Article

Chromatin interaction of TATA-binding protein is dynamically regulated in human cells

Department of Physiological Chemistry and Netherlands Proteomic Center, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, Netherlands.
Journal of Cell Science (Impact Factor: 5.33). 08/2010; 123(Pt 15):2663-71. DOI: 10.1242/jcs.064097
Source: PubMed

ABSTRACT Gene transcription in mammalian cells is a dynamic process involving regulated assembly of transcription complexes on chromatin in which the TATA-binding protein (TBP) plays a central role. Here, we investigate the dynamic behaviour of TBP by a combination of fluorescence recovery after photobleaching (FRAP) and biochemical assays using human cell lines of different origin. The majority of nucleoplasmic TBP and other TFIID subunits associate with chromatin in a highly dynamic manner. TBP dynamics are regulated by the joint action of the SNF2-related BTAF1 protein and the NC2 complex. Strikingly, both BTAF1 and NC2 predominantly affect TBP dissociation rates, leaving the association rate unchanged. Chromatin immunoprecipitation shows that BTAF1 negatively regulates TBP and NC2 binding to active promoters. Our results support a model for a BTAF1-mediated release of TBP-NC2 complexes from chromatin.

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    • "Our findings have important ramifications for transcriptional fidelity of mammalian cells. Fluorescence microscopy experiments indicated that the mammalian orthologs of Mot1p and NC2 collaborate to increase the off-rates of TBP from DNA in human cells (26,27). Recent genome-wide binding maps of human TBP and TFIIB revealed that the vast majority of binding events occur at non-coding transcription sites (52). "
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    ABSTRACT: Chromatin structure in transcribed regions poses a barrier for intragenic transcription. In a comprehensive study of the yeast chromatin remodelers and the Mot1p-NC2 regulators of TATA-binding protein (TBP), we detected synthetic genetic interactions indicative of suppression of intragenic transcription. Conditional depletion of Mot1p or NC2 in absence of the ISW1 remodeler, but not in the absence of other chromatin remodelers, activated the cryptic FLO8 promoter. Likewise, conditional depletion of Mot1p or NC2 in deletion backgrounds of the H3K36 methyltransferase Set2p or the Asf1p-Rtt106p histone H3-H4 chaperones, important factors involved in maintaining a repressive chromatin environment, resulted in increased intragenic FLO8 transcripts. Activity of the cryptic FLO8 promoter is associated with reduced H3 levels, increased TBP binding and tri-methylation of H3K4 and is independent of Spt-Ada-Gcn5-acetyltransferase function. These data reveal cooperation of negative regulation of TBP with specific chromatin regulators to inhibit intragenic transcription.
    Nucleic Acids Research 01/2014; 42(7). DOI:10.1093/nar/gkt1398 · 9.11 Impact Factor
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    • "FRAP studies were performed using a Zeiss 510 META confocal LSM (Zeiss, Oberkocken, Germany) as previously described [43]. GFP protein expression was induced with 0.5 μg/ml doxycycline for five hours. "
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    ABSTRACT: Background Recognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches. Results The PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA. Conclusion Concerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.
    Epigenetics & Chromatin 05/2013; 6(1):12. DOI:10.1186/1756-8935-6-12 · 4.46 Impact Factor
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    • "The effects seen upon loss of TBP–BTAF1 interaction differ from those seen upon knockdown of BTAF1 expression which has been shown to lower TBP mobility in the nucleus (43). In human cells, BTAF1 knockdown was shown to increase TBP occupancy at several promoters (43) and maybe also more globally in the genome (44). "
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    ABSTRACT: The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells.
    Nucleic Acids Research 02/2012; 40(4):1446-59. DOI:10.1093/nar/gkr802 · 9.11 Impact Factor
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