lines showed thinner flower pedicels, more cell number and small pedicel cell size. The cellulase activity in pedicel abscission zone of transgenic plants was higher than that of wild type. In addition, steady-state mRNA levels of five cell wall hydrolase genes coding for either functional or regulatory proteins were induced to higher levels in the transgenic lines. These results clearly demonstrate that PsJOINTLESS may affect pedicel abscission zone development.
... Human SRF and yeast MCM1 are known transcriptional activators whose DNA-binding and dimerization domains, like the binding domain of MEF2 here, include the MADS sequence (Norman et al. 1988;Christ and Tye 1991). Moreover, the target DNA sequences for MCM1 and SRF are also A/T-rich sites similar to, but distinct from, MEF2 (Hayes et al. 1988;Boxer et al. 1989;Passmore et al. 1989). ...
Article
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The MEF2 site is an essential element of muscle enhancers and promoters that is bound by a nuclear activity found, so far, only in muscle and required for tissue-specific transcription. We have cloned a group of transcription factors from human muscle that are responsible for this activity: They are present in muscle-specific DNA-binding complexes, have a target sequence specificity identical to that of the endogenous activity, and are MEF2 site-dependent transcriptional activators. These MEF2 proteins comprise several alternatively spliced isoforms from one gene and a related factor encoded by a second gene. All share a conserved amino-terminal DNA-binding domain that includes the MADS homology. MEF2 transcripts are ubiquitous but accumulate preferentially in skeletal muscle, heart, and brain. Specific alternatively spliced isoforms are restricted to these tissues, correlating exactly with the presence of endogenous MEF2 activity. Furthermore, MEF2 protein is detected only in skeletal and cardiac muscle nuclei and not in myoblast and nonmuscle cells. Thus, post-transcriptional regulation is important in the generation of tissue-specific MEF2 activity. Cardiac and smooth, as well as skeletal, muscles contain functionally saturating levels of MEF2 trans-activating factors that are absent in nonmuscle cells. Moreover, MEF2 is induced in nonmuscle cells by MyoD; however, MEF2 alone is insufficient to produce the full muscle phenotype. Implications for the molecular mechanisms of myogenesis are considered.
... The plant type II MADS-box transcription factors have a conserved so-called MIKC structure, in which four distinct domains can be recognised. The MADS-box (M) domain encodes a highly conserved region of 56 amino acids that is necessary for DNA-binding (Hayes et al. 1988). The moderately conserved K (keratin-like) domain in the central part of these proteins forms a coiledcoil structure and has been shown to be important for MADS-box homo-or heterodimerisation. ...
Article
MADS-box genes involved in flower development have been isolated and studied in a wide variety of plant species. However, most of these studies are related to dicot species like Antirrhinum majus, Arabidopsis thaliana and Petunia hybrida. Although the floral structures of typical monocot and dicot flowers differ substantially, previous studies indicate that MADS-box genes controlling floral organ identity in dicots can also be identified in monocot plants like rice and maize. To extend this study further to obtain a more global picture of monocot and dicot MADS-box gene evolution, we performed a phylogenetic study using MADS-box genes from A. thaliana and Oryza sativa. Furthermore, we investigated whether the identified orthologues of Arabidopsis and rice have a conserved expression profile that could indicate conservation of function.
... The FIL2 gone reveals two exons (371 bp and 799 bp) which are separated by a 264 bp intron. The promoter region displays a 10 bp TATA-box at position -24 upstream of the putative transcription start and an element at position -528, which resembles the consensus sequence of the binding site for MADS-box transcription factors (CArG-box; Hayes et al., 1988;Pollock and Treisman, 1991). ...
Article
The expression of the Antirrhinum gene FIL2 is affected in mutants of the homeotic transcription factor DEFICIENS. Northern and Western blot analyses showed that FIL2 in wild-type Antirrhinum flowers is expressed weakly in the petals and more abundantly in the reproductive organs; the gene is active in the filaments and anthers of stamens, and in the stigma and transmitting tissue of the carpels. The FIL2 protein is glycosylated with high mannose type glycan chains and is located in the middle lamella of the extracellular matrix. The amino acid sequence contains 10 tandem repeats, the composition of which is similar to the Leucine-Rich Repeat (LRR) motif found in mammals, Drosophila and yeast. The possiblity that FIL2 might be a component of a cellular signalling mechanism, involving LRR-mediated protein-protein interactions is discussed.
... It is likely that most floral MADS box genes play regulatory roles in controlling flower development, although the precise roles of many of these MADS box genes are still not clear. The MADS box encodes a 56 amino-acid region (Schwarz- Sommer etaL, 1990), termed the MADS domain, which is necessary for DNA-binding in vitro (Hayes etal., 1988; Mizukami et al., 1996; Norman et al., 1988; Passmore et aL, 1989). As shown in Figure 1, the plant MADS domain proteins also contain a second domain of moderate sequence similarity, which is absent from MADS domain proteins of other organisms. ...
Article
SummaryMADS domain (for MCM1, AG, DEFA and SRF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein—protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.
... Generally, MADS domain proteins bind to DNA sequences called CArGboxes (for C-A-rich-G). While most MADS domain proteins prefer the so-called serum response element (SRE)-type CArG-box, 59-CC(A/T) 6 GG-39, for binding (Hayes et al., 1988; Riechmann et al., 1996; de Folter and Angenent, 2006), some others, such as the mammalian MYOCYTE ENHANCER FACTOR2A (MEF2A) and the MIKC*-type proteins of all species investigated so far, preferentially bind to the N10-type CArG-box or MEF2 consensus binding site. This motif has the consensus sequence 59-CTA(A/T) 4 TAG-39 but might be more loosely defined as 59-C(A/T) 8 G-39 (Pollock and Treisman, 1991; Shore and Sharrocks, 1995; Verelst et al., 2007a; Zobell et al., 2010; Wu et al., 2011). ...
Article
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There are two groups of MADS intervening keratin-like and C-terminal (MIKC)-type MADS box genes, MIKC(C) type and MIKC* type. In seed plants, the MIKC(C) type shows considerable diversity, but the MIKC* type has only two subgroups, P- and S-clade, which show conserved expression in the gametophyte. To examine the functional conservation of MIKC*-type genes, we characterized all three rice (Oryza sativa) MIKC*-type genes. All three genes are specifically expressed late in pollen development. The single knockdown or knockout lines, respectively, of the S-clade MADS62 and MADS63 did not show a mutant phenotype, but lines in which both S-clade genes were affected showed severe defects in pollen maturation and germination, as did knockdown lines of MADS68, the only P-clade gene in rice. The rice MIKC*-type proteins form strong heterodimeric complexes solely with partners from the other subclade; these complexes specifically bind to N10-type C-A-rich-G-boxes in vitro and regulate downstream gene expression by binding to N10-type promoter motifs. The rice MIKC* genes have a much lower degree of functional redundancy than the Arabidopsis thaliana MIKC* genes. Nevertheless, our data indicate that the function of heterodimeric MIKC*-type protein complexes in pollen development has been conserved since the divergence of monocots and eudicots, roughly 150 million years ago.
... A number of these genes, including AP7, AP3, and AG (Yanofsky et al., 1990;Jack et al., 1992;Mandel et al., 1992), encode proteins with sequence similarity to the transcription factors MCM1 in yeast (Passmore et al., 1988) and the serum response factor in humans (Norman et al., 1988). This sequence similarity encompasses a highly conserved stretch of more than 50 amino acids, the "MADS domain," which has been shown to be a DNA binding motif (Hayes et al., 1988;Norman et al., 1988;Schwarz-Sommer et al., 1990). More recent studies have shown the products of the Arabidopsis AG gene and the Antirrhinum DfFlClfNS gene (another MADS-box gene affecting floral organ identity) to be sequence-specific DNA binding proteins (Mueller and Nordheim, 1991;Schwarz-Sommer et al., 1992). ...
Article
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Recent genetic and molecular studies in Arabidopsis and Antirrhinum suggest that mechanisms controlling floral devel- opment are well conserved among dicotyledonous species. To assess whether similar mechanisms also operate in more distantly related monocotyledonous species, we have begun to clone homologs of Arabidopsis floral genes from maize. Here we report the characterization of two genes, designated ZAG7 and ZAG2 (for Zea AG), that were cloned from a maize inflorescence cDNA library by low stringency hybridization with the AGAMOUS (AG) cDNA from Arabidopsis. ZAGl en- codes a putative polypeptide of 286 amino acids having 61% identity with the AGAMOUS (AG) protein. Through a stretch of 56 amino acids, constituting the MADS domain, the two proteins are identical except for two conservative amino acid substitutions. The ZAG2 protein is less similar to AG, with 49% identity overall and substantially less similarity than ZAGl outside the well-conserved MADS domain. Like AGI ZAG7 RNA accumulates early in stamen and carpel primordia. In contrast, ZAGP expression begins later and is restricted to developing carpels. Hybridization to genomic DNA with the full-length ZAGl cDNA under moderately stringent conditions indicated the presence of a large family of related genes. Mapping data using maize recombinant inbreds placed ZAG7 and ZAG2 near two loci that are known to affect maize flower development, Polytypic ear (Pt) and Tassel seed4 (Ts~), respectively. The ZAG1 protein from in vitro translations binds to a consensus target site that is recognized by the AG protein. These data suggest that maize contains a homolog of the Arabidopsis floral identity gene AG and that this gene is conserved in sequence and function.
... We next examined the DNA-binding surfaces of the pre-and postduplication MADS-box proteins. S. cerevisiae Arg80 and Mcm1 have very closely related DNA-binding specificities (19,20), indicating that, at most, a limited divergence in MADS-box DNA-binding specificity occurred following duplication. That the DNA-binding specificity did not change substantially is also supported by our observation that the preduplication AncMADS protein can complement the deletion of either postduplication gene (Fig. 2). ...
