Genes & Development

Published by Cold Spring Harbor Laboratory Press
Online ISSN: 0890-9369
Publications
Article
The stage-specific embryonic antigen SSEA-1 is a cell-surface oligosaccharide molecule expressed with temporal precision during the murine preimplantation period and implicated in adhesive events involving the process of compaction. We used a mammalian transient expression system to isolate a cloned human cDNA that determines expression of the SSEA-1 molecule. The cDNA sequence predicts a type II transmembrane protein with a domain structure similar to mammalian glycosyltransferases, but without primary sequence similarity to these enzymes. The carboxy-terminal domain of this protein was shown to be catalytically active as a fucosyltransferase when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. The enzyme is an exceptional glycosyltransferase in that it can use both type I and type II oligosaccharides as acceptor substrates to generate subterminal Fuc alpha(1,4)- and Fuc alpha(1,3)-linkages, respectively, in a manner analogous to the human Lewis blood group fucosyltransferase. Southern blot analysis shows that the cDNA corresponds to sequences syntenic to the Lewis locus on chromosome 19. These results indicate that this cDNA is the product of the human Lewis blood group locus, provide genetic confirmation of the hypothesis that this enzyme can catalyze two distinct transglycosylation reactions, and outline an approach to the isolation of other sequences that determine expression of developmentally regulated oligosaccharide antigens.
 
Article
The blood group i/I antigens were the first identified alloantigens that display a dramatic change during human development. The i and I antigens are determined by linear and branched poly-N-acetyllactosaminoglycans, respectively. In human erythrocytes during embryonic development, the fetal (i) antigen is replaced by the adult (I) antigen as a result of the appearance of a beta-1,6-N-acetylglucosaminyltransferase, the I-branching enzyme. Here, we report the cDNA cloning and expression of this branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans, thus introducing the I antigen in transfected cells. The cDNA sequence predicts a protein with type II membrane topology as has been found for all other mammalian glycosyltransferases cloned to date. The Chinese hamster ovary cells that stably express the isolated cDNA acquire I-branched structures as evidenced by the structural analysis of glycopeptides from these cells. Comparison of the amino acid sequence with those of other glycosyltransferases revealed that this I-branching enzyme and another beta-1,6-N-acetylglucosaminyltransferase that forms a branch in O-glycans are strongly homologous in the center of their putative catalytic domains. Moreover, the genes encoding these two beta-1,6-N-acetylglucosaminyltransferases were found to be located at the same locus on chromosome 9, band q21. These results indicate that the I-branching enzyme represents a member of a beta-1,6-N-acetylglucosaminyltransferase gene family of which expression is controlled by developmental programs.
 
Article
We report that the murine Hox-1.3 homeo domain protein is a nuclear phosphoprotein capable of binding to specific DNA sequences. DNase I protection of the Hox-1.3 gene promoter region with the Hox-1.3 protein identifies a binding site 144 bp upstream from the start of transcription. Both phosphorylated and nonphosphorylated forms bind DNA directly in a sequence-specific manner. Electrophoretic mobility shift assays were performed with a set of synthetic oligonucleotides representing either the DNase I-protected region of the Hox-1.3 gene or partially homologous sequences present in promoter regions of other characterized viral, yeast, and mammalian genes. From the results, we deduce a consensus binding motif of CPyPyNATTAT/GPy. Base substitutions in the core ATTA sequence severely reduce or abolish binding. In the SV40 enhancer, the Hox-1.3 binding motif overlaps both the octamer (Octa2) and the transactivator protein-1 (AP-1) binding sites. The Hox-1.3 binding motif also overlaps the nuclear factor III (NF-III) octamer motif in the adenovirus-2 origin of DNA replication. Overlap among DNA-binding sites suggests that regulation imparted by certain cis-elements may be integrated by these different factors.
 
Article
To test whether the mouse Hox-1.3 gene is a cognate of the Drosophila Sex combs reduced (Scr) gene, we inserted a hsp 70-Hox-1.3 fusion gene into the Drosophila genome. Transgenic flies displayed Scr-like homeotic transformations after ectopic expression of Hox-1.3 induced by heat shock. In larvae, the thoracic segments T2 and T3 are transformed toward T1. In adults, head structures are dramatically disrupted, including transformation of antenna towards leg. Transformations are not the result of ectopic activation of the endogenous Scr gene. Rather, Hox-1.3 appears to directly regulate Scr target genes, as demonstrated by the ectopic activation of fork head by Hox-1.3. The results suggest that mouse Hox-1.3 cannot only substitute functionally for Drosophila Scr in the determination of external structures but also can participate in the regulatory hierarchy of insect organogenesis.
 
Article
To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.
 
Article
E4BP4, a basic leucine zipper transcription factor, contains a DNA-binding domain closely related to DBP, HLF, and TEF, which are PAR proteins. Here, we show that the phase of e4bp4 mRNA rhythm is opposite to that of the dbp, hlf, and tef rhythms in the suprachiasmatic nucleus (SCN), the mammalian circadian center, and the liver. The protein levels of E4BP4 and DBP also fluctuate in almost the opposite phase. Moreover, all PAR proteins activate, whereas E4BP4 suppresses, the transcriptional activity of the reporter gene containing a common binding sequence in transcriptional assays in vitro. An electrophoretic mobility shift assay demonstrated that E4BP4 is not able to dimerize with the PAR proteins, but is able to compete for the same binding sites with them. Furthermore, we showed sustained low e4bp4 and high dbp mRNA levels in mCry-deficient mice. These results indicate that the E4BP4 and PAR proteins are paired components of a reciprocating mechanism wherein E4BP4 suppresses the transcription of target genes during the time of day when E4BP4 is abundant, and the PAR proteins activate them at another time of day. E4BP4 and the PAR proteins may switch back and forth between the on-off conditions of the target genes.
 
Article
Although most mRNAs initiate translation by 5' ribosome scanning, some small fraction of mammalian and viral mRNAs utilize either of two alternate mechanisms, known as internal ribosome entry and ribosome shunting. Ribosome shunting is a poorly understood form of initiation in which 40S ribosome subunits are loaded onto mRNA through interactions with the m7GTP cap, but then bypass large segments of the mRNA as directed by cis-acting RNA shunting elements and trans-acting protein factors. Here, we describe the molecular mechanism by which ribosome shunting occurs with high efficiency on adenovirus late mRNAs. We show that the viral 100k protein possesses a selective binding element for the 5' noncoding region (5'NCR) of viral late mRNAs (known as the tripartite leader), forms a complex with initiation factor eIF4G and poly(A)-binding protein (PABP), and strongly and selectively enhances the level of both factors and 40S ribosome subunits on viral mRNAs in polysomes. Mutational and biochemical studies demonstrate that the ability of 100k protein to bind both the tripartite leader and eIF4G are critical to promote a high level of ribosome shunting. A molecular mechanism for ribosome shunting is described by which enhanced binding of eIF4G and possibly PABP with 100k protein, and simultaneous interaction with the tripartite leader 5'NCR, drives 40S ribosome recruitment and initiation on mRNAs.
 
