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In the developing brain, the production of neurons from multipotent precursors must be carefully regulated in order to generate the appropriate numbers of various differentiated neuronal types. Inductive signals from extrinsic elements such as growth factors need to be integrated with timely expression of intrinsic elements such as transcription factors that define the competence of the cell. The transcriptional Mediator complex offers a mechanism to coordinate the timing and levels of intrinsic and extrinsic influences by acting as a rapid molecular switch for transcription of poised RNA pol II. The epithalamus is a highly conserved region of the vertebrate brain that differentiates early and rapidly in the zebrafish. It includes the pineal and parapineal organs and the habenular nuclei. Mutation of the Mediator complex subunit Med12 impairs the specification of habenular and parapineal neurons and causes a loss of differentiation in pineal neurons and photoreceptors. Although FGF ligands and transcription factors for parapineal and photoreceptor development are still expressed in the pineal complex of med12 mutants, FGF signaling is impaired and transcription factor expression is reduced and/or delayed. We find that the timely expression of one of these transcription factors, tbx2b, is controlled by Med12 and is vital for parapineal specification. We propose that the Mediator complex is responsible for subtle but significant changes in transcriptional timing and amplitude that are essential for coordinating the development of neurons in the epithalamus.
© 2013 Published by Elsevier Inc.
White blood cell count (WBC) is unique among identified inflammatory predictors of chronic disease in that it is routinely measured in asymptomatic patients in the course of routine patient care. We led a genome-wide association analysis to identify variants associated with WBC levels in 13,923 subjects in the electronic Medical Records and Genomics (eMERGE) Network. We identified two regions of interest that were each unique to subjects of genetically determined ancestry to the African continent (AA) or to the European continent (EA). WBC varies among different ancestry groups. Despite being ancestry specific, these regions were identifiable in the combined analysis. In AA subjects, the region surrounding the Duffy antigen/chemokine receptor gene (DARC) on 1q21 exhibited significant association (p value = 6.71e-55). These results validate the previously reported association between WBC and of the regulatory variant rs2814778 in the promoter region, which causes the Duffy negative phenotype (Fy-/-). A second missense variant (rs12075) is responsible for the two principal antigens, Fya and Fyb of the Duffy blood group system. The two variants, consisting of four alleles, act in concert to produce five antigens and subsequent phenotypes. We were able to identify the marginal and novel interaction effects of these two variants on WBC. In the EA subjects, we identified significantly associated SNPs tagging three separate genes in the 17q21 region: (1) GSDMA, (2) MED24, and (3) PSMD3. Variants in this region have been reported to be associated with WBC, neutrophil count, and inflammatory diseases including asthma and Crohn's disease.
Several transcriptional activators, called "classical" because each bears a natural acidic activating region attached to a DNA binding domain, are proteolytically unstable in yeast, and it has been suggested that this instability is required for transcriptional activation. Here we test the generality of that proposal by examining a set of activators (called "nonclassical") that lack activating regions. These activators (e.g., LexA-Gal11) comprise a LexA DNA binding domain fused to a component of the Mediator and are believed to insert the latter into the Mediator and recruit it (and, indirectly, other components required for transcription) to a gene bearing LexA sites. We find that three, and only three, Mediator subunits, all from its tail domain, work as activators when fused to LexA. All three are unstable, and for the case analyzed in detail, stabilization decreases activity. Thus, to the extent tested, both classical and nonclassical activators work most efficiently when proteolytically unstable.
(c) 2010 Elsevier Ltd. All rights reserved.
The objective of this research was to further characterize the promoter regions of the bovine and porcine fatty acid-binding protein 4 (FABP4) genes relative to those of other mammals. The DNA sequences of FABP4 promoters for the human, mouse, cow, pig, and dog were obtained from the genomic database of the National Center for Biotechnology Information and also from the sequencing of bovine and porcine genomic DNA clones obtained by 5' PCR racing of genomic DNA. Sequence alignments of these FABP4 promoters using the basic local alignment search tool of the National Center for Biotechnology Information revealed 3 highly conserved promoter regions across the species. Two computational bioinformatics databases and the literature identified the conserved transcription factor-binding sites for C/EBP, adapter primer-1, and boxes of CAAT and TATA in the first conserved proximal promoter region, a direct repeat 1-type PPAR responsive element in the second distal conserved region, and another PPAR responsive element in the third distal conserved promoter region of FABP4 in all 5 mammals. Five new short interspersed repetitive elements (SINE) in the bovine FABP4 promoter and 2 new SINE in the porcine were found, but these SINE did not disrupt the 3 conserved regions, indicating that important regulatory elements are maintained regardless of evolutionary pressure. In conclusion, the conserved cis-acting elements, especially the 2 key adipocyte transcription factors C/EBP and PPAR, may contribute greatly to adipogenic regulation and adipose tissue-specific expression of FABP4 in these mammals. This helps to further characterize and decipher important cis-acting elements that are important for adipocyte development in adipose and muscle tissue.
The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.
The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.