Article
Most models of gene duplication assume that the ancestral functions of the preduplication gene are independent and can therefore be neatly partitioned between descendant paralogs. However, many gene products, such as transcriptional regulators, are components within cooperative assemblies; here, we show that a natural consequence of duplication and divergence of such proteins can be competitive interference between the paralogs. Our example is based on the duplication of the essential MADS-box transcriptional regulator Mcm1, which is found in all fungi and regulates a large set of genes. We show that a set of historical amino acid sequence substitutions minimized paralog interference in contemporary species and, in doing so, increased the molecular complexity of this gene regulatory network. We propose that paralog interference is a common constraint on gene duplicate evolution, and its resolution, which can generate additional regulatory complexity, is needed to stabilize duplicated genes in the genome.
... From the N to the C terminus of the protein, four characteristic domains can be identified: the MADS-box (M), intervening (I), keratin-like (K), and C-terminal (C) domains. The MADS-box is a DNA binding domain of ‫ف‬ 58 amino acids that binds DNA at consensus recognition sequences known as CArG boxes [CC(A/T) 6 GG] (Hayes et al., 1988;Riechmann et al., 1996b). The interaction with DNA has been studied in detail for the human and yeast MADS-box proteins thanks to the resolved crystal structures (Pellegrini et al., 1995;Santelli and Richmond, 2000). ...
Article
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MADS-box transcription factors are key regulators of several plant development processes. Analysis of the complete Arabi-dopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function. Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member. We have divided this transcription factor family into five groups (named MIKC, M , M , M , and M) based on the phylogenetic relationships of the conserved MADS-box domain. This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins. MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants.
... 식물에서 HSF는 토마토 (Scharf et al., 1990), 애기장대 (Hubel & Schoffl, 1994), 콩 (Czarnecka-Verner et al., 1995), 옥수수 (Gagliardi et al., 1995) 등에서 특징되었으며, DNA 결합 도메인 분석과 올리고머화 도메인의 비교로 A와 B의 주요 클래스로 구분되었다 (Czarnecka-Verner et al., 1995;Nover et al., 1996;Czarnecka-Verner et al., 1997;Nover & Scharf, 1997 Ratcliffe et al., 2001;Scortecci et al., 2001;SHORT VEGETATIVE PHASE:SVP, Hartmann et al., 2000) 화기 분열조직의 결정(APETALA1:AP1, Mandel et al., 1992;FRUITFULL:FUL, Gu et al., 1998;CAULIFLOWER: CAL, Bowman et al., 1993), 화기의 형성(AP1, SEPALLATA: SEP1 to SEP3, Pelaz et al., 2000;APETALA3:AP3, Jack et al., 1992;PISTILLATA:PI, Goto & Meyerowitz, 1994;AGAMOUS: AG, Yanofsky et al., 1990), 과실 형성(SHATTERPROOF: SHP1 and SHP2, Liljegren et al., 2000;FUL), 종자 색소 침착 및 내피세포 발달(TRANSPARENT TESTA16, Nesi et al., 2002) 등의 연구가 진행되었다. 애기장대와 많은 식물 종에서 MADS-box의 넓은 다양상 (Kater et al., 2001;Fornara et al., 2003)과 MIKC-type MADS-box 유전자의 계통발생 학적 분석 (Purugganan et al., 1995;Theissen et al., 1996;Munster et al., 1997) (Hayes et al., 1988;Riechmann et al., 1996). I 도메인은 덜 보존되고 이량체화에 기여하며, K 도메인은 MADS-box 단백질의 이량체화를 촉진하는 꼬 인 코일 구조에 의해 특징된다 (Davies et al., 1996;Fan et al., 1997). ...
Article
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Transcription factors are essential for the regulation of gene expression in plant. They are binding to either enhancer or promoter region of DNA adjacent to the gene and are related to basal transcription regulation, differential enhancement of transcription, development, response to intercellular signals or environment, and cell cycle control. The mechanism in controlling gene expression of transcription can be understood through the assessment of the complete sequence for the maize genome. It is possible that the maize genome encodes 4,000 or more transcription factors because it has undergone whole duplication in the past. Previously, several transcription factors of maize have been characterized. In this review article, the transcription factors were selected using Pfam database, including many family members in comparison with other family and listed as follows: ABI3/VP1, AP2/EREBP, ARF, ARID, AS2, AUX/IAA, BES1, bHLH, bZIP, C2C2-CO-like, C2C2-Dof, C2C2-GATA, C2C2-YABBY, C2H2, E2F/DP, FHA, GARP-ARR-B, GeBP, GRAS, HMG, HSF, MADS, MYB, MYB-related, NAC, PHD, and WRKY family. For analyzing motifs, each amino acid sequence has been aligned with ClustalW and the conserved sequence was shown by sequence logo. This review article will contribute to further study of molecular biological analysis and breeding using the transcription factor of maize as a strategy for selecting target gene.
... In both networks, these regulators contain sequence-specific DNA binding domains such as homeodomains, MADS domains, and zinc fingers (Weirauch and Hughes, 2011). In some cases the cis-regulatory sequence recognized by a given transcription regulator has not changed significantly since the divergence of yeast and mammals (Hayes et al., 1988). Moreover, transcription regulators from one species (e.g. ...
Article
When transcription regulatory networks are compared among distantly related eukaryotes, a number of striking similarities are observed: a larger-than-expected number of genes, extensive overlapping connections, and an apparently high degree of functional redundancy. It is often assumed that the complexity of these networks represents optimized solutions, precisely sculpted by natural selection; their common features are often asserted to be adaptive. Here, we discuss support for an alternative hypothesis: the common structural features of transcription networks arise from evolutionary trajectories of "least resistance"-that is, the relative ease with which certain types of network structures are formed during their evolution. Copyright © 2015 Elsevier Inc. All rights reserved.
... MADS-domain proteins have been described to bind to DNA sequences with the consensus CC(A/T) 6 GG (Hayes et al. 1988) To answer that question Arabidopsis plants were transformed with the human MEF2A transcription factor which has a general preference for the CTA(A/T) 4 TAG motif (Pollock andTreisman 1991, Shore andSharrocks, 1995). Also, AGL15 and AGAMOUS, which are classical MIKC c genes that are normally not expressed in Arabidopsis pollen, were expressed under control of the AGL65 promoter in the mutant background. ...
... For example, the transcription regulator Mcm1 regulates many hundreds of genes in Saccharomyces cerevisiae (Messenguy and Dubois 2003). It is sufficiently conserved that a human homolog can substitute for it in vivo (Primig et al. 1991); the human and yeast proteins have retained a nearly identical DNA-binding specificity (Hayes et al. 1988). However, only 15% of the connections between this regulator and its deeply conserved target genes are observed in two other ascomycete species, Candida albicans and Kluyveromyces lactis (Tuch et al. 2008a). ...
Article
The rewiring of gene regulatory networks over evolutionary timescales produces changes in the patterns of gene expression and is a major source of diversity among species. Yet the molecular mechanisms underlying evolutionary rewiring are only beginning to be understood. Here, we discuss recent analyses in ascomycete yeasts that have revealed several general principles of network rewiring. Specifically, we discuss how transcription networks can maintain a functional output despite changes in mechanism, how specific types of constraints alter available evolutionary trajectories, and how regulatory rewiring can ultimately lead to phenotypic novelty. We also argue that the structure and "logic" of extant gene regulatory networks can largely be accounted for by constraints that shape their evolutionary trajectories.
... Other intermediate CArG boxes with a variable length A/T core may also be recognized in vivo (Nurrish and Treisman, 1995). The SRF-type CArG box is favored by many MADS complexes investigated thus far (Hayes et al., 1988;Riechmann et al., 1996;de Folter and Angenent, 2006). MADS proteins such as AGAMOUS-LIKE-15 (AGL15) have shown a preference for the longer MEF2-type binding site and associated intermediates (Tang and Perry, 2003). ...
Article
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In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve.
... This group, which is the biggest of the five, is characterized by a conserved domain structure (figure 2): All its members are composed of a MADS-box (M) domain, a Intervening (I) domain, a Keratin-like (K) domain and a C-terminal (C) domain (For the domain structure of the other clades see (Parenicová et al. 2003)). The N-terminal part of the 58 amino acid long MADS-box domain is composed of multiple β-sheets and has DNA binding activity (Riechmann et al. 1996a, Hayes et al. 1988) while a short C-terminal part of this domain is involved in dimerization. The less conserved I domain contributes to the specification of dimerization. ...
Article
Little is known about the molecular mechanisms involved reproductive organ development in seed plants and about the evolution of the different reproductive strategies of angiosperms (flowering plants, or "vessel seed" plants) and gymnosperms ("naked seed" plants). The development of the angiosperm flower has been extensively studied at the molecular level. Four classes of genes, the A-, B-, C-and E-genes, specify the identity of the floral organs in a combinatorial manner. These homeotic (= identity conferring) genes contain a conserved sequence motiv, the MADS-box, which encodes the DNA-binding MADS domain, and their protein products operate as heterotetrameric transcriptional regulators. Here, I compared the Bgenes APETALA 3 and PISTILLATA and C-gene AGAMOUS from the angiosperm thale cress (Arabidopsis thaliana) with their orthologs in the gymnosperm Norway spruce (Picea abies) (DEFICIENS AGAMOUS LIKE 13-I (DAL13-I) and DAL2, respectively) by analyzing with which of 107 other Arabidopsis and spruce MADS domain proteins their protein products can interact. The results for the Arabidopsis and spruce genes are in general highly similar. Interestingly, DAL2 interacted with the Arabidopsis proteins SEPALATA1, of which no ortholog has hitherto been described in spruce, and SUPPRESSOR OF CONSTANS 1 (SOC1) which is an important regulator of flowering time in Arabidopsis. The MADS-box genes, and the SEPALATA-genes in particular, are believed to be in part responsible for the appearance and radiation of angiosperms. The identification of gymnosperm orthologs of the angiosperm homeotic genes and their functional characterization could thus lead to a model of reproductive organ formation in gymnosperms and shed light on the evolution of the different reproductive strategies of seed plants.
