Fig 1.
SWI/SNF and MI2 family of ATP-dependent chromatin remodeling enzymes are involved in LTR repression.
(A) Four distinct classes of mammalian ATP-dependent chromatin remodeling complexes distinguished by their catalytic subunits, BRG-1/BRM (SWI/SNF), ISWI, INO80, or CHD3/4 (MI2). (B) RNAi depletion of ATPase subunit of each class of ATP-dependent chromatin remodeling complexes as indicated in J-Lat A2 cells containing an integrated latent LTR-Tat-IRES-GFP virus. Cells were nucleofected with control siRNA or siRNAs targeting INO80, CHD3, ISWI, BRG1, and BRM as indicated. GFP expression was monitored by FACS at indicated times post-transfection and is presented as % GFP-positive cells. (C) Western blot analysis to demonstrate depletion of individual ATPase subunits as indicated 3 and 6 d after transfection of siRNAs. RT-PCR analysis indicated stable depletion of INO80 mRNA up to 8 d after siRNA transfection. Error bars represent the SEM of three independent experiments. * p < 0.05, ** p < 0.01.
Fig 2.
The BAF complex represses basal transcription at the HIV promoter.
(A) Table of subunit composition of the two distinct SWI/SNF complexes, BAF and PBAF, in mammals. (B) GFP expression was monitored by flow cytometry at indicated times after siRNA transfection to measure HIV promoter activity. Results are presented as MFI for cells treated with a control siRNA or siRNAs specific for SWI/SNF subunits. (C) Same experiments as shown in (B) for clone D, with clone E, another Jurkat cell line containing an integrated LTR-GFP virus. Error bars represent the SEM of five independent experiments. * p<0.05. (D) Jurkat cells containing an integrated LTR-GFP virus (clone D) were transfected with control siRNA or siRNAs targeting various SWI/SNF complex subunits as indicated. Western blot analysis shows expression of each SWI/SNF subunit after its specific depletion 0, 2, 4, 6, 7, 8, 10, and 14 d after siRNA transfection with each specific antibody and β-actin loading control as indicated.
Fig 3.
Reactivation of latent HIV mediated by knockdown of BAF subunits.
(A) J-Lat A2 cells latently infected with LTR-Tat-IRES GFP virus were transfected with either control siRNA or siRNAs targeting various SWI/SNF subunits as indicated. GFP expression was monitored by FACS at the times indicated after transfection and is expressed as % GFP-positive cells. (B) J-Lat 11.1, clone of Jurkat cells containing a latent integrated full-length HIV virus harboring GFP in place of Nef, was transfected with either control siRNA or siRNAs targeting various SWI/SNF subunits as indicated. GFP expression was monitored by FACS at the times indicated after transfection and is expressed as % GFP-positive cells. Error bars represent the SEM of five independent experiments. * p < 0.05. (C) The BRG-1 complex remains intact following depletion of BAF or PBAF-specific subunits. BRG-1 was immunoprecipitated from J-Lat A2 cells transfected with control siRNA or siRNA targeting BAF250 or BAF180, and its associated proteins were examined by SDS-PAGE and Western blotting with antibodies against BRG-1 itself, the BAF or PBAF-specific subunits BAF250a, BAF180 and BAF200, the core subunit INI-1 and GAPDH as control.
Fig 4.
PBAF, recruited by K50K51 acetylated Tat, is a co-factor for Tat activation of the HIV promoter.
(A) J-Lat A2 cells containing an integrated LTR-Tat-FLAG-GFP were stimulated with PMA to induce expression of Tat-FLAG. Tat was immunoprecipitated from untreated or PMA-stimulated cell lysates and its associated proteins were examined by SDS-PAGE and Western blotting with antibodies against the BAF- or PBAF-specific subunits BAF250a and BAF180, and protein kinase D-1 and 14-3-3 as controls. (B) Tat co-immunoprecipitation with BAF180 is modulated by Tat acetylation. Tat (wild-type or K50R/L51R) was immunoprecipitated using anti-FLAG antibody and analyzed by Western blotting using antibody specific for BAF180. Tat acetylation levels were assessed using an anti-acetyl lysine antibody. All proteins were expressed at similar levels under the different experimental conditions as shown by the Inputs. (C) 1G5 Jurkat cells containing integrated LTR-Luciferase (LTR-Luc) were nucleofected with siRNAs against BAF180, BAF250, or with a control siRNA pool. Expression of BAF180, BAF250, and β-actin was analyzed by Western blotting after depletion of either BAF180 or BAF250. (D) Transactivation of the HIV promoter by Tat is reduced in the absence of BAF180. 48 h after siRNA depletion of BAF180 or BAF250, cells were re-transfected with either a control or Tat-expression vector (CMV-driven), and luciferase assay performed after 24 h. Error bars represent the SEM of three independent experiments. * p<0.05. (D).
