The single-minded gene functions as a master developmental regulator within the midline cell lineage of the embryonic central nervous system of Drosophila melanogaster. Genetic experiments suggest that Single-minded can function as a transcriptional activator. Regions of the Single-minded protein were fused to the DNA binding domain of the mammalian transcription factor Sp1 and shown to activate transcription from a reporter gene linked to Sp1 binding sites. Three independent activation domains were identified in the carboxy terminal region of Single-minded that include areas rich in serine, threonine, glutamine and proline residues. Germ line transformation experiments indicate that the carboxy terminal activation domains, the PAS dimerization domain, and the putative DNA binding basic domain of Single-minded are required for expression of CNS midline genes in vivo. These results define in vivo a functional activation domain within Single-minded and suggest a model in which Single-minded activates transcription through a direct interaction with promoter elements of CNS midline genes.
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"(4–6). Their Drosophila homologue, dSim, encodes a positively acting, master regulator of central nervous system (CNS) midline development (7–10). As with dSIM, SIM2 appears to have key neurological functions. "
[Show abstract][Hide abstract] ABSTRACT: Despite a growing number of descriptive studies that show Single-minded 2 (Sim2) is not only essential for murine survival, but also upregulated in colon, prostate and pancreatic tumours, there is a lack of direct target genes identified for this basic helix-loop-helix/PAS transcription factor. We have performed a set of microarray experiments aimed at identifying genes that are differentially regulated by SIM2, and successfully verified that the Myomesin2 (Myom2) gene is SIM2-responsive. Although SIM2 has been reported to be a transcription repressor, we find that SIM2 induces transcription of Myom2 and activates the Myom2 promoter sequence when co-expressed with the heterodimeric partner protein, ARNT1, in human embryonic kidney cells. Truncation and mutation of the Myom2 promoter sequence, combined with chromatin immunoprecipitation studies in cells, has lead to the delineation of a non-canonical E-box sequence 5'-AACGTG-3' that is bound by SIM2/ARNT1 heterodimers. Interestingly, in immortalized human myoblasts knock down of Sim2 results in increased levels of Myom2 RNA, suggesting that SIM2 is acting as a repressor in these cells and so its activity is likely to be highly context dependent. This is the first report of a direct SIM2/ARNT1 target gene with accompanying analysis of a functional response element.
Nucleic Acids Research 07/2008; 36(11):3716-27. DOI:10.1093/nar/gkn247 · 9.11 Impact Factor
"Sim1, a second mammalian homologue of sim, shares a high degree of sequence identity with Sim2 in the N-terminal bHLH and PAS dimerization domains (Ema et al., 1996; Fan et al., 1996). In general, bHLH-PAS family members heterodimerize to form transcriptional activators, an activity mediated through C-terminal domains (Franks and Crews, 1994; Jain et al., 1994). Sim1 and Sim2 may differ in this regard, as heterodimers between either protein and the aryl hydrocarbon receptor nuclear translocator (ARNT) have been shown to be transcriptional repressors in human embryonic kidney and COS-7 cell culture systems (Ema et al., 1996; Moffett et al., 1997). "
[Show abstract][Hide abstract] ABSTRACT: Sim2 is a member of the basic helix-loop-helix PAS transcription factor gene family and is evolutionarily related to the Drosophila single-minded gene, a key regulator of central nervous system midline development. In an effort to determine the biological roles of Sim2 in mammalian development, we disrupted the murine Sim2 gene through gene targeting. Mice homozygous for the disrupted allele (Sim2 -/-) exhibit a cleft of the secondary palate and malformations of the tongue and pterygoid processes of the sphenoid bone. These craniofacial malformations are the most probable cause of aerophagia (air swallowing with subsequent accumulation of air in the gastrointestinal tract) and postnatal death exhibited by Sim2 -/- mice. The developing palates of the Sim2 -/- mice are hypocellular, and at embryonic day 14.5 contain excess extracellular matrix component hyaluronan (HA) compared with heterozygotes and homozygous wild-type littermates. HA plays an important role in the regulation and mechanics of palate development. Its premature accumulation in Sim2 -/- animal palates suggests a regulatory role for Sim2 in HA synthesis and in the establishment of craniofacial architecture.
"HLF or HIF1α activates transcription of reporter plasmids harboring the Epo HRE in a hypoxia-dependent manner (see review article by Wenger and Gassmann, 1997; Ema et al., 1997; Tian et al., 1997; Figure 1B, lanes 3, 4, 11, 12). Since bHLH-PAS transcription factors carry the transactivation domains in the C-terminal half (Franks et al., 1994; Jain et al., 1994; Whitelaw et al., 1994; Hirose et al., 1996; Li et al., 1996), we investigated effects of truncation of the C-terminal half of HLF, HIF1α and Arnt on the hypoxiainducible gene expression (Figure 1A and B). Expression of one of the full-length HLF, HIF1α and Arnt with another of their C-terminal-truncated forms significantly decreased hypoxia-dependent transcriptional activation (Figure 1A and B, lanes 6, 8, 14, 16). "
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factor 1 (HIF1) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced -galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.
The EMBO Journal 03/1999; 18(7):1905-1914. DOI:10.1093/emboj/18.7.1905 · 10.43 Impact Factor