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. 2010 Sep 30;405(2):322-33.
doi: 10.1016/j.virol.2010.06.009.

Transcription through the HIV-1 nucleosomes: effects of the PBAF complex in Tat activated transcription

Rebecca Easley  1 , Lawrence Carpio, Luke Dannenberg, Soyun Choi, Dowser Alani, Rachel Van Duyne, Irene Guendel, Zachary Klase, Emmanuel Agbottah, Kylene Kehn-Hall, Fatah Kashanchi
Affiliations

Transcription through the HIV-1 nucleosomes: effects of the PBAF complex in Tat activated transcription

Rebecca Easley et al. Virology. .

Abstract

The SWI/SNF complex remodels nucleosomes, allowing RNA Polymerase II access to the HIV-1 proviral DNA. It has not been determined which SWI/SNF complex (BAF or PBAF) remodels nucleosomes at the transcription start site. These complexes differ in only three subunits and determining which subunit(s) is required could explain the regulation of Tat activated transcription. We show that PBAF is required for chromatin remodeling at the nuc-1 start site and transcriptional elongation. We find that Baf200 is required to ensure activation at the LTR level and for viral production. Interestingly, the BAF complex was observed on the LTR whereas PBAF was present on both LTR and Env regions. We found that Tat activated transcription facilitates removal of histones H2A and H2B at the LTR, and that the FACT complex may be responsible for their removal. Finally, the BAF complex may play an important role in regulating splicing of the HIV-1 genome.

