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Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.

ABSTRACT: Condensation of chromatin into higher order structures is mediated by intra- and interfiber nucleosome-nucleosome interactions. Our goals in this study were to determine the impact specific activator-dependent histone acetylation had on chromatin condensation and to ascertain whether acetylation-induced changes in chromatin condensation were related to changes in RNA polymerase II (RNAPII) activity. To accomplish this, an in vitro model system was constructed in which the purified transcriptional activators, Tax and phosphorylated CREB (cAMP-response element-binding protein), recruited the p300 histone acetyltransferase to nucleosomal templates containing the human T-cell leukemia virus type-1 promoter sequences. We find that activator-dependent p300 histone acetylation disrupted both inter- and intrafiber nucleosome-nucleosome interactions and simultaneously led to enhanced RNAPII transcription from the decondensed model chromatin. p300 histone acetyltransferase activity had two distinct components: non-targeted, ubiquitous activity in the absence of activators and activator-dependent activity targeted primarily to promoter-proximal nucleosomes. Mass spectrometry identified several unique p300 acetylation sites on nucleosomal histone H3 (H3K9, H3K27, H3K36, and H3K37). Collectively, our data have important implications for understanding both the mechanism of RNAPII transcriptional regulation by chromatin and the molecular determinants of higher order chromatin structure.

SUBMITTER: Szerlong HJ

PROVIDER: S-EPMC2952196 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.

Szerlong Heather J HJ Prenni Jessica E JE Nyborg Jennifer K JK Hansen Jeffrey C JC

The Journal of biological chemistry 20100818 42

Condensation of chromatin into higher order structures is mediated by intra- and interfiber nucleosome-nucleosome interactions. Our goals in this study were to determine the impact specific activator-dependent histone acetylation had on chromatin condensation and to ascertain whether acetylation-induced changes in chromatin condensation were related to changes in RNA polymerase II (RNAPII) activity. To accomplish this, an in vitro model system was constructed in which the purified transcriptiona ...[more]

PMID: 20720004

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Project description:Eukaryotic gene expression requires the coordination of multiple factors to overcome the repressive nature of chromatin. However, the mechanistic details of this coordination are not well understood. The SAGA family of transcriptional coactivators interacts with DNA-binding activators to establish regions of hyperacetylation. We have previously shown that, contrary to the prevailing model in which activator protein increases SAGA affinity for nucleosome substrate, the Gal4-VP16 activator model system augments the rate of acetylation turnover for the SAGA complex from budding yeast. To better understand how this stimulation occurs, we have identified necessary components using both kinetics assays and binding interactions studies. We find that Gal4-VP16-mediated stimulation requires activator binding to DNA flanking the nucleosome, as it cannot be reproduced in trans by activator protein alone or by exogenous DNA containing the activator binding site in combination with the activator protein. Further, activator-mediated stimulation requires subunits outside of the histone acetylation (HAT) module, with the Tra1 subunit being responsible for the majority of the stimulation. Interestingly, for the HAT module alone, nucleosome acetylation is inhibited by activator proteins due to non-specific binding of the activator to the nucleosomes. This inhibition is not observed for the yeast ADA complex, a small complex comprised mostly of the HAT module, suggesting that subunits outside of the HAT module in both it and SAGA can overcome non-specific activator binding to nucleosomes. However, this activity appears distinct from activator-mediated stimulation, as ADA complex acetylation is not stimulated by Gal4-VP16. Graphical abstract Image 1 Highlights • Stimulation of nucleosome acetylation by SAGA requires activator in cis• Tra1 mediates the majority of activator stimulation• The core HAT complex of SAGA is inhibited by activator due to non-specific binding• The related ADA complex is neither stimulated nor inhibited by activator

Dataset Information

Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.

Publications

Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.

Similar Datasets

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