... This gene family commonly possesses a highly conserved N-terminal DNA binding domain (MADS-box), a moderately conserved coiled-coil domain for protein reciprocity (K-box), a lightly conserved domain (I domain) between the MADS-box and K-box, and a non-conserved C-terminal. The MADS-box is a DNA binding domain of approximately 58 amino acids that binds DNA at consensus recognition sequences known as CArG boxes [CC(A/T)6GG] [2,3]. The I terminal is less conserved and contributes to the specification of dimerization. ...
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MADS-box transcription factors are important for plant growth and development, and hundreds of MADS-box genes have been functionally characterized in plants. However, less is known about the functions of these genes in the economically important allopolyploid oil crop, Brassica napus. We identified 307 potential MADS-box genes (BnMADSs) in the B. napus genome and categorized them into type I (Mα, Mβ, and Mγ) and type II (MADS DNA-binding domain, intervening domain, keratin-like domain, and C-terminal domain [MIKC]c and MIKC*) based on phylogeny, protein motif structure, and exon-intron organization. We identified one conserved intron pattern in the MADS-box domain and seven conserved intron patterns in the K-box domain of the MIKCc genes that were previously ignored and may be associated with function. Chromosome distribution and synteny analysis revealed that hybridization between Brassica rapa and Brassica oleracea, segmental duplication, and homologous exchange (HE) in B. napus were the main BnMADSs expansion mechanisms. Promoter cis-element analyses indicated that BnMADSs may respond to various stressors (drought, heat, hormones) and light. Expression analyses showed that homologous genes in a given subfamily or sister pair are highly conserved, indicating widespread functional conservation and redundancy. Analyses of BnMADSs provide a basis for understanding their functional roles in plant development.
... The MADS-box acronym is derived from the yeast MINICHROMOSOME MAINTENANCE 1 (MCM1) (Passmore et al., 1988), the Arabidopsis AGAMOUS (AG) (Yanofsky et al., 1990), the Antirrhinum DEFICIENS (DEFA) (Schwarz-Sommer et al., 1990), and the mammalian SERUM RESPONSE FACTOR (SRF) (Norman et al., 1988;Gramzow et al., 2010). All identified MADS-box proteins each contain a MADS-box domain of ~58 amino acid at the N-terminus that binds to a consensus CC[A/T] 6 GG sequence, termed as the "CarG-box" motif (Hayes et al., 1988;Riechmann et al., 1996). Interestingly, flowering plants (angiosperms) have more of these genes (e.g. ...
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The orchid family (Orchidaceae) represents the second largest angiosperm family, having over 900 genera and 27,000 species in almost all over the world. Orchids have evolved a myriad of intriguing ways in order to survive extreme weather conditions, acquire nutrients, and attract pollinators for reproduction. The family of MADS-box transcriptional factors have been shown to be involved in the control of many developmental processes and responses to environmental stresses in eukaryotes. Several findings in different orchid species have elucidated that MADS-box genes play critical roles in the orchid growth and development. An in-depth understanding of their ecological adaptation will help to generate more interest among breeders and produce novel varieties for the floriculture industry. In this review, we summarize recent findings of MADS-box transcription factors in regulating various growth and developmental processes in orchids, in particular, the floral transition and floral patterning. We further discuss the prospects for the future directions in light of new genome resources and gene editing technologies that could be applied in orchid research and breeding.
... Remarkably, MADS-box genes are present in all sorts of eukaryotes, from yeast to mammals. Apart from the DNA binding at so-called CArG-boxes, which are consensus DNA motifs whose core sequence is usually CC(A/T) 6 GG (Hayes et al., 1988;Riechmann, 1996), MADS-domain TFs possess the inherent ability to form homo-and heterodimers. This provides the key to explain the underlying molecular mechanism whereby organ identity genes exercise their partly overlapping functions, i.e., the physical interaction between the deriving TFs, which must dimerize in order to bind CArG-boxes (Schwarz-Sommer et al., 1992). ...
Thesis
Determination of floral organ identity in Arabidopsis thaliana largely comes down to a group of master regulators belonging to the family of homeotic MADS-domain transcription factors, which interact to form hetero-multimeric complexes and coordinate gene expression. How their limited number and conserved features still allow spatiotemporal specific selection and regulation of downstream gene targets remains uncertain. Growing evidence is revealing that floral MADS-domain proteins recruit additional co-factors to acquire DNA-binding specificity and dual regulatory activity. The co-adaptors SEUSS (SEU) and SEUSS-LIKE proteins (SLKs) build complexes with co-repressors LEUNIG (LUG), and LEUNIG_HOMOLOGUE (LUH) and, together, they have been shown to interact with certain MADS-domain proteins to exert regulatory effects, thus, possibly contributing to their functional specificity. Since the relevance of these interactions in floral organ development has only been demonstrated for specific cases, in this thesis we analysed genome-wide the position of SEU, SLK2, and LUH. The ChIP-Seq technique and a system for simultaneous induction of flower development enabled us to assess the putative binding sites of the co-regulators at distinct time points and compare them to publicly available binding profiles of MADS-domain transcription factors. This shows that co-regulators genomically co-locate to some extent with every investigated floral homeotic protein throughout the early stages of flower development, especially with APETALA1 during organ differentiation (stage 8), indicating that co-regulators may be recruited by organ-specific complexes in all whorls, and are particularly important for sepal and petal development. Comparing binding profiles at single genomic loci led to identification of novel genes potentially targeted by MADS proteins/co-regulator complexes. Using published, temporal gene expression data we determined the impact of SEU, SLK2, and LUH on target gene activity. Expression remains largely unaltered despite binding and there is no evident repressing tendency. Thus, the nature of this binding is unclear, but it may suggest that further factors are still required for regulation. Furthermore, we took advantage of the CRISPR/Cas9 gene-editing technology to remove a region comprising a putative SEU binding site upstream of the homeotic gene AGAMOUS. As a consequence, expression was considerably reduced, implying that this region is essential for AGAMOUS correct activity. Our preliminary study provides a clearer picture on the collaboration of MADS-domain transcription factors with co-regulators to govern genetic developmental programs underlying correct flower formation.
... MADS domain is composed of approximately 58 amino acid residues and is located at the N-terminus of these proteins. In terms of its function, MADS is responsible for binding to CArG-box in target genes, a type of motif with a consensus sequence of CC[A/T] 6 GG (Hayes et al. 1988;Teo et al. 2019). ...
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The identity of the floral organs is defined by a small group of transcriptional regulators, and the activities of these proteins can specify the feature of the different whorls in flowers. In the last thirty years, formation of the floral organs remained as the main research subject in plant, especially in Arabidopsis thaliana. By using A. thaliana as material, relevant research works have established a basic architecture for the development of floral organs in higher plants, and it was named as the ABCDE model. In accordance with this model, the identity of the different floral organs can be confirmed by the specific combinations of A-, B-, C-, D-, or E-class floral homeotic genes. SEP-like genes encode MADS transcription factors required for the development of all the four whorls of floral organs and for the determinacy of the floral meristems. In ABCDE model, these genes are defined as E-class floral homeotic genes. A great deal of studies have shown that the proteins encoding by these genes are the main regulators of flower development and act pivotal parts not only in organ identification but also in organ morphogenesis. In this paper, the functions of the E-class floral homeotic genes in dicotyledons are reviewed, and the progress that has been made in characterization of the floral organ identity factors in A. thaliana and other dicotyledons is discussed.
... Type II genes are believed to encode MIKC proteins, thus named for featuring a MADS (M) domain, an intervening (I) domain, a keratin-like (K) domain, and a characteristic C-terminal (C) domain (Kaufmann et al. 2005). All these TFs contain a characteristic amino-terminal domain (the MADS domain) of 60 residues that bind to target genes at a regulatory region that features a CC (A/T) 6 GG sequence called the CArG-box (Messenguyand Dubois 2003; Riechmann et al. 1996;Hayes et al. 1988). These MADS-box genes are believed to be homeotic, and they specify organ identity and, flowering time and several other reproductive processes (Dornelas et al. 2011). ...
Chapter
Orchids constitute the largest families within the flowering plants; they are one of the most highly evolved groups in the angiosperms; and because of their flashy flowers many species from several genera are very prominent horticultural commodities. Environmental factors such as temperature and daylength influence flowering; however, other factors play a major role such as the developmental stage, plant hormonal regulation and genetic factors. Research on model plants such as the thale cress (Arabidopsis thaliana) and rice (Oryza sativa) has allowed a broad understanding of the genetics and the molecular regulation of flowering in most plants. However, until very recently the specifics of floral regulation in orchids had remained quite obscure. In this review we have examined very recent work concerning flowering of orchids with a focus on the interplay between development, environmental cues, plant hormones, and gene networks. We have highlighted several examples of successful manipulation of flowering through biotechnology that may be very relevant for researchers and growers elsewhere.