Article
Gene regulation by AP-1 transcription factors in response to Jun N-terminal kinase (JNK) signaling controls essential cellular processes during development and in pathological situations. Here, we report genetic and molecular evidence that the histone acetyltransferase (HAT) Chameau and the histone deacetylase DRpd3 act as antagonistic cofactors of DJun and DFos to modulate JNK-dependent transcription during thorax metamorphosis and JNK-induced apoptosis in Drosophila. We demonstrate in cultured cells that DFos phosphorylation mediated by JNK signaling plays a central role in coordinating the dynamics of Chameau and DRpd3 recruitment and function at AP-1-responsive promoters. Activating the pathway stimulates the HAT function of Chameau, promoting histone H4 acetylation and target gene transcription. Conversely, in response to JNK signaling inactivation, DRpd3 is recruited and suppresses histone acetylation and transcription. This study establishes a direct link among JNK signaling, DFos phosphorylation, chromatin modification, and AP-1-dependent transcription and its importance in a developing organism.
 
Article
Our genetic analysis indicates that the unc-101 gene of Caenorhabditis elegans is required for many aspects of development and behavior, including negative regulation of vulval differentiation. We have cloned unc-101 and found that it encodes a homolog of the mammalian medium chains of clathrin-associated protein complexes located at the trans-Golgi and the plasma membrane, AP47 and AP50, respectively. Therefore, clathrin-mediated events might contribute to the negative regulation of vulval differentiation. Comparison of sequences, including a full-length sequence of a C. elegans AP50 homolog, reveals that UNC-101 is most closely related to AP47. Mouse AP47 and nematode UNC-101 proteins are functionally equivalent as assayed in transgenic nematodes. We have sequenced the mutant alleles of unc-101 identified in various genetic screens and shown that all but one are deletions or nonsense mutations, suggesting that these alleles severely reduce unc-101 function.
 
Article
Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate significantly from the average genomic pattern by being GC-rich, CpG-rich, and predominantly nonmethylated. Most, perhaps all, CGIs are sites of transcription initiation, including thousands that are remote from currently annotated promoters. Shared DNA sequence features adapt CGIs for promoter function by destabilizing nucleosomes and attracting proteins that create a transcriptionally permissive chromatin state. Silencing of CGI promoters is achieved through dense CpG methylation or polycomb recruitment, again using their distinctive DNA sequence composition. CGIs are therefore generically equipped to influence local chromatin structure and simplify regulation of gene activity.
 
Article
We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.
 
Article
A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.
 
The sequence between −230 and −265 is essential for embryonic and post-natal transcription of Blbp. Finer deletions within the −230 to −400 interval defines the sequence between −230 and −265 (deletion 12, arrow) as critical for expression in both the E12 forebrain and P6 cerebellum. Note that the other three constructs drove expression at one of the two locations assayed, suggesting that elements in those intervals are mostly involved in regional and/or temporal control of Blbp transcription. Representative whole-mount histochemical staining for-galactosidase activity of founders is shown at the bottom; the construct used and assay time point are indicated in the upper left of each panel. The strong expression in the developing olfactory bulbs seen at E12.5 for construct 9 was observed in two independent founders. (*) Construct 10 drove complete embryonic CNS expression but was noticeably weaker than the full-length 766-bp promoter.
Article
Radial glia function during CNS development both as neural progenitors and as a scaffolding supporting neuronal migration. To elucidate pathways involved in these functions, we mapped in vivo the promoter for Blbp, a radial glial gene. We show here that a binding site for the Notch effector CBF1 is essential for all Blbp transcription in radial glia, and that BLBP expression is significantly reduced in the forebrains of mice lacking the Notch1 and Notch3 receptors. These results identify Blbp as the first predominantly CNS-specific Notch target gene and suggest that it mediates some aspects of Notch signaling in radial glia.
 
Article
Males of the MyK-103 line of transgenic mice are fertile and sire litters of normal size, but they never transmit the transgene, whereas females transmit the transgene with normal frequency. The chromosome originally bearing the transgene can be transmitted through the male germ line, but only after the transgene is deleted or rearranged by intrachromosomal recombination. The transgene encodes a functional herpes simplex virus (HSV) thymidine kinase gene that causes sperm infertility when expressed in postmeiotic germ cells. Immunocytochemistry revealed clones of germ cells that fail to express HSV thymidine kinase. We postulate that these cells arose by transgene deletion in embryonic germ cells and postnatal spermatogonial stem cells and that they are responsible for the normal fertility of MyK-103 males. The frequency of recombination events at the integration locus suggests that it contains a hotspot for mitotic recombination.
 
Article
The embryonic expression of COUP-TFII, an orphan nuclear receptor, suggests that it may participate in mesenchymal-epithelial interactions required for organogenesis. Targeted deletion of the COUP-TFII gene results in embryonic lethality with defects in angiogenesis and heart development. COUP-TFII mutants are defective in remodeling the primitive capillary plexus into large and small microcapillaries. In the COUP-TFII mutant heart, the atria and sinus venosus fail to develop past the primitive tube stage. Reciprocal interactions between the endothelium and the mesenchyme in the vascular system and heart are essential for normal development of these systems. In fact, the expression of Angiopoietin-1, a proangiogenic soluble factor thought to mediate the mesenchymal-endothelial interactions during heart development and vascular remodeling, is down-regulated in COUP-TFII mutants. This down-regulation suggests that COUP-TFII may be required for bidirectional signaling between the endothelial and mesenchymal compartments essential for proper angiogenesis and heart development.
 
Article
Mammalian cells are believed to have a cell-intrinsic ability to increase glucose metabolism in response to hypoxia. Here we show that the ability of hematopoietic cells to up-regulate anaerobic glycolysis in response to hypoxia is dependent on receptor-mediated signal transduction. In the absence of growth factor signaling, hematopoietic cells fail to express hypoxia-inducible transcription factor (Hif-1alpha) mRNA. Growth factor-deprived hematopoietic cells do not engage in glucose-dependent anabolic synthesis and neither express Hif-1alpha mRNA nor require HIF-1alpha protein to regulate cell survival in response to hypoxia. However, HIF-1alpha is adaptive for the survival of growth factor-stimulated cells, as suppression of HIF-1alpha results in death when growing cells are exposed to hypoxia. Growth factor-dependent HIF-1alpha expression reprograms the intracellular fate of glucose, resulting in decreased glucose-dependent anabolic synthesis and increased lactate production, an effect that is enhanced when HIF-1alpha protein is stabilized by hypoxia. Together, these data suggest that HIF-1alpha contributes to the regulation of growth factor-stimulated glucose metabolism even in the absence of hypoxia.
 
Article
Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.
 
