TATA-binding Protein-free TAF-containing Complex (TFTC) and P300 Are Both Required for Efficient Transcriptional Activation

The Journal of Biological Chemistry. Sep, 2002  |  Pubmed ID: 12107188

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process.

Novel Subunits of the TATA Binding Protein Free TAFII-containing Transcription Complex Identified by Matrix-assisted Laser Desorption/ionization-time of Flight Mass Spectrometry Following One-dimensional Gel Electrophoresis

Proteomics. Feb, 2003  |  Pubmed ID: 12601814

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require the transcription factor II D (TF(II)D), a complex comprising the TATA binding protein (TBP) and TBP-associated factors. However, another multiprotein complex isolated more recently and called TFTC (TBP-free TAF(II )containing complex), was shown to mediate initiation of RNA polymerase II (Pol II) transcription in the absence of TF(II)D as well as specific acetylation of histone H3 in a nucleosomal context. Several subunits of the TFTC complex were already identified using classical methods such as Edman based microsequencing and Western blot analysis. In this article we present a mass spectrometry based proteomic approach to confirm previous results and to identify other possible subunits of the TFTC complex. The TFTC complex was separated on one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis and analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting. Identifications were realized after databank searches. This new characterization of TFTC complex confirmed the presence of already described subunits (TRRAP, GCN5, SAP130/KIA0017, TAF(II)150, TAF(II)135, TAF(II)100, TAF(II)80, TAF(II)20, SPT3 and PAF65beta). Moreover, a good coverage of these sequences was obtained. Interestingly, TAF(II)32 and PAF6alpha were also determined as potential novel subunits of TFTC. These results together show the suitability and the great potential of this method and offer new perspectives in fundamental studies of transcription factor complexes.

Dynamic Changes in Transcription Factor Complexes During Erythroid Differentiation Revealed by Quantitative Proteomics

Nature Structural & Molecular Biology. Jan, 2004  |  Pubmed ID: 14718926

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.

Heme Regulates the Dynamic Exchange of Bach1 and NF-E2-related Factors in the Maf Transcription Factor Network

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2004  |  Pubmed ID: 14747657

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 beta-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 beta-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.

Using Stable Isotope Tagging and Mass Spectrometry to Characterize Protein Complexes and to Detect Changes in Their Composition

Methods in Molecular Biology (Clifton, N.J.). 2007  |  Pubmed ID: 17484108

One of the primary goals of proteomics is the description of the composition, dynamics, and connections of the multiprotein modules that catalyze a wide range of biological functions in cells. Mass spectrometry (MS) has proven to be an extremely powerful tool for characterizing the composition of purified complexes. However, because MS is not a quantitative technique, the usefulness of the data is limited. For example, without quantitative measurements, it is difficult to detect dynamic changes in complex composition, and it can be difficult to distinguish bona fide complex components from nonspecifically copurifying proteins. In this chapter, we describe a strategy for characterizing the composition of protein complexes and their dynamic changes in composition by combining affinity purification approaches with stable isotope tagging and MS. The use of software tools for statistical analysis of the data is also described.

Activator-mediated Recruitment of the MLL2 Methyltransferase Complex to the Beta-globin Locus

Molecular Cell. Aug, 2007  |  Pubmed ID: 17707229

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.

Nucleosome and Transcription Activator Antagonism at Human Beta-globin Locus Control Region DNase I Hypersensitive Sites

Nucleic Acids Research. 2007  |  Pubmed ID: 17720709

Locus control regions are regulatory elements that activate distant genes and typically consist of several DNase I hypersensitive sites coincident with clusters of transcription activator binding sites. To what extent nucleosomes and activators occupy these sites together or exclusively has not been extensively studied in vivo. We analyzed the chromatin structure of human beta-globin locus control region hypersensitive sites in erythroid cells expressing embryonic and fetal globin genes. Nucleosomes were variably depleted at hypersensitive sites HS1-HS4 and at HS5 which flanks the 5' of the locus. In lieu of nucleosomes, activators were differentially associated with these sites. Erythroid-specific GATA-1 resided at HS1, HS2 and HS4 but the NF-E2 hetero-dimer was limited to HS2 where nucleosomes were most severely depleted. Histones H3 and H4 were hyperacetylated and H3 was di-methylated at K4 across the LCR, however, the H3 K4 MLL methyltransferase component Ash2L and histone acetyltransferases CBP and p300 occupied essentially only HS2 and the NF-E2 motif in HS2 was required for Ash2L recruitment. Our results indicate that each hypersensitive site in the human beta-globin LCR has distinct structural features and suggest that HS2 plays a pivotal role in LCR organization at embryonic and fetal stages of globin gene expression.

P38 MAPK Signaling Regulates Recruitment of Ash2L-containing Methyltransferase Complexes to Specific Genes During Differentiation

Nature Structural & Molecular Biology. Dec, 2007  |  Pubmed ID: 18026121

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 Mef2d. Furthermore, this interaction is modulated during differentiation through activation of the p38 MAPK signaling pathway via phosphorylation of Mef2d. Thus, we provide evidence that signaling pathways regulate the targeting of TrxG-mediated epigenetic modifications at specific promoters during cellular differentiation.