Fig 5.
Direct binding of distinct SWI/SNF complexes to the HIV promoter before and after transcriptional activation.
(A) Schematic representation of strategy to explore nucleosome position changes and enrichment of SWI/SNF at the HIV LTR in its repressed state and after PMA stimulation. J-Lat 11.1 cells at 0, 1, and 12 h after PMA addition were crosslinked and sonicated to yield fragments of approximately 150 bp. DNA accessibility was monitored by FAIRE while nucleosome occupancy was determined by histone H3 and H2B ChIPs. To determine enrichment of SWI/SNF complexes, we performed ChIPs with antibodies specific for BAF250, BAF180, BAF200, and BRG1. (B) PMA stimulation causes a reduction in histone density over HIV nuc-1 as determined by H3 and H2B ChIPs. Histone ChIPs are presented as percent immunoprecipitated DNA over input. (C) PMA stimulation is accompanied by an increase in DNA accessibility over the positioned nuc-1 of the HIV LTR. FAIRE results are presented as fold change respective to the unstimulated value (normalized to 1) for each primer pair. (D) BAF250a-specific BAF complex directly binds to nuc-1 in its repressed state, while BAF180 and BAF200-specific PBAF is recruited to nuc-1 upon PMA stimulation. SWI/SNF subunit ChIPs are presented as percent immunoprecipitated DNA over input. Immunoprecipitated DNA from ChIPs and phenol:chloroform extracted DNA from FAIRE were analyzed by qPCR using primer pairs specific for the HIV LTR nuc-0, DHS1, nuc-1, and nuc-2 regions, and control region amplifying upstream of the Axin2 gene. For all ChIP and FAIRE experiments, error bars represent the SEM of at least three independent experiments. * p < 0.05, ** p < 0.01. We depict results for J-Lat 11.1. Similar results were obtained for J-Lat A2 (S5 Fig).
Fig 6.
BAF represses HIV transcription by positioning nuc-1 of the HIV LTR.
(A) Location of the strictly positioned nucleosomes correlates negatively with the predicted histone binding affinity score (nucleosome score) of the DNA sequence encompassing the HIV LTR. Predicted nucleosome affinity for HIV nucleotide sequence 1–1800 was determined using the algorithm described [55]. Similar results were obtained with an alternative algorithm described (S6 Fig) [56,57]. Means and standard deviations for nucleosome score at insertion sites are indicated by blue (mean) and red lines (mean ± SD) and give reference to known genomic sites of HIV integration [58]. (B) Depletion BAF250 and BRG1 results in a peak in DNA accessibility over positioned nuc-1 and decreased accessibility over DHS1 of the HIV LTR. J-Lat 11.1 cells were nucleofected with either nontargeting siRNA or siRNAs targeting individual SWI/SNF subunits as indicated and subjected to FAIRE. FAIRE results are presented as fold change respective to value obtained for control siRNA transfected cells (normalized to 1) for each primer pair. (C) Depletion of BAF results in a reduction in histone density over the positioned LTR nuc-1 as determined by H3 and H2B ChIPs. (D) BAF180 and BAF250 are distinctly required for targeting of the BRG1 complex to the activated and silenced LTR nuc-1, respectively. Depletion of BAF250 abrogates targeting of the repressive BRG-1 complex while BAF180 depletion interferes with recruitment of BRG1 to nuc-1 in response to PMA stimulation. J-Lat 11.1 cells were nucleofected with either nontargeting siRNA or siRNAs targeting BAF250 or BAF180 as indicated. Depleted cells were then subjected to ChIPs using an antibody specific for BRG1. BRG1 ChIPs were analyzed by qPCR using primer pairs specific for the LTR nuc-1 and control region amplifying upstream of the Axin2 gene and are presented as percent immunoprecipitated DNA over Input. For all ChIP and FAIRE experiments, error bars represent the SEM of at least three independent experiments. * p < 0.05, ** p < 0.01. We depict results for J-Lat 11.1. Similar results were obtained for J-Lat A2 (S6 Fig).