RNase protection analysis to measure AP-2 mRNA levels in several murine cell lines and adult mouse tissues. Each lane represents protected RNA segments obtained after hybridizing 5 (jig cytoplasmic RNA with AP-2, Hox2.1, and Dhfi antisense RNA probes simultaneously for 16 hr at 63°C and digesting with RNase to remove unhybridized RNA (R2 probe for AP-2 was used; Fig. IB). The gel autoradiograph bands from one experiment have been cut into strips and arranged for easy reference. 
RNA in situ hybridization to 8.5-dpc mouse embryo sections. Serial paraffin sections (6 ixm) hybridized with AP-2 antisense probe B240 (Fig. IB) were photographed with bright-field illumination [left] to indicate morphology and dark-field illumination [right] to show hybridization signals. Toluidine blue-stained cell nuclei are visible in bright field; probe-specific hybridization signals (silver grains) appear white in dark field. [A] Lateral sagittal (longitudinal) section showing embryo and extraembryonic tissues. The diagonal lines represent the approximate planes of sections in C-E. [B] Close-up of A showing head and upper body of embryo. (C) Section passing coronally through the brain and transverse through the posterior neural plate in a presomitic region (dorsal is up; for orientation, see A). [D] Close-up of head region in C. (£) Frontal section through mid-body region showing neural tube and somites (S). (Mes) Mesencephalon (midbrain); (Rh) rhombencephalon (hindbrain); (S) approximate location of anterior-most somite; (PS) presomitic mesoderm; (hg) hindgut pocket; (AL) allantois; (A) amnion; (EC) extraembryonic coelom; (T) trophoblastic cells; (NC) presumed cranial neural crest cells; (HF) head neural folds; (HM) head mesenchyme; (NP) neural plate; (NF) neural folds; (TE) lateral thin ectoderm (presumptive epidermis); (YS) yolk sac. 
(See facing page for legend.) 
RNA in situ hybridization to 11.5-dpc mouse embryo sections. AP-2 expression in DRG [1], lateral CNS [2], and sympathetic ganglia (3) is noted. Approximate planes for sections in C and D are shown in A. [A] Sagittal section showing expression in DRG, frontonaso-prominence (FN), and lateral portions of the myelencephalon (My, posterior hindbrain) and spinal cord (sc). [See note in Fig. 5D regarding artifact signal from blood cells (be).] (Tel) Telencephalon; (Di) diencephalon; (Met) metencephalon (anterior hindbrain); (Mes) mesencephalon; (Ph) pharynx; (Mn) mandibular arch/tongue. [B] Parasagittal section through the lateral head and upper body, passing through the eye, mandibular (Mn) and hyoid (Hy) arches, pharynx, otic vesicle (ov, auditory vesicle), and heart (Ht). Dark-field views shows AP-2 signal in the lateral spinal cord and hindbrain (extending anteriorly to Met), in DRG (drg), sympathetic ganglia (3) near the heart, and facial-acoustic cranial ganglia (gVII-VIII) [part of signal here is due to extraneous material 
Article
We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.
 
Article
Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy.
 
Article
TFE3 is a DNA-binding protein that activates transcription through the muE3 site of the immunoglobulin heavy-chain enhancer. Its amino acid sequence reveals two putative protein dimerization motifs: a helix-loop-helix (HLH) and an adjacent leucine zipper. We show here that both of these motifs are necessary for TFE3 to homodimerize and to bind DNA in vitro. Using a dominant negative TFE3 mutant, we also demonstrate that both the HLH and the leucine zipper motifs are necessary and sufficient for protein-protein interactions in vivo. TFE3 is unable to form stable heterodimers with a variety of other HLH proteins, including USF, a protein that is structurally similar to TFE3 and binds a common DNA sequence. The analysis of "zipper swap" proteins in which the TFE3 HLH was fused to the leucine zipper region of USF indicates that dimerization specificity is mediated entirely by the identity of the leucine zipper and its position relative to the HLH. Hence, in this "b-HLH-zip" class of proteins, the leucine zipper functions in concert with the HLH both to stabilize protein-protein interactions and to establish dimerization specificity.
 
Article
To define homeo domain subregions that are important for embryonic targeting specificity of homeotic proteins, we generated a series of Deformed/Ultrabithorax chimeric genes in which parts of the Deformed homeo box region were substituted with Ultrabithorax sequences. Chimeric coding regions were attached to heat shock promoters and introduced into the Drosophila genome by P-element transformation. After heat-induced ectopic expression in embryos, we examined the cuticular phenotypes induced by the resulting chimeric proteins. We also tested the ability of the chimeric proteins to regulate transcription units that are normal targets of Deformed and Ultrabithorax. Our results indicate that specific amino acid residues at the amino end of the Ultrabithorax homeo domain are required to specifically regulate Antennapedia transcription; and in the context of a Deformed protein, these amino-end residues are sufficient to switch from Deformed- to Ultrabithorax-like targeting specificity. Although residues in the amino end of the homeo domain are also important in determining a Deformed-like targeting specificity, other regions of the Deformed homeo domain are also required for full activity.
 
Article
The idea that chromosomes have special terminal structures first arose as a consequence of experiments conducted by Muller, who found that treatment of Drosophila with X-rays rarely resulted in terminal deletions or inversions of the chromosomes (Muller 1938). Complementary experiments in maize by McClintock expanded upon the idea that telomeres, the physical ends of chromosomes, are required for chromosome stability, by contrasting the breakage-fusion-bridge cycle resulting from broken dicentric chromosomes with the stability of chromosomes with intact termini (McClintock 1941, 1942). With the dawn of molecular biology, telomeres in most eukaryotes are now known to be composed of short repeated G-rich sequences complexed with proteins to form a special heterochromatin-like structure. More recent experimental manipulation of chromosome termini and of the proteins that bind them have confirmed the early observations of Muller and McClintock, showing that a primary role of telomeres is to insulate the ends of chromosomes both from fusion with other ends and
 
Article
A large portion of the eukaryotic genome is transcribed as noncoding RNAs (ncRNAs). While once thought of primarily as "junk," recent studies indicate that a large number of these RNAs play central roles in regulating gene expression at multiple levels. The increasing diversity of ncRNAs identified in the eukaryotic genome suggests a critical nexus between the regulatory potential of ncRNAs and the complexity of genome organization. We provide an overview of recent advances in the identification and function of eukaryotic ncRNAs and the roles played by these RNAs in chromatin organization, gene expression, and disease etiology.
 
Article
The activity of the E2F transcription factor is controlled by physical association with the retinoblastoma protein (pRB) and two related proteins, p107 and p130. The pRB family members are thought to control different aspects of E2F activity, but it has been unclear what the respective functions of these proteins might be. To dissect the specific functions of pRB, p107, and p130 we have investigated how the expression of E2F-regulated genes is changed in cultures of primary cells lacking each of these family members. Whereas no changes were found in the expression of E2F-target genes in cells lacking either p107 or p130, deregulated expression of E2F targets was seen in cells lacking pRB and in cells lacking both p107 and p130. Surprisingly, the genes that were disregulated in these two settings were completely different. These findings show that pRB and p107/p130 indeed provide different functions in E2F regulation and identify target genes that are dependent on pRB family proteins for their normal expression.
 
Article
The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, we show that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos.
 
