Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter

Dahabieh, Matthew S.; Ooms, Marcel; Malcolm, Tom; Simon, Viviana; Sadowski, Ivan

doi:10.1016/j.virol.2011.07.002

Cited by 27 publications

(58 citation statements)

References 49 publications

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“…We concluded that PICH-1, PICH-2 and PICH-3 are TFIID-dependent complexes. Antibodies directed against TFII-I had a modest negative effect on PICH-3 formation (Figure 4A, lane 10 versus lane 7), consistent with a recent report that TFII-I can interact with the HIV core promoter [42]. The addition of antibodies against the mediator subunit, MED6 also reduced binding of PICH-3 (Figure 4A, lane 11 versus lane 7), suggesting PICH-3 depends on the mediator complex for its stable association with TASHET.…”

Section: Resultssupporting

confidence: 90%

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

et al. 2012

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BackgroundHIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown.ResultsUsing electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat.ConclusionOur data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

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Section: Resultssupporting

confidence: 90%

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

et al. 2012

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“…The virus contains eGFP expressed from an internal E1Fα promoter and dsRed expressed from the 5’ LTR as a fusion with p24Gag (Figure 2A). Since it was previously shown that YY1 is recruited to the LTR flanking the transcription start site through interaction with LSF [11], near an additional binding site for RBF-2 designated RBEI [23], we designed primers for ChIP to separately measure interaction of YY1 with the core promoter/RBEI region, the upstream RBEIII region, in addition to the enhancer region between these two elements (Figure 2B). We introduced a substitution of three nucleotides at the RBEIII site, shown to prevent binding of YY1 to the RBEIII oligonucleotide in vitro (Figure 2C).…”

Section: Resultsmentioning

confidence: 99%

An Upstream YY1 Binding Site on the HIV-1 LTR Contributes to Latent Infection

et al. 2013

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During HIV-1 infection a population of latently infected cells is established. This population is the major obstacle preventing total eradication of the virus from AIDS patients. HIV-1 latency is thought to arise by various mechanisms including repressive chromatin modifications. Transcription factors such as YY1 have been shown to facilitate repressive chromatin modifications by the recruitment of histone deacetylases. In this study, we identified a novel binding site for YY1 on the HIV-1 LTR, 120 nucleotides upstream of the transcription start site. We show that YY1 can bind to this site in vitro and in vivo and that binding to the LTR is dissociated upon T cell activation. Overexpression of YY1 causes an increase in the proportion of cells that produce latent infections. These observations, in combination with previous results, demonstrate that YY1 plays a prominent role in controlling the establishment and maintenance of latent HIV-1 provirus in unstimulated cells.

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“…Jurkat E6-1 (29), SupT1 (30), U937 (ATCC), HeLa (ATCC), and HEK293T (ATCC) cells were cultured under standard conditions as previously described (31).…”

Section: Construction Of a Panel Of Rgh Vectorsmentioning

confidence: 99%

“…Vesicular stomatitis virus G (VSV-G) pseudotyped viral stocks were created by transfecting HEK293T cells with viral molecular clones and pHEF-VSVg (24) in a 10:1 ratio as previously described (31). Purified, concentrated viral stocks were prepared by centrifugation of clarified supernatants from transfected HEK293T cells through a 6% iodixanolOptiPrep (Sigma) cushion.…”

Section: Construction Of a Panel Of Rgh Vectorsmentioning

confidence: 99%

A Doubly Fluorescent HIV-1 Reporter Shows that the Majority of Integrated HIV-1 Is Latent Shortly after Infection

Dahabieh

Ooms

Simon

et al. 2013

J Virol

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137

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Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter

Cited by 27 publications

References 49 publications

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

An Upstream YY1 Binding Site on the HIV-1 LTR Contributes to Latent Infection

A Doubly Fluorescent HIV-1 Reporter Shows that the Majority of Integrated HIV-1 Is Latent Shortly after Infection

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