Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Konesky, Kasey L.; Laybourn, Paul J.

doi:10.1016/j.ymeth.2006.11.004

Cited by 5 publications

(5 citation statements)

References 22 publications

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“…Assembly was performed at a 4:7 histone:DNA mass ratio. The degree of assembly was confirmed by micrococcal nuclease digestion (26). Micrococcal nuclease digestion of the chromatin-assembled 4TxRE promoter template is shown by Sharma et al (44).…”

Section: Methodsmentioning

confidence: 84%

“…For these studies, we assembled the Tax-responsive promoter fragment used in Fig. 1 into chromatin, using purified Drosophila core histones and the recombinant assembly proteins Acf1/ISWI and Drosophila NAP-1 (14,26,34). Micrococcal nuclease assays were performed to verify the assembly of nucleosomes on the immobilized promoter fragment.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant Drosophila histones were assembled into chromatin as previously described (14,16,26), except that the assembly was performed on the linear, bead-bound promoter fragments. Assembly was performed at a 4:7 histone:DNA mass ratio.…”

Section: Methodsmentioning

confidence: 99%

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The Human T-Cell Leukemia Virus Type 1 Tax Protein Confers CBP/p300 Recruitment and Transcriptional Activation Properties to Phosphorylated CREB

Geiger

Sharma

Kim

et al. 2008

Molecular and Cellular Biology

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The human T-cell leukemia virus-encoded oncoprotein Tax is a potent activator of viral transcription. Tax function is strictly dependent upon the cellular transcription factor CREB, and together they bind cAMP response elements within the viral promoter and mediate high-level viral transcription. Signal-dependent CREB phosphorylation at Ser 133 (pCREB) correlates with the activation of transcription. This activation has been attributed to recruitment of the coactivators CBP/p300 via physical interaction with the KIX domain. Here we show that the promoter-bound Tax/pCREB complex strongly recruits the recombinant, purified full-length coactivators CBP and p300. Additionally, the promoter-bound Tax/pCREB (but not Tax/CREB) complex recruits native p300 and potently activates transcription from chromatin templates. Unexpectedly, pCREB alone failed to detectably recruit the full-length coactivators, despite strong binding to KIX. These observations are in marked contrast to those in published studies that have characterized the physical interaction between KIX and pCREB and extrapolated these results to the full-length proteins. Consistent with our observation that pCREB is deficient for binding of CBP/p300, pCREB alone failed to support transcriptional activation. These data reveal that phosphorylation of CREB is not sufficient for CBP/p300 recruitment and transcriptional activation. The regulation of transcription by pCREB is therefore more complex than is generally recognized, and coregulators, such as Tax, likely play a critical role in the modulation of pCREB function.cAMP-response element binding protein (CREB) is one of the most widely studied transcription factors in metazoans. The basic leucine zipper region of CREB binds to cAMP response elements (CREs) and potentially regulates the expression of a significant percentage of genes in the human genome (13,21,53). The large number of cellular genes regulated by CREB exemplifies the critical role this archetypal transcription factor plays in vital cellular processes, such as development, differentiation, and cellular homeostasis. More than 300 distinct extracellular stimuli converge on several kinases, including protein kinases A and C, that phosphorylate CREB at serine 133 (Ser 133 ) to activate the transcription function of CREB (22). Phosphorylated CREB (pCREB) then recruits the cellular coactivator paralogues CREB-binding protein (CBP) and p300 to activate transcription (8,29,36,38).A significant number of studies support a model of CREB transcription function whereby the binding of pCREB to the KIX domain of CBP/p300 is concomitant with coactivator recruitment and transcriptional activation. This model is largely based on characterization of the physical interaction between pCREB and the isolated KIX domain (38, 39) and subsequent extrapolation of these data to the recruitment of the full-length proteins. Emerging evidence, however, indicates that CREB phosphorylation may not be sufficient for CBP/p300 recruitment and transcription function. First, stimuli ...