Targeted disruption of the murine StatSa gene. {A) Schematic representation of 12 kb of the murine StatSa gene is shown {top). The open box denotes a 5' noncoding exon, and the hatched boxes indicate protein coding exons. The double-headed arrow indicates the 6.9-kb BamHl fragment characteristic of the wild-type allele as defined using a labeled DNA fragment derived from the 5' flanking region of the StatSa gene (probe). (X) Xbal, (B) BamHl, (E) EcoRl, (K) Kpnl and (H) Hindlll sites are indicated. The targeting vector shows schematically the position of the PGK-neomycin gene {neo) and the PGK-thymidine kinase gene (TK), oriented as indicated by the arrows. The vector was designed such that the phosphoglycerate kinase-neo cassette replaced the first two protein coding exons of the StatSa gene together with a noncoding exon and promoter sequences. (B) DNA from wild-type, hemizygous, and homozygous null mice was screened by Southern blot analysis following BamHl digestion and probed with a 32 P-labeled probe derived from the S'-flanking region. 
StatSa-deficient mice display aberrant mammopoiesis. Mammary tissue was biopsied from hemizygous {B,D,F,H] and StatSa-deficient {A,C,E,G) mothers within 12 hr after parturition {A-C,G,H) or after 3 days of suckUng {E,F]. {A,B] Wholemount image; {C,D] sections of the whole mounts shown in A and B; {E,F) sections of tissue after 3 days of suckling. To maintain suckling in the StatSa-deficient mice, the mother was provided with a new litter every 12 hr. Whole mounts were stained with carmine red; sections were stained with hematoxyhn and eosin [C-F] or with anti-WAP antibodies {G,H]. 
Activation of StatSa and StatSb. Mammary tissue from postpartum mice was analyzed for the presence of StatSaStatSb heterodimers, tyrosine-phosphorylated StatSa, tyrosinephosphorylated StatSb, the presence of StatSa and StatSb, the presence of tyrosine-phosphorylated Stat3, and StatSb protein in a Western blot. (Lanes a,b) Mammary tissue from littermates within 12 hr after parturition; (lanes e,f] same littermates as shown in lanes a and b but after 3 days of suckling (the histology shown in Fig. 2 is from these mice). To guarantee suckling, the litter of the StatSa-/-female was switched every 12 hr with that of the StatSa +/-female. (Lanes c,d) Mammary tissue from a second set of littermates within 12 hr after parturition. 
Expression of milk protein genes. Mammary tissue from wild-type, hemizygous, and homozygous mice was biopsied within 12 hr after parturition (lanes a-f] or after 3 days of suckling (lanes g,h) and probed for the presence of RNA encoding StatSb, (3-casein, WAP, a-lactalbumin, and WDNMl. 
Protein composition in mammary tissue of Stat5adeficient mice. Protein extracts from mammary tissue biopsies of wild-type, hemizygous, and homozygous mice isolated within 12 hr after parturition (lanes a-e) or after 3 days of suckling (lanes f,g] were analyzed by Coomassie blue staining [A] and probed for the presence of WAP using anti-WAP antibodies (B). The molecular masses (in kD) of a standard run in the same gel are indicated at right. The data shown in Figs. 2-5 were from the same mice. 
Article
Prolactin (PRL) induces mammary gland development (defined as mammopoiesis) and lactogenesis. Binding of PRL to its receptor leads to the phosphorylation and activation of STAT (signal transducers and activators of transcription) proteins, which in turn promote the expression of specific genes. The activity pattern of two STAT proteins, Stat5a and Stat5b, in mammary tissue during pregnancy suggests an active role for these transcription factors in epithelial cell differentiation and milk protein gene expression. To investigate the function of Stat5a in mammopoiesis and lactogenesis we disrupted this gene in mice by gene targeting. Stat5a-deficient mice developed normally and were indistinguishable from hemizygous and wild-type littermates in size, weight, and fertility. However, mammary lobuloalveolar outgrowth during pregnancy was curtailed, and females failed to lactate after parturition because of a failure of terminal differentiation. Although Stat5b has a 96% similarity with Stat5a and a superimposable expression pattern during mammary gland development it failed to counterbalance for the absence of Stat5a. These results document that Stat5a is the principal and an obligate mediator of mammopoietic and lactogenic signaling.
 
Radiographic and histological analysis of mice. (A) X-rays, lateral view. (B) Histological sections of tibia, low power (4× magnification). Sections were stained both with hematoxylin and eosin and for TRAP activity. (C) Histological sections, TRAPstained osteoclasts in bone, high power (100×, note lacy ruffled border structures at the interface between osteoclasts and bone surface.) Genotypes of mice are indicated. Two examples of bones from src−/− mice rescued by the K295M transgene (TRAPsrc K295M ) are shown to demonstrate the variability in phenotype. The bottom sample from a src−/ −TRAPsrc K295M mouse is derived from the calvarium after treatment with IL-1 and is stained with hematoxylin and eosin only.
Article
The Src tyrosine kinase has been implicated in a wide variety of signal transduction pathways, yet despite the nearly ubiquitous expression of c-src, src-/- mice show only one major phenotype-osteopetrosis caused by an intrinsic defect in osteoclasts, the cells responsible for resorbing bone. To explore further the role of Src both in osteoclasts and other cell types, we have generated transgenic mice that express the wild-type and mutated versions of the chicken c-src proto-oncogene from the promoter of tartrate resistant acid phosphatase (TRAP), a gene that is expressed highly in osteoclasts. We demonstrate here that expression of a wild-type transgene in only a limited number of tissues can fully rescue the src-/- phenotype. Surprisingly, expression of kinase-defective alleles of c-src also reduces osteopetrosis in src-/- animals and partially rescues a defect in cytoskeletal organization observed in src-/- osteoclasts. These results suggest that there are essential kinase-independent functions for Src in vivo. Biochemical examination of osteoclasts from these mice suggest that Src may function in part by recruiting or activating other tyrosine kinases.
 
Article
The development of the nervous system is a dynamic process during which factors act in an instructive fashion to direct the differentiation and survival of neurons, and to induce axonal outgrowth, guidance to, and terminal branching within the target tissue. Here we report that mice expressing signaling mutants of the hepatocyte growth factor (HGF) receptor, the Met tyrosine kinase, show a striking reduction of sensory nerves innervating the skin of the limbs and thorax, implicating the HGF/Met system in sensory neuron development. Using in vitro assays, we find that HGF cooperates with nerve growth factor (NGF) to enhance axonal outgrowth from cultured dorsal root ganglion (DRG) neurons. HGF also enhances the neurotrophic activities of NGF in vitro, and Met receptor signaling is required for the survival of a proportion of DRG neurons in vivo. This synergism is specific for NGF but not for the related neurotrophins BDNF and NT3. By using a mild signaling mutant of Met, we have demonstrated previously that Met requires signaling via the adapter molecule Grb2 to induce proliferation of myoblasts. In contrast, the actions of HGF on sensory neurons are mediated by Met effectors distinct from Grb2. Our findings demonstrate a requirement for Met signaling in neurons during development.
 
Article
Over the past 10 years, the development of chromosome conformation capture (3C) technology and the subsequent genomic variants thereof have enabled the analysis of nuclear organization at an unprecedented resolution and throughput. The technology relies on the original and, in hindsight, remarkably simple idea that digestion and religation of fixed chromatin in cells, followed by the quantification of ligation junctions, allows for the determination of DNA contact frequencies and insight into chromosome topology. Here we evaluate and compare the current 3C-based methods (including 4C [chromosome conformation capture-on-chip], 5C [chromosome conformation capture carbon copy], HiC, and ChIA-PET), summarize their contribution to our current understanding of genome structure, and discuss how shape influences genome function.
 
Article
The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.
 
