Small Maf proteins serve as dual-function transcription factors through an exchange of their heterodimerization partners. For example, as heterodimers with hematopoietic cell-specific p45 NF-E2 or NF-E2-related factors (Nrf), they activate the -globin or antioxidative stress enzyme heme oxygenase 1 (HO-1) genes, respectively. In contrast, together with Bach1, they repress these same genes. However, the signals leading to this partner exchange are not known. Using chromatin immunoprecipitation assays in NIH 3T3 cells, we show that heme, an inducer of ho-1, promotes displacement of Bach1 from the MafK-occupied ho-1 enhancers, which is followed by Nrf2 binding to these elements. Whereas histone H3 at the ho-1 enhancers and promoter is hyperacetylated irrespective of gene activity, exposure of cells to heme results in de novo hyperacetylation and hypermethylation of histone H3 in the transcribed region. These data indicate that, under normal conditions, the chromatin structure of ho-1 is in a preactivation state, but transcription is repressed by Bach1. Heme induces switching of Maf dimers, resulting in ho-1 expression. Heme also promotes displacement of Bach1 from the -globin locus control region without affecting MafK binding in murine erythroleukemia cells. Thus, heme functions as a signaling molecule for gene expression in higher eukaryotes.
Cell-specific patterns of gene expression are established through the antagonistic functions of trithorax group (TrxG) and Polycomb group (PcG) proteins. Several muscle-specific genes have previously been shown to be epigenetically marked for repression by PcG proteins in muscle progenitor cells. Here we demonstrate that these developmentally regulated genes become epigenetically marked for gene expression (trimethylated on histone H3 Lys4, H3K4me3) during muscle differentiation through specific recruitment of Ash2L-containing methyltransferase complexes. Targeting of Ash2L to specific genes is mediated by the transcriptional regulator Mef 2d. Furthermore, this interaction is modulated during differentiation through activation of the p38 MAPK signaling pathway via phosphorylation of Mef 2d. Thus, we provide evidence that signaling pathways regulate the targeting of TrxG-mediated epigenetic modifications at specific promoters during cellular differentiation.Cell-specific gene expression programs are established during embryo-genesis through transient environmental signals. These programs are then stably maintained and passed to daughter cells through a process of cellular memory. Experiments in Drosophila melanogaster have identified PcG and TrxG proteins as the mediators of this memory 1 . The
Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.
During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.
Recently we identified a novel human (h) multiprotein complex, called TATA-binding protein (TBP)-free TAF IIcontaining complex (TFTC), which is able to nucleate RNA polymerase II transcription and can mediate transcriptional activation. Here we demonstrate that TFTC, similar to other TBP-free TAF II complexes (yeast SAGA, hSTAGA, and hPCAF) contains the acetyltransferase hGCN5 and is able to acetylate histones in both a free and a nucleosomal context. The recently described TRRAP cofactor for oncogenic transcription factor pathways was also characterized as a TFTC subunit. Furthermore, we identified four other previously uncharacterized subunits of TFTC: hADA3, hTAF II 150, hSPT3, and hPAF65. Thus, the polypeptide composition of TFTC suggests that TFTC is recruited to chromatin templates by activators to acetylate histones and thus may potentiate initiation and activation of transcription.Initiation of transcription of protein-encoding genes by RNA polymerase II requires transcription factor TFIID that is comprised of the TATA-binding protein (TBP) 1 and series of TBPassociated factors (TAF II s) (1). TFIID directs preinitiation complex assembly on both TATA-containing and TATA-less promoters. Previously, we have shown that functionally distinct TFIID complexes composed of both common and specific TAF II s exist in human HeLa cells (for review, see Ref. 2).We have isolated and partially characterized a novel human (h) multiprotein complex, which contains neither TBP nor TBPlike factor but is composed of several TAF II s and a number of uncharacterized polypeptides (3). This novel complex, called TBP-free TAF II -containing complex (TFTC) is able to direct preinitiation complex formation and initiation of transcription on both TATA-containing and TATA-less promoters in in vitro transcription assays and can mediate transcriptional activation by .Following the discovery of the TFTC complex, TAF II s have also been described in different histone acetyltransferase (HAT) complexes: the yeast SPT-ADA-GCN5 acetyltransferase (SAGA) complex and the human PCAF-GCN5 and the human STAGA complexes (4 -6). Histone acetylation and deacetylation have been strongly linked to the regulation of transcription (7). Yeast (y) Gcn5 has HAT activity and is a transcriptional coactivator required for correct expression of various genes (8,9). Transcriptional activators, such as VP16 or GCN4, interact directly with the SAGA complex and direct nucleosomal acetylation to potentiate transcriptional activation (10). The yeast SAGA complex consists of yGcn5 and various Ada (Ada1, Ada2, and Ada3) and Spt (Spt3, Spt7, Spt8, and Spt20) proteins (11). In addition to these proteins the SAGA complex also contains a distinct set of yTAF II s (TAF II 90, TAF II 68, TAF II 60, TAF II 25, and TAF II 17/20) (4). To date two human homologues of the yGcn5 have been identified. The first human homologue of yGcn5 is hGCN5 (called hGCN5-L), which is highly homologous to yeast GCN5 but contains an extended amino-terminal domain (12, 13). Furthermore, in huma...
Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineagesExpression of the basic helix-loop-helix transcription factor TAL1/SCL is required for erythrocyte differentiation; aberrant expression in lymphoid cells leads to oncogenic transformation. Here, global analysis of TAL1 binding in erythroid and malignant T cells identifies cell type specific functional interaction with the transcription factors RUNX and ETS1.
MLL-containing complexes methylate histone H3 at lysine 4 (H3K4) and have been implicated in the regulation of transcription. However, it is unclear how MLL complexes are targeted to specific gene loci. Here, we show that the MLL2 complex associates with the hematopoietic activator NF-E2 in erythroid cells and is important for H3K4 trimethylation and maximal levels of transcription at the beta-globin locus. Furthermore, recruitment of the MLL2 complex to the beta-globin locus is dependent upon NF-E2 and coincides spatio-temporally with NF-E2 binding during erythroid differentiation. Thus, a DNA-bound activator is important initially for guiding MLL2 to a particular genomic location. Interestingly, while the MLL2-associated subunit ASH2L is restricted to the beta-globin locus control region 38 kb upstream of the beta(maj)-globin gene, the MLL2 protein spreads across the beta-globin locus, suggesting a previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at a distance.
Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAF II s). In the presence of TBP-free TAF II complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identi®ed a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modi®cation of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modi®cation. Keywords: histone acetyltransferase/nucleotide excision repair/spliceosome assembly factor/TATA box-binding protein-associated factors (TAF II s)/xeroderma pigmentosum group E IntroductionTranscription initiation of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, which is comprised of the TATA boxbinding protein (TBP) and a series of TBP-associated factors (TAF II s) (Tansey and Herr, 1997;Bell and Tora, 1999). However, we have shown recently that initiation of Pol II transcription can occur in the absence of TFIID, in the presence of a novel human (h) multiprotein complex, termed TFTC for TBP-free TAF II complex (Wieczorek et al., 1998). TFTC is able to direct pre-initiation complex assembly on both TATA-containing and TATA-less promoters in vitro. TFTC contains neither TBP nor TBPlike factor, but is composed of several TAF II s (Wieczorek et al., 1998). The three-dimensional structure of TFTC has been determined, together with that of TFIID, at 3.5 nm resolution by electron microscopy and digital image analysis of single particles (Brand et al., 1999a). Human TFTC resembles a macromolecular clamp that contains ®ve globular domains organized around a solvent-accessible groove of a size suitable to bind DNA. TFIID contains only four domains, which are also organized around a solvent-accessible groove (Andel et al., 1999;Brand et al., 1999a). Comparison of the two three-dimensional models indicates that the structure of TFIID is almost included in that of TFTC, further con®rming some of the described functional similarities.TFTC, similarly to other TBP-free TAF II complexes, including yeast SAGA, hSTAGA and hPCAF/GCN5, contains the histone acetyltransferase (HAT) hGCN5 and is able to acetylate histone H3 in both a free and a nucleosomal context (Grant et a...
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