... Type II genes are believed to encode MIKC proteins, thus named for featuring a MADS (M) domain, an intervening (I) domain, a keratin-like (K) domain, and a characteristic C-terminal (C) domain (Kaufmann et al. 2005). All these TFs contain a characteristic amino-terminal domain (the MADS domain) of 60 residues that bind to target genes at a regulatory region that features a CC (A/T) 6 GG sequence called the CArG-box (Messenguyand Dubois 2003; Riechmann et al. 1996;Hayes et al. 1988). These MADS-box genes are believed to be homeotic, and they specify organ identity and, flowering time and several other reproductive processes (Dornelas et al. 2011). ...
Chapter
Orchids have flowers of unique beauty that are remarkable for their zygomorphic syndrome, which can be summarized as a floral architecture based on three categories of organs at the perianth: external tepals, internal lateral tepals, and a labellum or lip, a prominent central inner petal believed to be a specialized adaptation that attracts pollinators. These mesmerizing floral traits have enthralled researchers into the study of the orchid homologs of the MADS-box family of genes, which are transcriptional factors believed to be spatiotemporal determinants of organ identity during floral development. The identification of several putative members of the MADS-box family may clarify their potential role in orchid flower development, especially during the transition to flowering and during the patterning of orchid flower organs. Furthermore, we look into new technologies of genome analysis and gene editing in order to appraise potential applications for basic research purposes and for the breeding of new orchid varieties.
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We have identified oncogene-responsive sequences in the human c-fos promoter that mediate induction of transcription by several nonnuclear oncoproteins and the tumor promoter TPA. These sequences are regulated in a cell-specific manner. (i) In NIH 3T3 cells, the CArG box of the c-fos promoter is sufficient to mediate activation by oncogenes. (ii) In contrast, in HeLa cells, additional flanking sequences are also required, including the outer arm of the serum response element and the FAP site. We also show that the serum response factor, which binds to the CArG box, activates transcription in vivo in NIH 3T3 cells but not in HeLa cells. Finally, we present evidence that the intracellular level of the c-Fos protein could be a major determinant of cell-specific regulation of these oncogene-responsive elements of the c-fos promoter.
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Exactly how an organism adapts its transcriptional program in response to intra- and extracellular signals remains elusive. Development of computational approaches that use the large amounts of diverse intracellular data to unravel the cell's transcriptional program is one of today's main challenges in bioinformatics research. This thesis contributes to that field by investigating the transcriptional response of the yeast \textit{Saccharomyces cerevisiae} to multiple chemical and physical signals from its environment. In contrast to the commonly used shake-flask cultures, the gene expression data employed in this thesis originates from yeast grown in steady-state chemostat cultures. These chemostat cultures enable the accurate control, measurement and manipulation of individual cultivation parameters, such as growth rate, temperature and nutrient concentrations. A growth condition can thus be characterized by the combined settings of several cultivation parameters. Computational methods are developed that use these 'multifactorial' growth conditions to infer the effect of individual cultivation parameters and combinations of cultivation parameters on gene expression. The gene expression measurements are integrated with data about the binding potential of transcription factors (TF), the proteins that bind the DNA near a gene (promoter region) and possibly manipulate the rate at which the gene is transcribed. This integration enables us to investigate the effect of cultivation parameters on the activity of TFs. We present computational approaches that not only infer the activity of TFs as a function of the cultivation parameters, but also describe the combinatorial interplay between different TFs on gene promoters to regulate a gene's rate of transcription.
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We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis-acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non-permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.
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The first step in flower development is the transition of an inflorescence meristem into a floral meristem. Each floral meristem differentiates into a flower consisting of four organ types that occupy precisely defined positions within four concentric whorls. Genetic studies in Arabidopsis thaliana and Antirrhinum majus have identified early-acting genes that determine the identify of the floral meristem, and late-acting genes that determine floral organ identity. In Arabidopsis, at least two genes, APETALA1 and LEAFY, are required for the transition of an influorescence meristem into a floral meristem. We have cloned the APETALA1 gene and here we show that it encodes a putative transcription factor that contains a MADS-domain. APETALA1 RNA is uniformly expressed in young flower primordia, and later becomes localized to sepals and petals. Our results suggest that APETALA1 acts locally to specify the identity of the floral meristem, and to determine sepal and petal development.
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The mammalian transcription factor SRF and the yeast regulatory protein MCM1 contain DNA binding domains that are 70% identical; moreover, both proteins can bind the serum response element in the human c-fos promoter. Here we present an analysis of MCM1 sequence specificity by selection of sites from random sequence oligonucleotides. In this assay the MCM1 DNA binding domain selects binding sites containing the consensus (NotC)CCY(A/T)(A/T)(T/A)NN(A/G)G, distinct from the SRF binding consensus CC(A/T)6GG. Carboxylethylation interference analysis of a set of selected sites suggests that MCM1 contacts DNA in its major groove throughout one helical turn. These differences in specificity are largely due to sequence differences between the N terminal basic parts of the SRF and MCM1 DNA binding domains. Comparison of the relative binding affinities of MCM1 and SRF for a panel of representative binding sites showed that many high affinity MCM1 sites have negligible affinity for SRF and vice versa. Thus MCM1 and SRF have significantly different sequence specificities.
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The c-fos protooncogene is transcriptionally activated by a wide variety of agents including serum, growth factors, and phorbol esters. This induction is rapid and transient and is mediated through a number of identified promoter elements. Growth hormone (GH) is also known to induce transcription of c-fos in a variety of cell types including NIH 3T3 fibroblasts and 3T3-F442A preadipocytes. To identify DNA sequences in the c-fos gene regulated by GH, this study sought to determine whether induction of c-fos by GH involves previously identified c-fos promoter elements. A plasmid containing a growth factor-sensitive fragment of the upstream region of the c-fos promoter from -361 to -264 bp was tested for GH sensitivity. The fragment was cloned upstream of a human c-fos reporter [designated FOS by Human Gene Mapping 11 (1991)], which included basal promoter elements. In transiently transfected mouse NIH 3T3 fibroblasts, the promoter fragment conferred GH sensitivity on the human c-fos reporter. To identify a specific GH-sensitive DNA sequence in this promoter, a serum response element (SRE)-reporter plasmid was tested and found to be stimulated by GH. GH was effective in inducing expression through the SRE over a range of physiological GH concentrations. Since GH was recently found to synergize with serum factors in inducing c-fos transcription, the effect of GH and serum on SRE function was examined for insight into the mechanism for such synergism. The combined effect of GH and serum to induce reporter expression through the SRE was greater than the added effects of GH and serum, indicating that the synergism between GH and serum in inducing c-fos involves the SRE sequence. These studies identify the SRE as one specific DNA sequence in the c-fos promoter functionally regulated by GH. It is notable that GH is effective at physiological concentrations. Furthermore, synergism in c-fos induction between GH and serum factors is evident through the SRE.
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The mammalian transcription factor SRF (serum-response factor) and the related Saccharomyces cerevisiae transcription factor MCM1 are the prototypes of a new class of dimeric DNA-binding proteins. Their function is regulated in part by the interactions of their DNA-binding domains with accessory proteins. Recent work has advanced the functional characterization of the contributions of SRF and MCM1, and their accessory proteins to transcriptional activation.
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The serum response element (SRE) plays an essential role in the transcriptional regulation of proto-oncogene c-fos. A ternary complex, consisting of transcription factors p67SRF and p62TCF bound to the SRE is present in several cell types and its formation has been correlated with inducibility of the gene in different cells by serum, epidermal growth factor and phorbol esters. Interaction of p62TCF with the SRE in vitro exhibits both a degree of sequence specificity and a strict dependence on the presence of bound p67SRF. A 90 amino acid DNA-binding domain of p67SRF (coreSRF) suffices for ternary complex formation. DNase I footprinting and UV-mediated DNA-protein crosslinking experiments presented here show that direct DNA contacts are made by p62TCF with the 5' sequence of the SRE and thus explain the sequence dependence of ternary complex formation. Additionally, analysis of ternary complex formation by chimaeras of coreSRF and the related yeast protein ArgRI as well as comprehensive mutagenesis of non-conserved residues between the two proteins has yielded a coreSRF mutant specifically unable to interact with p62TCF and demonstrates that an extended structure in coreSRF is required for this interaction. Thus p62TCF exhibits dual component specificity in ternary complex formation over the c-fos SRE.
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DNA elements with the CC(A/T)6GG, or CArG, motif occur in promoters that are under different regulatory controls. CArG elements from the skeletal actin, c-fos, and myogenin genes were tested for their abilities to confer tissue-specific expression on reporter genes when the individual elements were situated immediately upstream from a TATA element. The c-fos CArG element, also referred to as the serum response element (SRE), conferred basal, constitutive expression on the test promoter. The CArG motif from the myogenin gene was inactive. The skeletal actin CArG motif functioned as a muscle regulatory element (MRE) in that basal expression was detected only in muscle cultures. Muscle-specific expression from the 28-bp MRE and the 2.3-kb skeletal actin promoter was trans repressed by the Fos and Jun proteins. The expression and factor-binding properties of a series of synthetic CArG elements were analyzed. Muscle-specific expression was conferred by perfect 28-bp palindromes on the left and right halves of the skeletal actin MRE. Chimeric elements of the skeletal actin MRE and the c-fos SRE differed in their expression properties. Muscle-specific expression was observed when the left half of the MRE was fused to the right half of the SRE. Constitutive expression was conferred by a chimera with the right half of the MRE fused to the left half of the SRE and by chimeras which exchanged the central CC(A/T)6GG sequences. At least three distinct proteins specifically bound to these CArG elements. The natural and synthetic CArG elements differed in their affinities for these proteins; however, muscle-specific expression could not be attributed to differences in the binding of a single protein. Furthermore, the MRE did not bind MyoD or the myogenin-E12 heterodimer, indicating that muscle-specific expression from this element does not involve a direct interaction with these helix-loop-helix proteins. These data demonstrate that the conserved CArG motifs form the core of a family of functionally different DNA regulatory elements that may contribute to the tissue-specific expression properties of their cognate promoters.