Genotypes and phenotypes of embryos from Nf2 heterozygous intercrosses A. Genotypes Number of embryos 
Nf2 homozygous mutant embryos. Morphology of E7.25 wild-type (A) and homozygous mutant (B) embryos. Arrow in A depicts embryonic-extraembryonic boundary. (C-E) Saggital sections through an E7.5 wild-type (C) and Nf2 homozygous mutant (D,E) embryos exhibiting underdeveloped ectoplacental cones and a lack of organized extraembryonic ectoderm. The embryonic ectoderm in D continued to expand and folded back on itself, whereas the columnar visceral endoderm folded into a series of pleats at the extraembryonic-embryonic juncture probably because of the failed development of underlying extraembryonic structures. The embryo in E is misoriented with respect to the mesometrial-antimesometrial axis of the deciduum (mesometrium is toward the top of the figure in each case), and exhibits variable regions of squamous and columnar visceral endoderm. Note the lack of a distinct mesodermal cell population in either mutant embryo. (F,G)Wildtype E6.5 embryo (F) and Nf2 mutant E6.5 embryo (G). The proamniotic cavity in G has formed, but neither organized extraembryonic ectoderm nor ectoplacental cavity has formed. The cell death apparent in the embryo in G is not a consistent feature of Nf2 mutant embryos. Arrows in F and G indicate embryonic-extraembryonic juncture. (ec) Ectoplacental cone; (eed) extraembryonic ectoderm; (ed) embryonic ectoderm; (cve) columnar visceral endoderm; (sve) squamous visceral endoderm; (m)mesometrial; (a) antimesometrial. 
Nf2 mRNA expression in gastrulating embryos. In situ hybridization of E7.5 wild-type (A-C) and early mutant (DF) embryos using probes corresponding to the 3' half of the mouse Nf2 mRNA (A,B} and to the deleted exons 2-4 (C,E,F). In wild-type embryos, high levels of Nf2 expression are detected with both probes throughout the embryo proper, the extraembryonic ectoderm and ectoplacental cone (A-C) and at lower levels in trophoblast giant cells and parietal endoderm (A,B). The arrowhead in A marks the extraembryonic-embryonic boundary. (D-F) 
Features of Nf2 homozygous mutant chimeric embryos. (A) Typical chimeric embryo undergoing gastrulation with high contribution to the embryo proper. The arrow marks the embryonic-extraembryonic boundary; Nf2-deficient (bluestaining) cells representing embryonic mesoderm are migrating into the extraembryonic portion of the embryo to contribute to the formation of the amnion and the allantois. (B) Saggittal section of a similar embryo, depicting Nf2-deficient contribution to all three germ layers in the embryo proper. (C) Chimeric embryo at E8.0 that has developed well beyond gastrulation with Nf2 mutant contribution to the ectodermal, endodermal and mesodermal lineages. (m) Mesoderm/mesenchyme; (en) endoderm; (ec) ectoderm; (nf) neural folds; (s) somite. 
Article
The neurofibromatosis type II (NF2) tumor suppressor encodes a putative cytoskeletal associated protein, the loss of which leads to the development of Schwann cell tumors associated with NF2 in humans. The NF2 protein merlin belongs to the band 4.1 family of proteins that link membrane proteins to the cytoskeleton and are thought to be involved in dynamic cytoskeletal reorganization. Beyond its membership in this family, however, the function of merlin remains poorly understood. In order to analyze the function of merlin during embryogenesis and to develop a system to study merlin function in detail, we have disrupted the mouse Nf2 gene by homologous recombination in embryonic stem cells. Most embryos homozygous for a mutation at the Nf2 locus fail between embryonic days 6.5 and 7.0, exhibiting a collapsed extraembryonic region and the absence of organized extraembryonic ectoderm. The embryo proper continues to develop, but fails to initiate gastrulation. These observations are supported by the expression patterns of markers of the extraembryonic lineage and the lack of expression of mesodermal markers in the mutant embryos. Mosaic studies demonstrate that merlin function is not required cell autonomously in mesoderm, and support the proposition that merlin function is essential for the development of extraembryonic structures during early mouse development.
 
Article
The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.
 
Article
The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that regulate exit from the stem cell compartment, we constitutively expressed c-Myc in primary human keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell types, activation of c-Myc in keratinocytes caused a progressive reduction in growth rate, without inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched for stem or transit amplifying cells on the basis of beta1 integrin expression and by use of this method to fractionate cells prior to c-Myc activation, we found that c-Myc acted selectively on stem cells, driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis reconstituted on a dermal equivalent led to premature execution of the differentiation program. The transcriptional regulatory domain of c-Myc was required for these effects because a deletion within that domain acted as a dominant-negative mutation. Our results reveal a novel biological role for c-Myc and provide new insights into the mechanism regulating epidermal stem cell fate.
 
Article
Vertebrate Hox genes are required for the proper organization of structures along the rostrocaudal axis. Hoxd-11 is expressed in the posterior part of the embryo, up to the level of prevertebra 27, and its expression boundary is reproduced by a Hoxd-11/lacZ transgene. Expression of this transgene anterior to prevertebra 27 is prevented by the silencing activity of a cis-acting element, region IX. Using transgenic mice, we show that Hoxd-11 repression by region IX is necessary to position the sacrum properly. This silencing activity depends on phylogenetically conserved sequences able to bind in vitro retinoic acid receptors and COUP-TFs. ES cells were used to generate mice carrying a subtle mutation that abolishes binding of nuclear receptors to region IX. Mutant mice display an anterior shift of their lumbosacral transition inherited as a codominant trait. In mutant embryos, expression of both Hoxd-11 and Hoxd-10 mRNAs in the prevertebral column is anteriorized. These results illustrate the sharing, in cis, of a single regulatory element in order to establish the expression boundaries of two neighboring Hoxd genes.
 
Article
We investigated the roles of Caenorhabditis elegans MRE-11 in multiple cellular processes required to maintain genome integrity. Although yeast Mre11 is known to promote genome stability through several diverse pathways, inviability of vertebrate cells that lack Mre11 has hindered elucidation of the in vivo roles of this conserved protein in metazoan biology. Worms homozygous for an mre-11 null mutation are viable, allowing us to demonstrate in vivo requirements for MRE-11 in meiotic recombination and DNA repair. In mre-11 mutants, meiotic crossovers are not detected, and oocyte chromosomes lack chiasmata but appear otherwise intact. gamma-irradiation of mre-11 mutant germ cells during meiotic prophase eliminates progeny survivorship and induces chromosome fragmentation and other cytologically visible abnormalities, indicating a defect in repair of radiation-induced chromosome damage. Whereas mre-11 mutant germ cells are repair-deficient, they retain function of the meiotic G(2) DNA damage checkpoint that triggers germ cell apoptosis in response to ionizing radiation. Although mre-11/mre-11 animals derived from heterozygous parents are viable and produce many embryos, there is a marked drop both in the number and survivorship of embryos produced by succeeding generations. This progressive loss of fecundity and viability indicates that MRE-11 performs a function essential for maintaining reproductive capacity in the species.
 
Article
Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.
 
