Activator-dependent p300 Acetylation of Chromatin in Vitro

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 acetylationinduced 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,ubiquitousactivityintheabsenceofactivatorsandactivatordependent 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.The genomes of eukaryotes are assembled into a complex nucleoprotein architecture called chromatin. In the first level of chromatin structure, 147 bp of DNA are wrapped 1.65 times around an octamer of core histones to form the nucleosome (1). Nucleosomal arrays make up the next level of chromatin architecture and consist of histone octamers spaced repetitively at ϳ20 -60-bp intervals along a DNA molecule. When proteins other than core histones bind to nucleosomal arrays, chromatin is formed. Chromatin undergoes a series of hierarchical conformational changes, leading to formation of higher order levels of condensed structure (see below). The transcription of genes by RNA polymerase II (RNAPII) 2 must occur within this dense chromatin environment. Hence, a long-standing question in the field is the extent to which higher order chromatin fiber architecture influences gene expression (Refs. 2-4; for review, see Refs. 5-10).Nucleosomal arrays and chromatin in solution in vitro are in equilibrium between multiple architectural states; that is, a primary structure having an extended "beads-on-a-string" conformation, secondary folded structures resulting from intrafiber nucleosome-nucleosome interactions, and tertiary oligomeric structures resulting from reversible interfiber nucleosome-nucleosome interactions (5, 9). Folding and oligomerization both are induced by increasing concentrations of cations, e.g. 1-10 mM MgCl 2 . In addition, transitions between the different condensed states require the amino-terminal "tail" domains (NTDs) of the core histones (5, 11). ...

Definition of a FoxA1 Cistrome That Is Crucial for G1 to S-Phase Cell-Cycle Transit in Castration-Resistant Prostate Cancer

“…S6C and S6D). Our findings are consistent with previous in vitro findings that activator-dependent histone H3 acetylation leads to an open chromatin structure through disruption of both inter- and intrafiber internucleosome interactions (41, 42). Thus, although histone modifications and nucleosome depletion are not required for FoxA1 to open in vitro -reconstituted condensed chromatin (4), our findings suggest that in vivo FoxA1 binding requires both active histone H3K4 methylation and other collaborating transcription factors capable of inducing histone acetylation and/or nucleosome disruption.…”

BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia

“…p300 and CBP are known to acetylate several lysine residues on core histones, which includes H3K27 and H4K8 (McManus and Hendzel, 2003; Szerlong et al, 2010). Since BRD4 occupancy tends to encompass acetylated nucleosomes that flank TF binding sites, we considered whether p300/CBP maintains histone acetylation near BRD4-occupied enhancers and promoters.…”

Responses of the embryonic epigenome to maternal diabetes

“…Based on the reported effects of excess glucose on histone acetylation (Wellen et al, 2009), we limited our focus on this particular modality of the epigenome. Within the spectrum of histone modifications, we chose H4K5Ac as a mark correlated with active promoters (Rosenfeld et al, 2009) and H3K27Ac as a mark found to be associated with tran-scriptional enhancers (Creyghton et al, 2010; Szerlong et al, 2010). Our results show that in chromatin of neurulation-stage embryos, genomic patterns of histone acetylation marks are affected by the metabolic status of the mother, supporting the hypothesis that the embryonic epigenome is the target of adverse maternal metabolic conditions.…”

“…H3K27 acetylation is a histone mark that is co-localized with such transcriptional enhancers (Creyghton et al, 2010). In fact, p300, an enzyme generating the H3K27Ac mark, is regarded as a transcriptional activator (Szerlong et al, 2010); the corresponding gene Ep300 is an NTD gene (Yao et al, 1998). Furthermore, we find that the Ep300 gene itself is responsive to maternal diabetes, showing decreased expression in embryos at ED 10.5 from dams of the FVB inbred mouse strain with chemically induced diabetes (−1.38-fold; p = 0.006; Parlikova et al, 2009) and in ED 8.5 embryos of nonobese diabetic dams with spontaneously occurring diabetes (−.49-fold; p = 0.00013; Salbaum and Kappen, unpublished data) compared with embryos from normoglycemic dams.…”