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In recent years, isolation of several genes affecting flower development in Antirrhinum majus made this species a major model system to study this important developmental process. Genes like SQUAMOSA and FLORICAULA are involved in determination of the floral meristem. Their mutation results in the development of bract-forming shoots at positions where normally flowers would develop. The phenotypes obtained upon mutation of the genes found to affect floral organogenesis fall into three major categories. In each category, always the floral organs in two adjacent whorls become homeotically transformed. Based on this observation a simple genetic model has been proposed to explain the establishment of floral organ identity in the four concentric whorls of the flower. The model hypothesizes the independent induction of two developmental pathways specifying floral organ identity after the formation of sepals as the basic type of organ following induction of a floral meristem. One of these pathways is under the control of the PLENA gene, the other is controlled by the DEFICIENS and GLOBOSA genes. These genes, as well as SQUAMOSA, encode transcription factors sharing a conserved DNA binding domain: the MADS-box. In vitro DNA-binding studies complemented with molecular genetic analysis of the respective mutants show that the DEF and GLO proteins may act together in the form of a heterodimer in the regulation of their target genes as well as in autoregulation. The possible interactions between other MADS-box proteins and their role in flower development is under current investigation.
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We demonstrate that Saccharomyces cerevisiae cells possess a 33-41-kilodalton protein with DNA-binding properties remarkably similar to those of the immunoglobulin enhancer-binding protein NF-muE3. We further show that the muE3-binding site functions as an upstream activating sequence in yeast cells, stimulating transcription from a truncated CYC1 promoter. These data suggest that the yeast protein, designated YEB-3, and NF-muE3 are functionally related and perhaps evolutionarily conserved.
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Deficiens, a homeotic gene involved in the genetic control of flower development, codes for a putative transcription factor. Upon mutation of the gene, petals are transformed to sepals and stamens to carpels, indicating that deficiens is essential for the activation of genes required for petal and stamen formation. In a search for putative target genes of deficiens, several stamen- and petal-specific genes were cloned that are expressed in wild type but not in the deficiens globiferamutant. In this report the molecular characterization of two of these genes, tap1 and fil1, is presented. They are transiently expressed during flower development. In situ hybridization data demonstrate that tap1 is expressed in the tapetum of the anthers and fill in the filament of the stamen and at the bases of the petals. Both genes encode small proteins with N-terminal hydrophobic domains suggesting that they are secreted. We discuss possible functions of the gene products and their relationship to the deficiens gene.
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The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.
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The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500-fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre-existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the ‘conditioning’ of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma-specific expression are all mediated by the cis-elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ-specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue-specific response to MeJa, in conjunction with developmental control, is discussed.
Article
The c-fos serum response element (SRE) is necessary and sufficient for induction of the c-fos gene in response to serum and growth factors. This activation is dependent upon serum response factor (SRF), a transcriptional activator which binds the SRE. A factor, p62TCF, which binds in conjunction with SRF to the SRE and which is activated by mitogen-activated protein kinase, has also been implicated in c-fos regulation. By using a reporter gene system with weak SRE mutations that is dependent upon overexpression of SRF for serum induction, we have found that there are at least two pathways for serum induction that converge on the SRE. Loss of TCF binding by mutations in SRF and the SRE did not reduce serum induction of the reporter genes. We have found a pathway for serum induction that is sensitive to mutations in the A/T-containing central sequence of the SRE and which is independent of TCF. When this pathway was mutated, activation was dependent upon TCF binding, demonstrating that TCF can also function in serum induction. Both of the signalling pathways required a minimal domain of SRF. This domain, spanning SRF's DNA binding domain, was sufficient for serum induction when fused to a heterologous transcriptional activation domain.
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Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.
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The Saccharomyces cerevisiae MCM1 protein, which is essential for viability, participates in both transcription activation and repression as well as DNA replication. However, neither the full network of genes at which MCM1 acts nor whether MCM1 itself mediates a regulatory response is known. Thus far, sites of MCM1 action have been identified by chance during analysis of particular genes. To identify a more complete set of genes on which MCM1 acts, we isolated a library of yeast genomic sequences to which MCM1 binds and then identified known genes within this library. Fragments of genomic DNA, bound to bacterially expressed MCM1 protein, were collected on a nitrocellulose filter, cloned, and analyzed. This selected library contains a large number of genes. As expected, it is enriched for strong MCM1 binding sites and contains cell-type-specific genes known to require MCM1. In addition, it also includes sequences upstream (or near the 5' end) of a number of identified yeast genes that have not yet been shown to be controlled by MCM1. These include genes whose products are involved in (i) the control of cell cycle progression (CLN3, CLB2, and FAR1), (ii) synthesis and maintenance of cell wall or cell membrane structures (PMA1, PIS1, DIT1,2, and GFA1), (iii) cellular metabolism (PCK1, MET2, and CCP1), and (iv) production of a secreted glycoprotein which is heat shock inducible (HSP150). The previously unidentified MCM1 binding site in the essential PMA1 gene is required for expression of a PMA1:lacZ fusion gene, providing evidence that one site is functionally important. We speculate that MCM1 coordinates decisions about cell cycle progression with changes in cell wall integrity and metabolic activity. The presence in the library of three genes involved in cell cycle progression reinforces the idea that one of the functions of MCM1 is indeed analogous to that of the mammalian serum response factor.
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Cytoplasmic calcium is a nearly universal second messenger in eukaryotes. In many cell types, elevated intracellular calcium interacts synergistically with inducers of protein kinase C to elicit activation of complete biological programs normally induced by extracellular signals. In T cells, elevated cytoplasmic calcium is a critical mediator of activation in response to stimulation of the antigen receptor, and in some T-cell lines, treatment with a combination of calcium ionophore and protein kinase C activator mimics authentic antigen treatment. The synergistic interaction of calcium and protein kinase C in T cells is also observed at the level of gene expression. Here we examine the molecular mechanisms through which these agents exert synergistic control over the expression of the c-fos proto-oncogene in a T-cell hybridoma. We find that the principal effect of calcium is on the elongation of c-fos transcripts. This step constitutes the major control of c-fos mRNA accumulation in these cells. In addition, calcium regulates the initiation of c-fos transcription. This effect requires the serum response element of the c-fos gene and an additional sequence immediately 3' to this element. Thus, calcium regulates c-fos expression through at least two distinct molecular pathways.
Article
We used a yeast genetic screen to isolate cDNAs that encode a protein, SRF accessory protein-1 (SAP-1), that is recruited to the c-fos serum response element (SRE) as part of a ternary complex that includes serum response factor (SRF). SAP-1 requires DNA-bound SRF for ternary complex formation and makes extensive DNA contacts to the 5′ side of SRF, but does not bind DNA autonomously. Ternary complex formation by SAP-1 requires only the DNA-binding domain of SRF, which can be replaced by that of the related yeast protein MCM1. We isolated cDNAs encoding two forms of SAP-1 protein, SAP-1a and SAP-1b, which differ at their C termini. Both SAP-1 proteins contain three regions of striking homology with the elk-1 protein, including an N-terminal ets domain. Ternary complex formation by SAP-1 requires both the ets domain and a second conserved region 50 amino acids to its C-terminal side. SAP-1 has similar DNA binding properties to the previously characterized HeLa cell protein .
Article
A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989). When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription. The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene. A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.
Article
The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.
Article
The FUS1 gene of Saccharomyces cerevisiae is transcribed in a and alpha cells, not in a/alpha diploids, and its transcription increases dramatically when haploid cells are exposed to the appropriate mating pheromone. In addition, FUS1 transcription is absolutely dependent on STE4, STE5, STE7, STE11, and STE12, genes thought to encode components of the pheromone response pathway. We now have determined that the pheromone response element (PRE), which occurs in four copies within the FUS1 upstream region, functions as the FUS1 upstream activation sequence (UAS) and is responsible for all known aspects of FUS1 regulation. In particular, deletion of 55 bp that includes the PREs abolished all transcription, and a 139-bp fragment that includes the PREs conferred FUS1-like expression to a CYC1-lacZ reporter gene. Moreover, three or four copies of a synthetic PRE closely mimicked the activity conferred by the 139-bp fragment, and even a single copy of PRE conferred a trace of activity that was haploid specific and pheromone inducible. In the FUS1 promoter context, four copies of the synthetic PRE inserted at the site of the 55-bp deletion restored full FUS1 transcription. Sequences upstream and downstream from the PRE cluster were important for maximal PRE-directed expression but, by themselves, did not have UAS activity. Other yeast genes with PREs, e.g., STE2 and BAR1, are more modestly inducible and have additional UAS elements contributing to the overall activity. In the FUS1 promoter, the PREs apparently act alone to confer activity that is highly stimulated by pheromone.
Article
Expression of a mammalian major histocompatibility complex (MHC) class I gene is in part regulated by a silencer DNA sequence element which binds a complex of silencer factors. This negative regulatory system is shown to be strikingly similar to the yeast alpha 2 mating-type repression system. A moderate DNA sequence homology exists between the MHC class I silencer DNA element and the yeast alpha 2 operator. Mammalian silencer factors specifically bind to the yeast alpha 2 operator DNA and also specifically interact with a yeast alpha 2-binding protein. Furthermore, the alpha 2 operator functions as a silencer element in mammalian cells when placed upstream of a MHC class I promoter.
Chapter
Tightly regulated gene activity provides the molecular basis for ensuring properly controlled growth and division of eukaryotic cells. A comprehensive understanding of the regulatory mechanisms that direct gene activity of both proliferating and non-proliferating cells will provide important insight into the origin and causes of proliferative disorders in human pathology, including cancer.