Article
Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcriptional silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Ty1 retrotransposons marked with a weakened URA3 gene (Ty1-mURA3) efficiently integrated into rDNA. The mURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. MET15 and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and MET15 silencing, but deletion of SIR1 or SIR3 did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.
 
pl5 prevents p27 binding to cyclin D-cdk4/6. (A) Increasing concentrations of recombinant pI5 were incubated for 20 min on ice with insect cell lysates (I pi) containing cdk4 and cyclin D2. The resulting complexes were immunoprecipitated with a cdk4 antibody. The kinase activity associated with these precipitates was measured using [^-'^^PJATP and GST-Rb fusion protein as substrate, and was quantitated using a Phosphorlmager. The cdk4 kinase activity obtained in the absence of pI5 is set as 100%. [B] Insect cell lysates (I jil) containing re­ combinant cyclin D2 and cdk4 (lanes 1-8) or cdk4 alone (lane 9) were incubated for 20 min on ice with the indicated concentra­ tions of recombinant pI5. Recombinant p27 (790 ng) was then added and the incubation continued for 20 min. (C) MvILu cells (R-1B/L17 subclone) were transiently transfected with pCMVbased vectors encoding cdk4, cdk4-R24C, p27, and pi5. Wholecell extracts were subjected to Western immunoblotting with cdk4-, p27-, and pl5-specific antibodies. A cdk4-specific anti­ body was used to capture the complexes, which were subjected to Western immunoblotting with cyclin D2 (A), p27 {B,C), or pi5 [A-C] antibodies. For unknown reasons, the level of expres­ sion of cdk4-R24C was lower than that of wild-type cdk4. How­ ever, note that the ratio of cdk4 to p27, in lanes 3 and 4, was the same with both cdk4 constructs. 
Prior occupancy of cyclin D-cdk4 by pl5 prevents p27 binding. [A] Cyclin D2-cdk4 (lanes 1-6] or cdk4 (lanes 7,8) were incubated for 20 min with no p27 (lane 1) or with a fixed amount of recombinant p27 (90 ng; lanes 2-8). The indicated concentrations of pl5 were then added, and incubations contin­ ued for 20 min. {B] Cyclin D2-cdk4 (lanes 1-8] was incubated without (lane 1] or with recombinant pl5 (200 ng; lanes 2-8] for the indicated time. Then, recombinant p27 (790 ng; lanes 1-8] was added and the incubation continued for an additional 10 min. (C) Cyclin D2-cdk4 was incubated without pl5 (lanes 4,9] or with pi5 added to the lysate 1 min prior (lanes 1,5], at the same time (lanes 2,6), 1 min after (lanes 3,7), or 5 min after (lane 8] p27 addition. Once p27 or p27(N) was added, the incubation continued for 10 min. (D) Cyclin D2-cdk4 (lanes 1-8] or cdk4 (lane 9) was incubated in the absence (lanes 1-4] or presence (lanes 5-9] of 4 mM ATP and 7 mM MgClj for 10 min. pl5 was not added (lanes 4,8,9) or added to the complexes 1 min prior (lanes 1,5], at the same time (lanes 2,6] or 1 min after (lanes 3,7] p27 addition. Once p27 was added the incubation continued for 10 min. [E] Cyclin D2-cdk4 (lanes 1-7] and cdk4 (lane 8) were incubated without (lanes 1,5-8) or with (lanes 2-4] recombinant pi5 for 10 min prior to incubation with the indicated concen­ trations of p27 (lanes 2-8] for 10 min. In all the experiments shown, 1 IJ1 of insect cell lysate was used. Cdk4 was immunoprecipitated, and the amount of associated p27 or cyclin D2 was determined by Western immunoblotting of these precipitates with p27 or cyclin D2 antibodies. 
Subcellular location of pi5, p27, p27(l-151), cyclin D, and cdk4. MvlLu cells (subclone R-1B/L17J were transfected transiently with vectors encoding pi5, p27, p27(l-151), cyclin Dl, or cdk4, or cotrans­ fected with these vectors, as indicated. Transfections with the empty vector pCMVS were used as a negative control. The cells were stained with antibodies against pi5, p27, cyclin Dl, or cdk4, as in­ dicated, followed by staining with a fluorescein-conjugated anti-rabbit antibody (pl5 and cdk4) or fluorescein-conjugated anti-mouse antibody (p27 and cyclin Dl). 
Schematic representation of the p27 and pl5 constructs. The cycHn-cdk binding domain and the putative bipartite NLS of p27 are indicated. The hatched box represents the SV40 T antigen nuclear lo­ calization signal (PKKKRKV) that was added to the carboxyl terminus of p27(l151) and wild-type pi5. 
Article
In dividing cells, p27(Kip1) is predominantly bound to cyclin D-cdk4 without inhibiting this kinase. Upon being induced by TGF-beta or with a conditional expression system in lung epithelial cells, p15(Ink4b) binds to and inhibits the cyclin D-dependent kinases, prevents p27 binding to these cdk complexes, and promotes p27 binding and inhibition of cyclin-cdk2. In vitro, however, p15 prevents p27 binding only if it has access to cyclin D-cdk4 first. We present evidence that the different subcellular location of p15 and p27 ensures the prior access of p15 to cdk4. In the cell, p15 is localized mostly in the cytoplasm, whereas p27 is nuclear. p15 prevails over p27 or a p27 construct consisting of the cdk inhibitory domain tagged with a nuclear localization signal. However, when p15 and p27 are forced to reside in the same subcellular location, either the cytoplasm or the nucleus, p15 no longer prevents p27 from binding to cdk4. These properties allow p15 and p27 to coordinately inhibit cdk4 and cdk2.
 
Article
Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the human hsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. We demonstrate that activation of hsp70 leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor alpha-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to alpha-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. We suggest that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation.
 
Article
During development of the central nervous system, oligodendrocyte progenitor cells (O-2A) undergo an orderly pattern of cell proliferation and differentiation, culminating in the ability of oligodendrocytes to myelinate axons. Here we report that p27(Kip1), a cyclin-dependent kinase inhibitor, is an important component of the decision of O-2A cells to withdraw from the cell cycle. In vitro, accumulation of p27 correlates with differentiation of oligodendrocytes. Furthermore, only a fraction of O-2A cells derived from p27-knockout mice differentiate successfully compared to controls. Inability to differentiate correlates with continued proliferation, suggesting that p27 is an important component of the machinery required for the G1/G0 transition in O-2A cells. In vivo, expansion of O-2A precursors before withdrawal, in part, leads to a greater number of oligodendrocytes. Together these data indicate a role for p27 during the decision to withdraw from the cell cycle in the oligodendrocyte lineage.
 