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Section: Methodsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

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The Human T-Cell Leukemia Virus Type 1 Tax Protein Confers CBP/p300 Recruitment and Transcriptional Activation Properties to Phosphorylated CREB

Geiger

Sharma

Kim

et al. 2008

Molecular and Cellular Biology

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“…Earlier studies on in vitro reconstitution of yeast and mammalian nucleosomes suggested that mammalian nucleosomes bind DNA more readily (80% chromatin assembly), and that yeast nucleosomes are comparatively unstable (~16% assembly) (Lee et al, 1982). Furthermore, it has long been known that in vitro transcription using human chromatin (or any higher eukaryote chromatin) inhibits transcription (Konesky and Laybourn, 2007; Lorch et al, 1987), but a recent paper using native yeast chromatin showed enhanced in vitro transcription compared to naked DNA (Nagai et al, 2017). Our study may provide a means to reconcile these opposing observations.…”

Section: Discussionmentioning

confidence: 99%

Resetting the Yeast Epigenome with Human Nucleosomes

Truong

Boeke

2017

Cell

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Summary Humans and yeast are separated by a billion years of evolution, yet their conserved histones retain central roles in gene regulation. Here, we “reset” yeast to use core human nucleosomes in lieu of their own – a rare event taking twenty days – which initially only worked with variant H3.1. The cells adapt by acquiring suppressor mutations in cell-division genes, or by acquiring certain aneuploid states. Converting five histone residues to their yeast counterparts restored robust growth. We reveal that humanized nucleosomes are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy, globally reduce RNA content, and slow adaptation to new conditions by delaying chromatin remodeling. These humanized yeasts (including H3.3) pose fundamental new questions about how chromatin is linked to many cell processes, and provides a platform to study histone variants via yeast epigenome reprogramming.

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“…The assembled chromatin was stored in a buffer containing 0.1 M NaCl, 0.1% Nonidet P-40, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM DTT, and 20% glycerol at 4°C. Micrococcal nuclease assays were performed as described (34).…”

Section: Methodsmentioning

confidence: 99%

The coactivators CBP/p300 and the histone chaperone NAP1 promote transcription-independent nucleosome eviction at the HTLV-1 promoter

Sharma

Nyborg

2008

Proc. Natl. Acad. Sci. U.S.A.

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The human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma. The multifunctional virally encoded oncoprotein Tax is responsible for malignant transformation and potent activation of HTLV-1 transcription. Tax, in complex with phosphorylated cAMP response element binding protein (pCREB), strongly recruits the cellular coactivators CREB binding protein (CBP)/p300 to the viral promoter concomitant with transcriptional activation. Although the mechanism of activator/coactivator-mediated transcriptional activation is poorly understood, the recruitment of CBP/p300 by regulatory factors appears to function, in part, by promoting changes in chromatin architecture that are permissive to transcriptional activation. Here, we show that CBP/p300 recruitment promotes histone acetylation and eviction of the histone octamer from the chromatin-assembled HTLV-1 promoter template in vitro. Nucleosome disassembly is strictly acetyl-CoA dependent and is not linked to ATP utilization. We find that the histone chaperone, nucleosome assembly protein 1 (NAP1), cooperates with CBP/p300 in eviction of the acetylated histones from the chromatin template. These findings reveal a unique mechanism in which the DNA-bound Tax/pCREB complex recruits CBP/p300, and together with NAP1, the coactivators cooperate to dramatically reduce nucleosome occupancy at the viral promoter in an acetylation-dependent and transcription-independent fashion.Tax ͉ cAMP response element binding protein ͉ acetylation ͉ chromatin ͉ histone acetyltransferase

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Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Cited by 5 publications

References 22 publications

The Human T-Cell Leukemia Virus Type 1 Tax Protein Confers CBP/p300 Recruitment and Transcriptional Activation Properties to Phosphorylated CREB

The Human T-Cell Leukemia Virus Type 1 Tax Protein Confers CBP/p300 Recruitment and Transcriptional Activation Properties to Phosphorylated CREB

Resetting the Yeast Epigenome with Human Nucleosomes

The coactivators CBP/p300 and the histone chaperone NAP1 promote transcription-independent nucleosome eviction at the HTLV-1 promoter

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