Article
MADS-box genes are important transcription factors affecting overall development, but their role in sweet potato [Ipomoea batatas (L.) Lam.] has not been fully studied. This study isolated six novel MADS-box genes (IbSOC1, IbFUL1, IbAGL6, IbSVP1, IbSVP2, and IbSVP3) from sweet potato [Ipomoea batatas (L.) Lam. cv. Annouimo] during the early root differentiation stage using the de novo transcriptome assembly sequencing method. At the early root differentiation (between 0 and 3 days after transplanting), the IbSOC1, IbFUL1, and IbSVP2 genes decreased rapidly, whereas the IbSVP3 gene decreased gradually. In the early stages of root formation (0–30 days), the levels of IbSVP1 and IbSVP3 expression were steady, but the levels of IbSOC1 expression decreased gradually. The expression of six novel genes was also conducted in the tuberous root formation stage (30–90 days), and the IbSVP3 gene increased significantly according to the formation of the tuberous root. Six novel MADS-box genes that were believed to influence the entire root formation of sweet potato were isolated from the sweet potato. This study provides a genetic basis for further research on sweet potato root formation and development.
Article
Full-text available
The yeast trans-activator protein GAL4, when expressed in HeLa cells, stimulates transcription from several class B (II) eukaryotic promoters containing GAL4 binding sites either as the full UASG or as synthetic 17-mers. The characteristics of this activation are indistinguishable from those of the SV40 enhancer. Transcription was similarly stimulated from either complex promoter regions containing multiple upstream elements or from a simple promoter region composed of only a TATA box. Addition of a 17-mer GAL4 binding site to the SV40 enhancer resulted in a synergistic enhancement of transcription in the presence of GAL4. Furthermore, chimeras of the human estrogen receptor DNA binding domain and either GAL4 or GCN4 activating "acidic" regions can activate a promoter region controlled by an estrogen-responsive enhancer. Together, these data indicate that the molecular mechanisms responsible for transcriptional enhancement have been conserved from yeast to man.
Article
Full-text available
The yeast alpha 2 protein, the product of the MAT alpha 2 gene, is a regulator of yeast cell type; it turns off transcription of the a-specific genes by binding to an operator located upstream of each gene. In this paper we describe the domain structure, subunit organization, and some unusual features of the way this protein contacts its operator. We show that the protein is folded into two domains. The carboxy-terminal domain binds specifically to the operator; the amino-terminal domain contains dimerization contacts. The alpha 2 dimer differs from those of the phage repressors in that it is flexible and therefore is able to bind tightly to differently spaced operator half-sites. In the natural operator, the centers of the operator half-sites are two and one-half turns of DNA apart, exposing them on opposite sides of the DNA helix. We show that the design of alpha 2 allows a dimer to reach across its operator such that it occupies the two half-sites but leaves the middle of the operator available to other proteins.
Article
Full-text available
We have investigated the sequence requirements for induction of the human c-fos gene by epidermal growth factor (EGF), 12-O-tetradecanoyl-13-acetate (TPA), and the calcium ionophore A23187 by transfecting c-fos promoter mutants into HeLa and A431 cells. Induction by both EGF and TPA in HeLa cells required the presence of the c-fos enhancer located at -317 to -298 relative to the mRNA cap site. A23187, however, did not induce expression of the transfected gene, even though it strongly induced expression of the endogenous gene, suggesting that it has different requirements for induction than do EGF and TPA. We have also investigated the role of promoter sequences downstream of the enhancer in general expression and induction of c-fos. A sequence between -97 and -76, which includes an 8-base-pair perfect direct repeat, was needed for efficient general expression but not for induction of the gene. A factor in nuclear extracts that bound specifically to this sequence was detected by a gel mobility shift assay. A 7-base-pair sequence, located between -63 and -57 relative to the mRNA cap site and previously shown to be important for general expression of mouse c-fos, was also important for general expression of the human gene. In addition, this element was important for inducibility by EGF and TPA, since induction was significantly reduced when internal deletion mutants that retained the enhancer but lacked the -63 to -57 sequence element were analyzed in transfecting assays.
Article
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Transcription of the c-fos proto-oncogene is rapidly induced in the rat pheochromocytoma PC12 cell line by a wide variety of stimuli, including polypeptide growth factors, phorbol esters, and calcium ion fluxes. We have mapped the upstream sequence requirements for this activation in PC12 cells by analysis of promoter deletion mutants in a transient expression assay. Two distinct pathways of c-fos induction are defined that differ in their requirement for cis-acting DNA sequences. Calcium activation of c-fos transcription is dependent on a DNA element located approximately 60 base pairs upstream of the transcription start site. This region is highly conserved between human, mouse, and chicken c-fos genes and contains a sequence that resembles the consensus for a cyclic AMP response element. The dyad symmetry element at position -300, which is necessary for serum responsiveness of c-fos, appears to be unimportant for calcium activation of the gene. The dyad symmetry element is, however, an essential cis-acting sequence for c-fos inducibility by nerve growth factor, epidermal growth factor, fibroblast growth factor, and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. Studies in vivo and in vitro with various mutants of the dyad symmetry element indicate that c-fos activation by polypeptide growth factors and 12-O-tetradecanoyl activation by polypeptide growth factors and 12-O-tetradecanoyl phorbol-13-acetate is mediated by a common transcription factor, and that this factor is identical to the previously described serum response factor. In vitro DNA-binding assays suggest that the quantity of serum response factor-binding activity remains unchanged during c-fos transcriptional activation.
Article
Full-text available
We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).
Article
Based on nucleotide sequence determination, we have identified two new yeast genes FUN80 and FUN81 located on chromosome XIII. They are both essential for cellular growth but their function is still unknown. FUN80 is closely linked to the ARGRI (or ARG80) gene while FUN81 is located next to the ARGRII (or ARG81) gene. Interestingly, the proteins encoded by these two genes have a long stretch of acidic amino acids within their C-terminal portions.
Article
Transcription of the c-fos gene is transiently activated to generate large amounts of unstable c-fos RNA when quiescent fibroblasts are stimulated by polypeptide mitogens or whole serum. A cloned human c-fos gene (c-fosH) transfected into mouse fibroblasts is regulated in a similar manner. An element essential for transcription activation is located between nucleotides -332 and -276 relative to the mRNA cap site. This element has properties similar to those of previously characterized transcription enhancer elements. However, replacement of the 5' activating element by enhancers from SV40 or Moloney murine leukemia virus does not allow regulated c-fosH expression. The study of fusion genes showed that in addition to the 5' activating element, transient accumulation of c-fosH RNA following serum stimulation requires sequences at the 3' end of the c-fosH gene.
Article
Transcription of the c-fos proto-oncogene is rapidly induced by serum growth factors. A short c-fos DNA element, the serum response element (SRE), is required for this response to serum. However, serum activates a series of distinct intracellular signaling pathways, and it is not known to which of these pathways the SRE responds. To address this question, mutations have been introduced into the SRE of an otherwise intact c-fos promoter/enhancer. These mutations strongly reduce the binding of a nuclear factor to this site. Plasmids carrying either a wild-type or mutant c-fos SRE were transfected into fibroblasts and tested for their response to whole serum, purified recombinant c-sis protein, the protein kinase C activator phorbol myristate acetate, and activators of the cyclic AMP (cAMP) second messenger system. Assays were carried out under normal conditions and after chronic phorbol ester-treatment to deplete phorbol ester activatable protein kinase C activity from transfected cells. The results show that the SRE is necessary and sufficient for response to both protein kinase C-dependent and -independent intracellular signaling pathways but not for response to the cAMP pathway.
Article
GAL4, a protein that activates transcription in yeast, is shown to activate the mouse mammary tumor virus promoter in mammalian cells. Activation depends upon a GAL4-binding sequence inserted upstream of the gene. Deletion mutants of GAL4 bearing one or both of the "activating regions" required for activation in yeast also activate transcription in mammalian cells. A derivative of GAL4 that binds to DNA but cannot activate transcription in yeast also fails to activate transcription in mammalian cells. We also show that GAL4 and the glucocorticoid receptor activate the mouse mammary tumor virus promoter synergistically.
Article
The consensus recognition element for the mammalian transcription factor AP-1 is very similar to that of the transcriptional activator GCN4. Here, we show that the AP-1 recognition element (ARE) found in the SV40 enhancer can activate transcription from a heterologous promoter in S. cerevisiae. This activation, however, is not dependent on the presence of GCN4 as evidenced by ARE-dependent transcription in a gcn4 yeast strain. A previously unknown yeast transcription factor that is probably responsible for this activation was identified and highly purified. The yeast factor, designated yAP-1, shares remarkably similar biochemical and DNA-binding characteristics with mammalian AP-1. These data suggest that the yeast and mammalian AP-1 are evolutionarily conserved and perhaps functionally related. Also note-worthy is that GCN4 can bind to a GCN4 recognition element (GCRE) and to the ARE with approximately equal affinities; yAP-1, however, has a much lower affinity for the GCRE than the ARE, suggesting that yAP-1 can discriminate between these elements in vivo.
Article
The SV40 promoter is expressed well in the fission yeast S. pombe, and it initiates transcription at the same site as in mammalian cells. The majority of the enhancer sequences, however, do not contribute to this activity. DNAase I footprint analysis of the promoter revealed the presence of an AP-1-like factor in S. pombe cells that protects a region of the promoter almost identical to that protected by human AP-1. The specificity of binding of the yeast and mammalian AP-1 proteins was found to be similar. We have found two AP-1-like binding activities in budding yeast cells, one of which appears quite distinct from the binding activity of the product of the budding yeast GCN4 gene. We also demonstrate that in fission yeast the AP-1 binding site can act as an upstream activating sequence. The DNA-protein complexes containing the mammalian AP-1 and fission yeast AP-1-like factors are sensitive to phosphatase treatment, indicating that they may be phosphorylated.