Overexpression of miR-11 suppresses dE2f1-induced apoptosis in transgenic animals. (A) Third instar larval eye discs of various genotypes were incubated with BrdU for 90 min at room temperature, followed by fixing and staining with antibodies recognizing BrdU (red) and active caspase (C3) (green). Analysis was performed on a minimum of 10 larvae of each genotype. The position of the MF is marked with an arrowhead. (B,C) Flies carrying Act88FGal4 and UAS-dE2f1 (B) or UAS-ced3 (C) transgenes were crossed to either a wildtype chromosome (Canton S) or UAS-miR11. (D) RNA was extracted from third instar larval eye discs from GMR-Gal4 crosses described in A, and transcriptional profiles were determined by Affymetrix gene expression microarrays (see the Materials and Methods). Differentially expressed genes were compared for enrichment of GOBP categories with a false discovery rate (FDR) standard cutoff of 0.05 for differentially expressed genes relative to GMR-Gal4/+. Heat map color values represent statistical significance of enrichment (see the scale bar).
Generation and initial characterization of mir-11 mutant. (A) Genomic organization of the mir-11 region. (B) Genomic DNA was extracted from wild type (Canton S); the targeting line used for homologous recombination, which contains wild-type and mir-11 mutant intron sequences (targeting line); or the homozygous mir-11 mutant (D mir-11) flies. PCR was performed using primers binding to the regions indicated to follow the presence and loss of the mir-11 gene. (C) RNA was extracted from wildtype (Canton S), mir-11 mutant, or GMR>mir-11 larvae. The dme-miR-11 TaqMan assay was used to measure mature miR-11 expression. (D-F) Clones of mir-11 mutant tissue were generated using eyFLP. Wild-type tissue is marked by GFP (green). (D) Third instar larval eye discs were dissected, and dE2F1 expression (magenta) was detected by immunofluorescence. (E) To mark proliferating cells, eye discs were incubated with BrdU for 60 min prior to fixation and antibody staining. (F) Apoptotic cells were labeled with the C3 antibody, which recognizes the active forms of caspases.
mir-11 mutant cells are highly sensitive to dE2F1-dependent irradiation-induced apoptosis and have a higher level of expression of hid and rpr. (A,B) Wild-type (A) or mir-11 mosaic mutant (B) third instar larvae were exposed to 40 Gy of irradiation, and eye discs were harvested at the indicated times following exposure. Apoptosis was detected using the C3 antibody (red), which recognizes active caspases. Clones of mir11 mutant tissue were generated using ey-FLP. Wild-type tissue is marked by GFP (green). Yellow arrows indicate patches of apoptotic cells that appear in the mir-11 mutant cells at an earlier time point than in the adjacent wild-type tissue. At 4 h after irradiation, the stripe of C3positive cells anterior to the MF is expanded in the mir-11 mutant tissue. The position of the MF is marked with the arrowhead. (C) C3-positive cells in wild-type or mutant tissue were counted at the indicated times after irradiation. An average of a minimum of six eye discs was counted. Standard deviations are indicated. (D) Canton S or mir-11 D1 third instar larvae were exposed to 40 Gy of irradiation and then allowed to develop. The number of adults to emerge was counted, and the percentage of survival was calculated. The average and standard deviation of three separate experiments are shown. (*) P = 0.032 in a paired t-test. (E) RNA was extracted from wild-type or mir11 mosaic mutant larval eye discs 1 h following irradiation (40 Gy), and rpr, hid, grim, and skl expression was measured by qRT-PCR. b-tubulin and RpP0 were used as controls in normalization. Expression of rpr and hid is elevated in mir-11 mutant mosaic eye discs before and after irradiation. (**) P < 0.001; (*) P < 0.05 in paired t-tests.
Article
The E2F family of transcription factors regulates the expression of both genes associated with cell proliferation and genes that regulate cell death. The net outcome is dependent on cellular context and tissue environment. The mir-11 gene is located in the last intron of the Drosophila E2F1 homolog gene dE2f1, and its expression parallels that of dE2f1. Here, we investigated the role of miR-11 and found that miR-11 specifically modulated the proapoptotic function of its host gene, dE2f1. A mir-11 mutant was highly sensitive to dE2F1-dependent, DNA damage-induced apoptosis. Consistently, coexpression of miR-11 in transgenic animals suppressed dE2F1-induced apoptosis in multiple tissues, while exerting no effect on dE2F1-driven cell proliferation. Importantly, miR-11 repressed the expression of the proapoptotic genes reaper (rpr) and head involution defective (hid), which are directly regulated by dE2F1 upon DNA damage. In addition to rpr and hid, we identified a novel set of cell death genes that was also directly regulated by dE2F1 and miR-11. Thus, our data support a model in which the coexpression of miR-11 limits the proapoptotic function of its host gene, dE2f1, upon DNA damage by directly modulating a dE2F1-dependent apoptotic transcriptional program.
 
Cell shape changes and localization of Xcad-11. (A) Analysis of CNC migration in vitro. Control cells injected with mGFP (200 pg) showed extensive protrusion activity. CNC from Xcad-11-depleted embryos exhibited membrane blebbing. CoMOinjected cells showed the same cell behavior as wild-type cells. Bar, 10 mm. (B) Protrusion formation of CNC in vivo. Wild-type cells showed extensive protrusive activity while Xcad-11MO-injected cells were unable to form filopodia or lamellipodia. The right-hand scheme displays the experimental procedure. The inserts show an outside view of transplanted embryos at stage 26. Anterior is to the right side. Bar, 20 mm. (C) F-actin staining of wild-type and Xcad11MO-injected CNC explants. In wild type, F-actin was enriched in small (arrowhead) and large (arrows) lamellipodia. In Xcad-11MOtreated explants, no F-actin was seen in blebs (arrowheads). Bar, 10 mm. (D) Localization of Xcad-11 in CNC explants. Embryos were injected with 1 ng of Xcad-11-EGFP and mCherry (200 pg). (Bottom left) Xcad-11-EGFP was visible in filopodia and lamellipodia, while mCherry labeled cell membranes. The boxed area is enlarged in the bottom left panel. Z-stack revealed localization of Cad11-EGFP at the substrate-facing cell surface. Bar, 10 mm.
Article
Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.
 
Mechanisms of lysine demethylation by LSD1 and JMJC proteins. ( A ) LSD1 demethylates H3K4me2/me1 via an amine oxidation reaction using FAD as a cofactor. The imine intermediate is hydrolyzed to an unstable carbinolamine that subsequently degrades to release formaldehyde. ( B ) The JMJC proteins use ␣ KG and iron (Fe) as cofactors to hydroxylate the methylated histone substrate. Fe(II) in the active site activates a molecule of dioxygen to form a highly reactive oxoferryl [Fe(IV) = O] species to react with the methyl group. The resulting carbinolamine intermediate spontaneously degrades to release formaldehyde. Throughout the figure, the wavy line indicates attachment to the peptide backbone. 
Phylogenetic tree of the JmjC family of demethylases. The names, synonyms, substrate specificities, and domain structures of the proteins are provided. The lists of synonyms may be longer, but due to space limitations only the most relevant are provided. Putative oncoproteins are in red and putative tumor suppressors are in green. (JmjC) Jumonji C domain; (JmjN) Jumonji N domain; (PHD) plant homeodomain; (Tdr) Tudor domain; (Arid) AT-rich interacting domain; (Fbox) F-box domain; (C5HC2) C5CHC2 zinc-finger domain; (CXXC) CXXC zinc-finger domain; (TPR) tetratricopeptide domain; (LRR) leucine-rich repeat domain; (TCZ) treble-clef zinc-finger domain; (PLAc) cytoplasmic phospholipase A2 catalytic subunit.
Model for the involvement of demethylases/ methyltransferases in transcriptional regulation of developmental genes. Histone methyltransferases and demethylases are found in the same complex, which methylates one mark while removing the opposing mark. The methylation pattern at a specific gene is determined by the equilibrium between activities of the two opposing complexes, exemplified here by the activating MLL2/UTX complex and the repressive PRC2/ RBP2 complex (Agger et al. 2007; Pasini et al. 2008). Analogously, it has been shown that repressive complexes carrying K9 methyltransferase activity (G9a) may also contain H3K4 demethylase activities (Tahiliani et al. 2007). Correspondingly, it may be envisioned that H3K9 demethylases may form a part of activating complexes carrying methyltransferases to activating marks as H3K4.
The involvement of demethylases in AR-mediated transcription. When bound to its ligands, androgen (A), the AR translocates to the nucleus to interact with histone demethylases on androgen-responsive elements (ARE) on specific genes. Through its interaction with JMJD2C, LSD1 or JMJD1A demethylation is triggered, removing the repressive H3K9 methylation and leading to the transcriptional induction of these androgen-responsive genes. Repressive complexes (RCO), possibly featuring H3K9-methyltransferase (KMT), HDAC, and H3K4 demethylase (JARID1) activities, may potentially act to prevent ligand-independent activation. 
Demethylases and histone modifications in senescence. (A) JMJD3 and p53 are transcribed from the same genomic region: chromosome 17p13.1. In response to oncogenic, replicative, or other types of stress (DNA damage, drug treatment, etc.) the tumor suppressors p53 and INK4A-ARF are induced, triggering senescence. JMJD3 or UTX may be involved in the transcriptional activation of the INK4A-ARF tumor suppressor locus by removing repressive K27 methylation from the gene (see B). In turn, p16 and p14ARF lead to the induction of pRB and p53, respectively, triggering senescence and/or apoptosis. (B) The compacted, transcriptionally silent chromatin structure of the INK4A-ARF locus serves to ensure cell cycle progression in normal "unstressed" cells and in some malignant cells. (Left panel) This silent state is maintained by H3K27 methylation mediated by the PcG proteins, and possibly also by the repressive activities of JARID1 proteins and other chromatin modifiers. When cells with intact tumor suppressor pathways are subjected to oncogenic stimuli or other types of stress, PcG proteins are displaced from the INK4A-ARF locus and K27 demethylases are possibly recruited to remove the repressive K27 methylation, leading to the expression of the INK4AARF locus and the induction of senescence. (C, left panel) In normal "unstressed" cells the transcription factor E2F mediates transcription, while the tumor suppressor pRB is maintained in an inactive phosphorylated state as a result of low p16 levels. During senescence induction, p16 is induced, causing hypophoshorylation of pRB. (Right panel) pRB subsequently recruits chromatin modifiers as HDAC, SUV39H1 (setting H3K9 methylation), SUV4H20 (setting H4K20 methylation), HP1, and possibly JARID1 proteins to silence euchromatic E2F target promoters and form SAHFs.
Article
The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer.
 