Analysis of Epigenetic Modifications of Chromatin at Specific Gene Loci by Native Chromatin Immunoprecipitation of Nucleosomes Isolated Using Hydroxyapatite Chromatography

Nature Protocols. 2008  |  Pubmed ID: 18323811

Chromatin immunoprecipitation (ChIP) is routinely used to examine epigenetic modification of histones at specific genomic locations. However, covalent modifications of histone tails can serve as docking sites for chromatin regulatory factors. As such, association of these regulatory factors with chromatin could cause steric hindrance for antibody recognition, resulting in an underestimation of the relative enrichment of a given histone modification at specific loci. To overcome this problem, we have developed a native ChIP protocol to study covalent modification of histones that takes advantage of hydroxyapatite (HAP) chromatography to wash away chromatin-associated proteins before the immunoprecipitation of nucleosomes. This fast and simple procedure consists of five steps: nuclei isolation from cultured cells; fragmentation of chromatin using MNase; purification of nucleosomes using HAP; immunoprecipitation of modified nucleosomes; and qPCR analysis of DNA associated with modified histones. Nucleosomes prepared in this manner are free of contaminating proteins and permit an accurate evaluation of relative abundance of different covalent histone modifications at specific genomic loci. Completion of this protocol requires approximately 1.5 d.

Chromodomain-mediated Spreading on Active Genes

Nature Structural & Molecular Biology. Jan, 2009  |  Pubmed ID: 19125168

Dual Role for the Methyltransferase G9a in the Maintenance of Beta-globin Gene Transcription in Adult Erythroid Cells

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2009  |  Pubmed ID: 19822740

Using a proteomics screen, we have identified the methyltransferase G9a as an interacting partner of the hematopoietic activator NF-E2. We show that G9a is recruited to the beta-globin locus in a NF-E2-dependent manner and spreads over the entire locus. While G9a is often regarded as a corepressor, knocking down this protein in differentiating adult erythroid cells leads to repression of the adult beta(maj) globin gene and aberrant reactivation of the embryonic beta-like globin gene E(y). While in adult cells G9a maintains E(y) in a repressed state via dimethylation of histone H3 at lysines 9 and 27, it activates beta(maj) transcription in a methyltransferase-independent manner. Interestingly, the demethylase UTX is recruited to the beta(maj) (but not the E(y)) promoter where it antagonizes G9a-dependent H3K27 dimethylation. Collectively, these results reveal a dual role for G9a in maintaining proper expression (both repression and activation) of the beta-globin genes in differentiating adult erythroid cells.

UTX Mediates Demethylation of H3K27me3 at Muscle-specific Genes During Myogenesis

The EMBO Journal. Apr, 2010  |  Pubmed ID: 20300060

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.

Crosstalk Between Histone Modifications Maintains the Developmental Pattern of Gene Expression on a Tissue-specific Locus

Epigenetics : Official Journal of the DNA Methylation Society. May, 2010  |  Pubmed ID: 20424518

Genome wide studies have provided a wealth of information related to histone modifications. Particular modifications, which can encompass both broad and discrete regions, are associated with certain genomic elements and gene expression status. Here we focus on how studies on the beta-globin gene cluster can complement the genome wide effort through the thorough dissection of histone modifying protein crosstalk. The beta-globin locus serves as a model system to study both regulation of gene expression driven at a distance by enhancers and mechanisms of developmental switching of clustered genes. We investigate recent studies, which uncover that histone methyltransferases, recruited at the beta-globin enhancer, control gene expression by long range propagation on chromatin. Specifically, we focus on how seemingly antagonistic complexes, such as those including MLL2, G9a and UTX, can cooperate to functionally regulate developmentally controlled gene expression. Finally, we speculate on the mechanisms of chromatin modifying complex propagation on genomic domains.

Differential Genomic Targeting of the Transcription Factor TAL1 in Alternate Haematopoietic Lineages

The EMBO Journal. Feb, 2011  |  Pubmed ID: 21179004

TAL1/SCL is a master regulator of haematopoiesis whose expression promotes opposite outcomes depending on the cell type: differentiation in the erythroid lineage or oncogenesis in the T-cell lineage. Here, we used a combination of ChIP sequencing and gene expression profiling to compare the function of TAL1 in normal erythroid and leukaemic T cells. Analysis of the genome-wide binding properties of TAL1 in these two haematopoietic lineages revealed new insight into the mechanism by which transcription factors select their binding sites in alternate lineages. Our study shows limited overlap in the TAL1-binding profile between the two cell types with an unexpected preference for ETS and RUNX motifs adjacent to E-boxes in the T-cell lineage. Furthermore, we show that TAL1 interacts with RUNX1 and ETS1, and that these transcription factors are critically required for TAL1 binding to genes that modulate T-cell differentiation. Thus, our findings highlight a critical role of the cellular environment in modulating transcription factor binding, and provide insight into the mechanism by which TAL1 inhibits differentiation leading to oncogenesis in the T-cell lineage.

Quantitative Proteomic Analysis of Dystrophic Dog Muscle

Journal of Proteome Research. May, 2011  |  Pubmed ID: 21410286

Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis of DMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α). Subsequently, we were able to showed that PGC1-α expression is dramatically reduced in GRMD compared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.

Crystal Structure of the Trithorax Group Protein ASH2L Reveals a Forkhead-like DNA Binding Domain

Nature Structural & Molecular Biology. Jul, 2011  |  Pubmed ID: 21642971

Absent, small or homeotic discs-like 2 (ASH2L) is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. Here we report that ASH2L binds DNA using a forkhead-like helix-wing-helix (HWH) domain. In vivo, the ASH2L HWH domain is required for binding to the β-globin locus control region, histone H3 Lys4 (H3K4) trimethylation and maximal expression of the β-globin gene (Hbb-1), validating the functional importance of the ASH2L DNA binding domain.