Article
Serum-induced transcription of the proto-oncogene fos is under negative feedback regulation mediated by the fos protein. The fos promoter region responsive to repression is also required for serum inducibility and binds a nucleoprotein complex in which the nuclear factor AP-1 is associated with fos protein.
Article
This chapter reviews the biology of Platelet-derived growth factor (PDGF) with an emphasis on understanding the relationship between growth factors and cell transformation. It also discusses the regulation of gene expression by PDGF. PDGF has been shown to stimulate the growth of fibroblasts in culture from chickens through humans. In vivo, PDGF is thought to play a role in the maintenance of the vascular lining, though direct evidence is lacking. Through the study of the molecular biology of PDGF, the role of oncogenes in cell transformation and their relationship to growth factors is beginning to be understood. It appears that the deregulation of molecular events that are normally controlled by growth factors can lead to cell transformation. Several known oncogenes can be associated with specific molecular events regulated by growth factors. Some oncogenes, such as the sis gene, may code for a growth factor that is capable of stimulating cell growth through an autocrine-type mechanism. Other oncogenes may code for functions similar to events inside the cell that are regulated by growth factors. It seems quite likely that some oncogenes will either turn out to be growth factor receptors or mimic the functions of them. This may be a likely explanation for the coincidence that a large number of oncogenes as well as growth factor receptors are tyrosine kinases.
Article
We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.
Article
The c-fos gene is rapidly and transiently activated in quiescent BALB/c-3T3 cells in response to serum, platelet-derived growth factor or conditioned medium from v-sis-transformed cells. This activation occurs at the level of transcription and in the absence of new protein synthesis. Using a gel electrophoresis DNA-binding assay, we have found a DNA-binding activity in BALB/c-3T3 cells that is induced within 20 min of treatment with conditioned medium from v-sis-transformed cells. A DNA methylation interference assay has shown that this factor binds to a sequence approximately 346 base pairs upstream of the transcription initiation site of the human c-fos gene. Insulin, epidermal growth factor, and phorbol 12-myristate 13-acetate fail to induce this DNA-binding factor. Protein synthesis inhibitors do not block the induction of this activity. We propose that this factor preexists in an inactive form in quiescent cells and that its binding activity is activated in response to appropriate extracellular inducers.
Article
The jun oncoprotein, which causes sarcomas in chickens, and the DNA-binding domain of yeast GCN4, which coordinately regulates the expression of amino acid biosynthetic genes, show significant homology. In yeast cells deleted for the GCN4 gene, GCN4 function can be conferred by a hybrid protein in which the GCN4 DNA-binding domain is replaced by the homologous region of jun. Moreover, in strains containing various mutations of the GCN4 binding site in the HIS3 promoter, HIS3 expression is affected similarly by the hybrid protein and by GCN4. These results indicate that the jun oncoprotein binds the same DNA sequences as GCN4, and strongly suggest that jun is derived from a normal cellular transcription factor (possibly AP-1, which recognizes similar sequences). This provides direct evidence for the idea that alterations in the machinery for proper gene expression can lead to the oncogenic state.
Article
We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences (ARSs). These mutants are known as mcm for their defect in the maintenance of minichromosomes. We have now characterized in more detail one ARS-specific mutation, mcm1-1. This Mcm1 mutant has a second phenotype; MAT alpha mcm1-1 strains are sterile. MCM1 is non-allelic to other known alpha-specific sterile mutations and, unlike most genes required for mating, it is essential for growth. The alpha-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, alpha-factor. In addition, transcripts of the MF alpha 1 and STE3 genes, which encode the alpha-factor precursor and the alpha-factor receptor, respectively, are greatly reduced in this mutant. These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of alpha-specific genes. We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7. The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts. MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1. A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the alpha-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.
Article
Trans-acting factors that mediate B-cell specific transcription of immunoglobulin genes have been postulated based on an analysis of the expression of exogenously introduced immunoglobulin gene recombinants in lymphoid and non-lymphoid cells. Two B-cell-specific, cis-acting transcriptional regulatory elements have been identified. One element is located in the intron between the variable (V) and constant (C) regions of both heavy and kappa light-chain genes and acts as a transcriptional enhancer. The second element is found upstream of both heavy and kappa light-chain gene promoters. This element directs lymphoid-specific transcription even in the presence of viral enhancers. We have sought nuclear factors that might bind specifically to these two regulatory elements by application of a modified gel electrophoresis DNA binding assay. We report here the identification of a human B-cell nuclear factor (IgNF-A) that binds to DNA sequences in the upstream regions of both the mouse heavy and kappa light-chain gene promoters and also to the mouse heavy-chain gene enhancer. This sequence-specific binding is probably mediated by a highly conserved sequence motif, ATTTGCAT, present in all three transcriptional elements. Interestingly, a factor showing similar binding specificity to IgNF-A is also present in human HeLa cells.
Article
We have identified a factor in nuclear extracts that binds to the c-fos enhancer. Treatment of A431 cells with epidermal growth factor results in a rapid increase in the level of transcription and a concomitant increase in binding of the factor to the enhancer. Surprisingly, as transcription decreases rapidly, the enhancer-binding activity remains elevated. In addition, although HeLa cells exhibit no detectable transcription of the c-fos gene, they contain significant amounts of binding activity, comparable to those in induced A431 cells. These results suggest that regulation of c-fos transcription involves more than simply an increased level of a factor capable of binding the enhancer. Finally, transcription of c-fos in A431 cells is markedly induced by the tumor promoter TPA and the calcium ionophore A23187, yet neither induced an increased level of the enhancer-binding activity. These agents thus appear to activate c-fos transcription via a mechanism distinct from that used by epidermal growth factors.
Article
The heat shock transcription factor (HSTF) has been purified to apparent homogeneity from S. cerevisiae and D. melanogaster by sequence-specific DNA-affinity chromatography. A synthetic oligonucleotide containing an hsp83-like heat shock element (HSE) was prepared and ligated into concatamers and covalently coupled to Sepharose. This DNA-affinity resin allowed the rapid isolation of a yeast and a Drosophila protein with the same apparent molecular weight (70 kd). The yeast HSTF will bind to both its own and the Drosophila HSEs. Similarly, the Drosophila HSTF will bind to both its own and the yeast HSEs. The yeast and Drosophila HSTFs were subjected to preparative SDS gel electrophoresis, and the 70 kd polypeptides were eluted, renatured, and observed to generate the identical footprint pattern as the native HSTFs. Affinity-purified Drosophila HSTF was further shown to stimulate specific HSE-dependent transcription from a Drosophila hsp70 gene in vitro.
Article
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Article
The c-fos proto-oncogene is rapidly inducible by a variety of extracellular stimuli. In order to dissect the intracellular signalling pathways responsible for c-fos induction, we have used a gel electrophoresis DNA-binding assay to identify trans-acting factors that bind to c-fos regulatory regions. We have identified a factor in Balb/c-3T3 cells that binds to an oligonucleotide, containing sequences -351 to -337 of the human c-fos gene. This factor is inducible in quiescent cells within 30 min of addition of conditioned medium from v-sis transformed cells. Cycloheximide fails to block this induction. This result suggests that the factor is present in an inactive form in quiescent cells and is activated only in response to the appropriate inducer. Fibroblast growth factor (FGF) and the tumor promoter phorbol myristate acetate (TPA) induce the c-fos gene but not this DNA-binding activity. We propose from this that there are multiple regulatory regions upstream of c-fos each capable of responding to a different set of stimuli.
Article
STE3 mRNA is present only in Saccharomyces cerevisiae alpha cells, not in a or a/alpha cells, and the transcript level increases about fivefold when cells are treated with a-factor mating pheromone. Deletions in the 5' noncoding region of STE3 defined a 43-base-pair (bp) upstream activation sequence (UAS) that can impart both modes of regulation to a CYC1-lacZ fusion when substituted for the native CYC1 UAS. UAS activity required the alpha 1 product of MAT alpha, which is known to be required for transcription of alpha-specific genes. A chromosomal deletion that removed only 14 bp of the STE3 UAS reduced STE3 transcript levels 50- to 100-fold, indicating that the UAS is essential for expression. The STE3 UAS shares a 26-bp homology with the 5' noncoding sequences of the only other known alpha-specific genes, MF alpha 1 and MF alpha 2. We view the homology as having two components--a nearly palindromic 16-bp "P box" and an adjacent 10-bp "Q box." A synthetic STE3 P box was inactive as a UAS; a perfect palindrome P box was active in all three cell types. We propose that the P box is the binding site for a transcription activator, but that alpha 1 acting via the Q box is required for this activator to bind to the imperfect P boxes of alpha-specific genes. Versions of the P box are also found upstream of a-specific genes, within the binding sites of the repressor alpha 2 encoded by MAT alpha. Thus, the products of MAT alpha may render gene expression alpha or a-specific by controlling access of the same transcription activator to its binding site, the P box.
Article
The S. cerevisiae HAP2 and HAP3 gene products have been shown to recognize CCAAT-containing transcription elements as a HAP2/HAP3 multisubunit heterologous complex, or heteromer. Recently, we have demonstrated that the human sequence-specific DNA-binding protein, CP1, also recognizes CCAAT-containing transcription elements as a heteromer. Mammalian cells contain at least three distinct CCAAT-binding proteins, all of which appear to be multisubunit complexes composed of heterologous subunits. One of these proteins, CP1, has DNA-binding properties that are virtually identical to the yeast HAP2/HAP3 complex. These two proteins bind to the same target sequences, make the same DNA contacts, and are affected in a similar manner by mutations in the CCAAT element. Most surprisingly, the subunits of CP1 and HAP2/HAP3 are functionally interchangeable. That is, the yeast/human hybrid complexes that are formed retain the ability to specifically recognize CCAAT elements.
