TaxAbolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1Promoter

Konesky, Kasey L.; Nyborg, Jennifer K.; Laybourn, Paul J.

doi:10.1128/jvi.00631-06

Cited by 6 publications

(5 citation statements)

References 66 publications

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“…46), which activates RNAPII-dependent transcription (47). p300 recruitment to the promoter and RNAPII transcriptional activation is associated with acetylation of core histones in vivo (48,49) and in vitro (38,44,48,50). Thus, the HTLV-1 promoter provides an excellent system to study the mechanistic basis for how activator-dependent p300 acetylation affects gene expression.…”

Section: Resultsmentioning

confidence: 99%

“…In Vitro Transcription-In vitro transcription reactions were performed as described (38), except HTLV-1 chromatin or free DNA templates (4 nM) were used in place of the bead-bound chromatin. Briefly, 30-l reaction mixtures containing Tax (130 nM), pCREB (130 nM), p300 (10 nM), and/or acetyl-CoA (100 M) as indicated in transcription buffer (1 mM dithiothreitol, 25 mM Tris-HCl (pH 7.9), 50 mM KCl, 6.25 mM MgCl 2 , 0.5 mM EDTA, 10% glycerol) were supplemented with ϳ40 g (total protein) of nuclear extract for 30 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

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Activator-dependent p300 Acetylation of Chromatin in Vitro

Szerlong

Prenni

Nyborg

et al. 2010

Journal of Biological Chemistry

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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). ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Activator-dependent p300 Acetylation of Chromatin in Vitro

Szerlong

Prenni

Nyborg

et al. 2010

Journal of Biological Chemistry

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“…As a potent activator of transcription, the human T cell leukemia virus type 1 (HTLV-1)-encoded Tax protein serves as an outstanding model for studies on transcriptional regulation in a chromatin context in higher eukaryotes (1)(2)(3)(4)(5). Tax and Ser 133 -phosphorylated cAMP response element binding protein (pCREB) together bind the HTLV-1 promoter at three conserved cAMP response elements (CREs) called viral CREs (vCREs).…”

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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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“…Experiments should be performed a minimum of three times. For examples of several transcriptional experiments see Figure 6 from Konesky et al (20).…”

Section: Biochemical Functional Assays On Chromatin Templates Transcrmentioning

confidence: 99%

“…As an example, see the footprint of the distal and middle vCREs see Fig. 7 (reproduced from (20)). Following primer extension, the DNA is precipitated and resolved on an 8% sequencing gel.…”

Section: Deoxyribonuclease I (Dnase I) Primer Extension Footprintingmentioning

confidence: 99%

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Konesky

Laybourn

2007

Methods

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We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how the degree of chromatin assembly in the absence and presence of histone H1 is measured using topological analysis and the use of micrococcal nuclease digestion performed to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further we describe the use sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).

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TaxAbolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1Promoter

Cited by 6 publications

References 66 publications

Activator-dependent p300 Acetylation of Chromatin in Vitro

Activator-dependent p300 Acetylation of Chromatin in Vitro

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

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

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