Purification of CREB to apparent homogeneity: Removal of truncation products and contaminating nucleic acid

Journal of Biological Chemistry

et al. 2010

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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). ...

“…Recombinant Ser133-phosphorylated CREB 327 (pCREB) (27,28) and Tax-His 6 (29) were purified as described (30). Nuclear extract from CEM cells was prepared as described (31).…”

Section: Methodsmentioning

confidence: 99%

Activator-dependent p300 Acetylation of Chromatin in Vitro

Szerlong

Prenni

Journal of Biological Chemistry

et al. 2010

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“…Recombinant CREB327 (27) and Tax-His6 (28) were purified as described. CREB was phosphorylated at Ser-133 using the catalytic subunit of PKA.…”

Section: Methodsmentioning

confidence: 99%

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

Sharma

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

2008

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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

“…Bacterially expressed Tax-His 6 (61) was purified to Ͼ98% homogeneity as previously described (16). CREB 327 was purified to apparent homogeneity, free of contaminating nucleic acids, as recently described (30). Full-length His 6 -tagged p300 was expressed from recombinant baculovirus in Sf9 cells and purified as previously described (14,26).…”

Section: Methodsmentioning

confidence: 99%

The Proto-Oncogene Bcl3, Induced by Tax, Represses Tax-Mediated Transcription via p300 Displacement from the Human T-Cell Leukemia Virus Type 1 Promoter

Kim

Sharma

2008

J Virol

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The etiology of human T-cell leukemia virus type 1 (HTLV-1)-induced adult T-cell leukemia is linked to the expression of the viral oncoprotein Tax. Although the mechanism of retroviral transformation is unknown, Tax interferes with fundamental cellular processes, including proliferation and apoptosis, and these events may directly link Tax to early steps in malignant progression. In this study, we examined the interplay between Tax and the potent proto-oncogene B-cell chronic leukemia protein 3 (Bcl3). Bcl3 is a critical regulator of cell survival and proliferation and is overexpressed in HTLV-1-infected cells. We found that Tax Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus etiologically linked to an unusually aggressive and ultimately fatal malignancy called adult T-cell leukemia. Adult T-cell leukemia is characterized by the presence of a chromosomally integrated HTLV-1 provirus in highly aneuploid T cells (33). However, the majority of HTLV-1-infected individuals remain lifelong asymptomatic carriers despite the fact that up to 70% of their CD4 ϩ T cells carry integrated provirus (3, 4). The molecular events that lead to HTLV-1-dependent transformation of the infected T cell remain enigmatic.HTLV-1 encodes a number of nonstructural proteins that participate in the life cycle of the virus. These include p12, p13, p30, HBZ, Rex, and Tax (for a review, see reference 34). Of these regulatory proteins, the oncoprotein Tax has been well characterized as a critical player in both viral and cellular transcription, as well as malignant transformation. Tax is a strong transcriptional activator required for efficient expression of the viral genome. Tax activates HTLV-1 transcription through three conserved cyclic AMP response elements (CREs) located in the transcriptional control region of the virus. These elements, called viral CREs (vCREs) serve as the binding site for a complex composed of Tax and the cellular transcription factor CREB (25,29,32). Together, the Tax/ CREB complex recruits the cellular coactivators CBP/p300 via direct interaction with their conserved KIX domain (14, 28).Phosphorylation of CREB at serine 133 (pCREB) is essential for strong p300 recruitment by the promoter-bound Tax/CREB complex. As such, pCREB is required for transcriptional activation by Tax (14). Consistent with these observations, Tax has been found to promote CREB phosphorylation in the infected cell, purportedly to ensure sufficient pCREB availability for Tax activation of viral gene expression (24,55,56).Mitotic replication has been established as a major vehicle of viral transmission within an infected individual. The Tax protein is required for the promotion of clonal proliferation and is directly linked to malignant transformation (1). Tax triggers changes in a variety of intracellular signal transduction pathways and deregulates gene expression though interaction with many different cellular proteins (17). For example, Tax-mediated activation of the NF-B pathway results in the upregulation of a large num...