Nf2 heterozygous mice are cancer prone. ( A ) Survival curve showing the decreased survival of Nf2 heterozygotes on an F 1 (C57BL/6 × 129/Sv) background (blue diamonds), compared to that of their wild-type F 1 siblings (black squares). ( B ) Histological section through an osteosarcoma in a Nf2 heterozygote. The tumor (os) is growing out of a vertebral bone and pressing on the spinal cord (sc) and associated spinal ganglion (g). This tumor is very well differentiated, with a high ratio of calcified bone matrix to cell nucleus (100×). ( C ) Histological section of a hepatocellular carcinoma in an Nf2 heterozygote. Note the trabecular nature of the tumor itself (hc), manifested as nests of cells with spaces in between. Normal liver on the right (li) is being compressed by the tumor (200×). ( D ) Loss of Nf2 heterozygosity in tumors from Nf2 +/− animals. Southern blotting of paired tail [(g) germ line] and tumor (t) samples. ( Top band) The wild-type allele; ( bottom band) the mutant allele. 
Tumors that arise in Nf2 heterozygous mice exhibit a very high rate of metastasis. (A) A fairly undifferentiated osteosarcoma metastasis (os) in the lung (lu) of a Nf2 +/− mouse. Some mineralization is present, but there is abundant cellularity. This is the typical histological appearance of an osteosarcoma metastasis (200×). (B) Highly differentiated metastasis in a Nf2 +/− mouse. This is an unusual form of metastasis frequently seen in Nf2 heterozygotes. The mineralized bone has formed a collar as in a long bone, and some lymphocytes appear to have homed to the ''pseudobone marrow cavity'' in the center (asterisk) (100×). (C) A highly differentiated osteosarcoma metastasis (os) in the liver (li) of a Nf2 +/− mouse. Again, there appears to be lymphocyte homing and formation of ''bone marrow.'' Two large blood vessels are also present within the metastasis and probably provided the route of entry for the tumor cells into the lung (100×). (D) A metastasis from a hepatocellular carcinoma (li) to the lung. This mouse also had an osteosarcoma that metastasized to the liver metastasis (os; center). The normal lung tissue is compressed in the upper corners (lu) (100×).
Generation of Nf2 +/−;p53 +/− compound heterozygotes in cis and in trans. (A) Map of mouse chromosome 11, depicting the locations of the Nf2 and p53 loci, as well as the D11MIT20 SSLP marker used for allelotyping. (B) Scheme for the generation of Nf2 +/−;p53 +/− trans and cis mice. (C) Survival of mice carrying both Nf2 and p53 mutations either in cis on the same chromosome 11, or in trans, on opposite chromosomes 11, compared to the survival of either singly heterozygous parental strain. (D) Absence of both the Nf2 and p53 wild-type alleles in tumors from Nf2 +/−;p53 +/− cis mice. The same DNAs were digested with either StuI (for Nf2 LOH) or StuI plus EcoRI (for p53 LOH) restriction enzymes, and probed with a Nf2-specific probe (left) or a p53-specific probe [right; (pg) pseudogene]. Residual wild-type signals present in the tumor samples are likely the result of contaminating normal tissue. The intensities of residual wild-type p53 vs. wild-type Nf2 signals appear unequal here; however, most tumors exhibit equivalent levels of contaminating tissue. (E) Frequency of metastasis associated with Nf2-deficient or Nf2 plus p53-deficient osteosarcoma or fibrosarcoma.
Histological appearance of the primary tumors from which Nf2-or p53-deficient fibrosarcoma cell lines were derived (left) and histological sections of the lungs of recipient animals into which each of these cell lines was injected (right). The total number of metastases produced by each cell line is indicated below. The lungs of animals injected with Nf2-deficient tumor cells (A,B) are covered with metastases (arrows indicate selected metastases), whereas none are detected in the lungs of animals injected with p53-deficient tumor cells (C,D). Note that the lung in A has hemorrhaged; blood fills much of the alveolar spaces (200×).
Article
A role for the membrane/cytoskeleton interface in the development and progression of cancer is established, yet poorly understood. The neurofibromatosis type II (NF2) tumor suppressor gene encodes a member of the ezrin/radixin/moesin (ERM) family of membrane/cytoskeleton linker proteins thought to be important for cell adhesion and motility. We report that in contrast to the narrow spectrum of benign tumors in human NF2 patients, Nf2 heterozygous mice develop a variety of malignant tumors. Using the fact that Nf2 is linked to the p53 tumor suppressor locus in the mouse we have also investigated the effects of genetic linkage of cancer-predisposing mutations on tumorigenesis and examined the genetic pathway to tumor formation involving Nf2 loss. Importantly, we observed a very high rate of metastasis associated with Nf2 deficiency, with or without loss of p53 function, and we provide experimental evidence supporting a role for Nf2 loss in metastatic potential. Together, our results suggest an important role for the NF2 tumor suppressor, and perhaps the ERM family in tumor formation and metastasis.
 
Top-cited authors
Charles J. Sherr
  • St. Jude Children's Research Hospital
James M Roberts
  • Fred Hutchinson Cancer Research Center
Danny Reinberg
  • NYU Langone Medical Center
Stephen Elledge
  • Brigham and Women's Hospital
Steven Mcknight
  • University of Texas Southwestern Medical Center