Fig 7.
BAF250 depletion induces change in chromatin structure at the HIV LTR as detected by High Resolution MNase nucleosomal mapping.
(A) Diagram showing the PCR amplicons used at the HIV LTR covering nucleotides 40–902 corresponding to Nuc0, DHS1, Nuc-1, DHS2, and Nuc-2. PCR products are 100 ± 10 bp in size and are spaced approximately 30 bp apart. (B) Change in chromatin structure of the HIV LTR in J-Lat 11.1 cells upon PMA stimulation. The chromatin profile of the HIV LTR was determined at 0 (red line), 1 h (light green line), and 12 h (dark green line) post-PMA stimulation by normalizing the amount of the MNase digested PCR product to that of the undigested product using the ΔC(t) method (y-axis), which is plotted against the midpoint of the corresponding PCR amplicon (x-axis). The x-axis represents base pair units with 0 as the start of LTR Nuc-0. Error bars represent the average of three independent experiments. (C) A loosely positioned nucleosome between the positioned Nuc-0 and nuc-1 is indicated in pink. (D) Depletion of BAF250 induces restructuring of the HIV LTR chromatin profile. The chromatin profile of the HIV LTR was determined in J-Lat 11.1 cells nucleofected with either control siRNA (brown line) or siRNA targeting BAF250 (blue line). The chromatin profile of untransfected cells are also provided for comparison (red line). Error bars represent the SEM of three independent experiments.
Fig 8.
BAF promotes the establishment of latent HIV infections.
(A) Western blot analysis demonstrates depletion of BAF/PBAF subunits as indicated 96 h after siRNA transfection. (B) Schematic representation of protocol for comparison of productive and latent infections in Jurkat cells containing or depleted of remodeling complex subunits. Jurkat cells were first nucleofected with control siRNA or siRNA targeting individual SWI/SNF subunits as indicated. After 48 h, cells were infected with retroviral particles containing the vector LTR-Tat-IRES-GFP. The percentage of productive infections (GFP-positive cells obtained after infection (12.2%) (left panel)), FACS-sorted GFP-negative cells (middle panel), or percent latent infections (GFP-positive cells obtained after PMA treatment of sorted GFP-negative cells (0.64%) (right panel)) are shown for Jurkat cells nucleofected with control siRNA. (C) Depletion of BAF/PBAF subunits as indicated does not significantly affect the percentage of productive HIV infections. (D) Depletion of the core SWI/SNF subunits BRG1 and INI-1 and the BAF-specific subunit BAF250 significantly decreases the incidence of latent HIV infections (the percent GFP positive cells obtained after PMA stimulation of GFP negative cell population) while depletion of PBAF-specific subunits BAF200 or BAF180 had no significant effect on latency establishment.
Fig 9.
Model for SWI/SNF regulation of HIV LTR transcription.
BAF uses energy from ATP hydrolysis to actively counteract intrinsic histone-DNA sequence preferences within HIV LTR. (A–B) BAF pulls/pushes a preferred nucleosome over DHS1 onto DNA sequences less favorable for nucleosome formation immediately downstream of the transcription start site (TSS), leading to positioning of nuc-1 and transcriptional repression. (C) Upon activation, BAF dissociates from the LTR resulting in re-positioning of the nucleosomes to thermodynamically more favorable positions leading to de-repression of HIV transcription. (D) Upon Tat expression, p300, recruited to the LTR, acetylates Tat. (E) p300-acetylated Tat then selectively recruits the PBAF complex, which uses energy from ATP hydrolysis to actively re-position nucleosomes formed downstream of TSS enabling efficient transcription elongation.