Article
The yeast alpha 2 protein is a cell-type-specific transcriptional repressor. It acts by binding to an operator located upstream of each of its target genes. In this paper, we describe a protein (GRM) that is present in all cell types and binds cooperatively with alpha 2 resulting in an unusual arrangement of the two proteins at the operator. A dimer of alpha 2 occupies the two ends of the operator and straddles the GRM protein, which binds to the center of the operator. Using mutant operators, we show that the recognition sequences for both GRM and alpha 2 are required for repression of a test promoter in vivo. Finally, we deduce that the GRM/alpha 2 cooperativity is mediated through a protein-protein interaction between GRM and the N-terminal domain of alpha 2. This conclusion follows from the observation that the isolated C-terminal domain of alpha 2 can co-occupy the operator with GRM but does not bind cooperatively with GRM.
Article
Most class B (II) promoter regions from higher eukaryotes contain the TATA box and upstream and enhancer elements. Both the upstream and enhancer elements and their cognate factors have regulatory functions, whereas the TATA sequence interacts with the TATA box factor BTF1 to position RNA polymerase B and its ancillary initiation factors (STF, BTF2 and BTF3) to direct the initiation of transcription approximately 30 base pairs downstream. In many respects, class B promoter regions from the unicellular eukaryote Saccharomyces cerevisiae are similarly organized, containing upstream activating sequences that bear many similarities to enhancers. Although they are essential for initiation, the yeast TATA sequences are located at variable distances and further from the start sites (40-120 base pairs), whose locations are primarily determined by an initiator element. The basic molecular mechanisms that control initiation of transcription are known to be conserved from yeast to man: the yeast transcriptional transactivator GAL4 can activate a minimal TATA box-containing promoter in human HeLa cells, and a human inducible enhancer factor, the oestrogen receptor, can activate a similar minimal promoter in yeast. This striking evolutionary conservation prompted us to look for the presence in yeast of an activity that could possibly substitute for the human TATA box factor. We report here the existence of such an activity in yeast extracts.
Article
Saccharomyces cerevisiae contains a protein which is functionally similar to the mammalian TATA element-binding transcription factor, TFIID. The yeast factor substitutes for TFIID in a mammalian RNA polymerase II in vitro transcription system, forms a stable preinitiation complex on the Adenovirus-2 major late promoter, and binds specifically to the TATA boxes of the viral promoter and the yeast CYC1 promoter. Interestingly, the yeast factor promotes initiation at a distance from the TATA element typical of a mammalian system.
Article
The product of the BAR1 gene of Saccharomyces cerevisiae is synthesized only in the a cell type and inactivates alpha-factor, the mating pheromone made by alpha cells. The MAT alpha 2 protein represses the transcription of a-cell-specific genes, including BAR1, in alpha and a/alpha diploid cells. Transcription of BAR1 in a cells in stimulated upon exposure to alpha-factor. Deletion analysis of the 5' noncoding region of the BAR1 gene revealed that the major upstream activation site (UAS) overlaps the 31 bp operator sequence required for MAT alpha 2 repression. This result has implications for the negative control of transcription in yeast. The deletion analysis also indicated that the sequence TGAAACA mediates alpha-factor stimulation.
Article
We show by electrophoresis mobility shift and by DNAase I footprinting assays that the alpha 1 product of the yeast alpha mating-type locus binds to homologous sequences within the control regions of the three known alpha-specific genes. Binding requires both alpha 1 and a second yeast protein(s) (called PRTF) that is present in all three cell types (a, alpha, and a/alpha); neither protein binds alone. Binding and competition experiments using synthetic oligonucleotides indicate that PRTF binds to only part of the homology found at alpha-specific genes and imply that alpha 1 binds to the remainder. Our results suggest that alpha 1 renders gene expression alpha-specific by creating a binding site for PRTF. Similar experiments lead to the idea that PRTF also plays a role in transcription of a-specific genes. Perhaps a-specificity is achieved through the occlusion of the PRTF binding site by alpha 2, the negative regulator encoded by the alpha mating-type locus.
Article
Transient transcriptional activation of the c-fos gene following serum stimulation of susceptible cells requires a conserved DNA element located 300 bp 5' to the mRNA cap site. A DNA-binding gel electrophoresis assay was used to detect a protein(s) in HeLa cell nuclear extracts that specifically binds to the 5' activating element. The protein recognizes a region of dyad symmetry within the 5' activating element, defined by binding competition, dimethylsulphate (DMS) interference and DNAase I and DMS protection studies. A single 22 bp synthetic copy of the dyad symmetry element will both compete efficiently for protein binding and restore serum regulation to c-fosH genes that lack the 5' activating element.
Article
In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene.
Article
Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution.
Article
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Article
The product of the yeast MAT alpha 2 gene, alpha 2 protein, negatively regulates expression of a class of yeast cell type specific genes, the a-specific genes. In this paper we show that alpha 2 is a sequence-specific DNA binding protein. It recognizes a 32 base pair DNA sequence (operator) located upstream of an a-specific gene, STE6. A strongly homologous sequence is present upstream of each of the other four known a-specific genes. A synthetic STE6 operator, when placed in a test promoter (CYC1), brings the promoter under negative control by alpha 2 in vivo. This operator brings about repression when it is placed between the CYC1 upstream activation sequence (UAS) and the transcription start and when it is placed upstream of the UAS, outside the promoter. Thus, the operator need not overlap with essential promoter sequences to permit repression by alpha 2.
Article
Transcription of the c-fos proto-oncogene is greatly increased within minutes of administering purified growth factors to quiescent 3T3 cells. This stimulation is the most rapid transcriptional response to peptide growth factors yet described, and implies a role for c-fos in cell-cycle control. Transformation by c-fos may result from a temporal deregulation of this control.
Article
Complementary DNA clones of genes induced by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells were isolated; one such clone contains a domain having nucleotide sequence homology with the third exon of c-fos. This nucleotide sequence homology is reflected in the predicted amino acid sequences of the gene products. Under low stringency conditions, the mouse v-fos gene cross-hybridizes with the PDGF-inducible complementary DNA clone. However, the messenger RNA transcripts of mouse c-fos and the new fos-related gene can be distinguished by gel electrophoresis and by S1 nuclease analysis. Expression of the authentic c-fos gene is induced by PDGF and superinduced by the combination of PDGF and cycloheximide.
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The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promoter, but not with a CAP-insensitive mutant. Formation of a stable “open” or “melted-in” complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.
Article
We show that expression of the iso-1-cytochrome c gene of Saccharomyces cerevisiae, CYC1, is tightly regulated by levels of intracellular heme. Expression is reduced at least 200-fold in cells grown under conditions of heme deficiency. Studies on the regulation of a CYC1-lacZ fused gene and direct determination of mRNA levels indicate that this control is transcriptional. Furthermore, we show that the heme regulatory site in the CYC1 promoter region is an upstream activation site (UASc) centered about 275 bp upstream from the region of transcriptional initiation. The latter region is required for optimal transcription and contains three TATA box sequences and six prominent mRNA initiation sites that span 34 bp. Substitution of the UASc with the UAS of the yeast GAL10 gene results in activation of the normal set of CYC1 transcripts. In this case, however, transcription is independent of regulation by heme, suggesting that in the wildtype, heme controls initiation per se and not translation or mRNA stability.
Article
The upstream activation site (UAS) of the yeast CYC1 gene is shown to contain two homologous subsites, UAS1 and UAS2. Each site, when placed upstream of the transcriptional initiation region of the yeast LEU2 gene, activates LEU2 transcription which is regulated by catabolite repression. UAS1 is responsible for most of the transcription under glucose repressed conditions, while UAS1 and UAS2 contribute equally to lactate derepressed transcription. A single point mutation in UAS2 increases its activity in glucose 10- to 20-fold. Several experiments indicate that UAS1 and UAS2 are regulated distinctly at the molecular level. First, UAS1 but not UAS2 is fully depressed in glucose by increasing the levels of intracellular heme. Second, trans-acting regulatory mutations, hap1-1 and hap2-1, selectively abolish the activity of UAS1 or UAS2. HAP1 appears to encode a protein that mediates catabolite repression of UAS1 by responding to intracellular heme levels.
Article
We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.
Article
Stimulation of fibroblasts with serum or purified growth factors leads to a dramatic induction of expression of both c-fos mRNA and protein within a few minutes, followed by activation of c-myc. This suggests that c-fos induction is a primary event and the earliest known effect on gene expression by growth factors.
Induc-tion of c-fos gene and protein by growth factors precedes ac-tivation of c-myc A Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MATa cells Inducible binding of a factor to the c-fos enhancer Purification of the c-los enhancer binding protein
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  • R Bravo
  • J Burckhardt
  • T Curran
  • S Passmore
  • G T Maine
  • R Elble
  • C Christ
  • B.-K Tye
  • R Prywes
  • R G Roeder
Muller, R., R. Bravo, J. Burckhardt, and T. Curran. 1984. Induc-tion of c-fos gene and protein by growth factors precedes ac-tivation of c-myc. Nature 312: 716-720. Passmore, S., G.T. Maine, R. Elble, C. Christ, and B.-K. Tye. 1988. A Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MATa cells. J. Mol. Biol. (in press). Prywes, R. and R.G. Roeder. 1986. Inducible binding of a factor to the c-fos enhancer. Cell 47: 777-784. • 1987. Purification of the c-los enhancer binding protein