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Figures

Figure 1
Figure 1. BRG1 associated complexes involved in HIV-1 transcription
A) Brahma-related gene-1 or (BRG1) is a catalytic subunit found in a variety of chromatin remodeling complexes including SWI/SNF complexes BAF and PBAF as well as WINAC, NCoR, mSin3A/HDAC, and NUMAC. The various complexes share other subunits that have a variety of activities: Green: core components; Pink: nuclear receptor association; Gold: DNA replication; Light Green: actin-related complexes; Blue: Ini1/Baf47/SNF5; Purple: other functions. As chromatin remodelers, these complexes can either act as transcriptional activators, repressors, or both. B) CEM cells were transfected (electroporation) with HIV-LTR-CAT (5 μg), CMV-Tat (3 μg), and with either siRNA against Baf250, Baf180, Baf200, Caf1, HDAC3, Cam1, or mSin3a (100 nM). Samples were incubated for 48 hours and subsequently processed for CAT assay. Fifty micrograms of total extracts were used for each CAT assay. Bottom panel shows the western blot from siRNA treated cells. C) Similar to Panel B, with titration (50, 100, and 150 nM) of siBaf200 (lanes 3-5) and siBaf250 (lanes 6-8). Upper insert: Western blot of siBaf200 and siBaf250 treated CEM cells (50μg of total extract) after 48 hours. Experiments in panel B and C are average representative of three independent experiments. D) Cells treated with various siRNAs were processed after 48 hrs and western blot with anti-Flag antibody. Cells, as in panel B, were transfected with HIV-LTR-CAT, and Flag-Tat 101 (3 μg) construct and 50μg of total extract were used for western blot.
Figure 1
Figure 1. BRG1 associated complexes involved in HIV-1 transcription
A) Brahma-related gene-1 or (BRG1) is a catalytic subunit found in a variety of chromatin remodeling complexes including SWI/SNF complexes BAF and PBAF as well as WINAC, NCoR, mSin3A/HDAC, and NUMAC. The various complexes share other subunits that have a variety of activities: Green: core components; Pink: nuclear receptor association; Gold: DNA replication; Light Green: actin-related complexes; Blue: Ini1/Baf47/SNF5; Purple: other functions. As chromatin remodelers, these complexes can either act as transcriptional activators, repressors, or both. B) CEM cells were transfected (electroporation) with HIV-LTR-CAT (5 μg), CMV-Tat (3 μg), and with either siRNA against Baf250, Baf180, Baf200, Caf1, HDAC3, Cam1, or mSin3a (100 nM). Samples were incubated for 48 hours and subsequently processed for CAT assay. Fifty micrograms of total extracts were used for each CAT assay. Bottom panel shows the western blot from siRNA treated cells. C) Similar to Panel B, with titration (50, 100, and 150 nM) of siBaf200 (lanes 3-5) and siBaf250 (lanes 6-8). Upper insert: Western blot of siBaf200 and siBaf250 treated CEM cells (50μg of total extract) after 48 hours. Experiments in panel B and C are average representative of three independent experiments. D) Cells treated with various siRNAs were processed after 48 hrs and western blot with anti-Flag antibody. Cells, as in panel B, were transfected with HIV-LTR-CAT, and Flag-Tat 101 (3 μg) construct and 50μg of total extract were used for western blot.
Figure 2
Figure 2. Involvement of PBAF complex on HIV-1 activated transcription
A) GST-Tat (wild-type 86 and 101) were purified over a glutathione column and eluted. To obtain acetylated Tat, GST-Tat (2 μg) was incubated with p300 and acetyl-CoA (Agbottah et al., 2006), washed, and incubated with HeLa phosphocellulose nuclear fraction (0.75 M, 100 μg) that contained both BAF and PBAF complexes. Samples were incubated overnight at 4°C in presence of EtBr (100 ng/ml), washed, bound complexes were run on a SDS/PAGE gel (4-20%) and western blotted for the presence of Baf200, BRG1, Baf250 and actin using specific antibodies. Lane 1 and 4, GST; lane 2, wild-type acetylated Tat 86; lane 3, wild type Tat 86; lane 5, wild-type acetylated Tat 101; and lane 6, wild-type Tat 101. B) Similar experiment as panel A, where acetylated Tat 86 and a Tat 50/51 mutant were used for the pull downs. Lane 1 (input) is counts from western blot of input (20 μg) of HeLa phosphocellulose nuclear fraction (0.75 M); lane 2 is counts from acetylated GST-Tat 86 pull down and lane 3 is counts from GST-Tat 86 50/51 mutant pull down. Band intensities were obtained using a Molecular dynamics phosphoroImager instrument. Western blots were with antibodies for Baf200, BRG1, Baf250 and actin.
3
3. Importance of Baf200 in Tat transactivation and HIV-1 viral production
A) Effect of knockdown of BAF or PBAF components on Tat transactivation. TZM-bl cells containing an integrated HIV-1 LTR-luciferase reporter construct were transfected with either no plasmid, Tat plasmid (0.5 μg), or siRNA for BRG1, Baf250, Baf180, Baf200 or Scr (luciferase scrambled) (75 nM). Luciferase levels were determined on day 4. Cells at day 4 were processed for westerns using various Baf and actin antibodies (lower insert). B) Baf180 mutant cell line (HCC1143) were transfected with pNL4-3 (20μg) either alone or with siBaf200 (75 nM). Supernatants were collected for presence of virus in days 2, 3 and 4. Cells (day 4) were also processed for westerns using Baf200 and actin antibodies (lower insert). C) HLM-1 cells (containing wild type virus with a mutation in Tat) were transfected with CMV-Tat (3 ug) either alone or in combination with various siRNAs (75 nM). Supernatants were collected at day 4 and used for RT assay. Cells were processed for western blots after 4 days. Experiments in all panels were performed at least three times.
3
3. Importance of Baf200 in Tat transactivation and HIV-1 viral production
A) Effect of knockdown of BAF or PBAF components on Tat transactivation. TZM-bl cells containing an integrated HIV-1 LTR-luciferase reporter construct were transfected with either no plasmid, Tat plasmid (0.5 μg), or siRNA for BRG1, Baf250, Baf180, Baf200 or Scr (luciferase scrambled) (75 nM). Luciferase levels were determined on day 4. Cells at day 4 were processed for westerns using various Baf and actin antibodies (lower insert). B) Baf180 mutant cell line (HCC1143) were transfected with pNL4-3 (20μg) either alone or with siBaf200 (75 nM). Supernatants were collected for presence of virus in days 2, 3 and 4. Cells (day 4) were also processed for westerns using Baf200 and actin antibodies (lower insert). C) HLM-1 cells (containing wild type virus with a mutation in Tat) were transfected with CMV-Tat (3 ug) either alone or in combination with various siRNAs (75 nM). Supernatants were collected at day 4 and used for RT assay. Cells were processed for western blots after 4 days. Experiments in all panels were performed at least three times.
Figure 4
Figure 4. Remodeling of the nucleosome on the HIV-1 promoter
A) ChIP assays of 4 core histones were performed from TNF-α-induced OM10.1 cells. ChIP assays were performed using H2A, H2B, H3 and H4 antibodies (10 ug ab/reaction). OM10.1 cells (a total of 5 × 107) were induced with TNF-α (10 ng/ml) for 2 hours and subsequently plated for 24hrs. Specific DNA sequences in the immunoprecipitates were detected by PCR, using primers specific for HIV-1 LTR nuc-0 (-415 to -255), HIV-1 LTR nuc-1 (+10 to +165) and Env (+8990 to +9120). The HIV-1 proviral DNA in OM10.1 cells was derived from clade B LAI strain. B) Similar to panel A (ChIP assay from OM10.1 cells), except using antibodies against H2A, FACT, and Pol II. Antibody against the Spt16 subunit of FACT was used for ChIP assay. C) HLM-1 cells were transfected with control empty vector (3 ug) and with CMV-Tat (3 ug) for 48hrs. Samples were processed for ChIP assay using antibodies (10 ug/reaction) against H2A, FACT, and Pol II. Recovered DNA was processed for qPCR. Enriched DNA was quantified using an ABI Prism 7100 instrument and SYBR green (Applied Biosystems, Foster City, CA, USA) and normalized to control empty vector HLM-1 cells. Results for FACT qPCR were similar in presence or absence of Tat. Values are average of three independent experiments.
Figure 4
Figure 4. Remodeling of the nucleosome on the HIV-1 promoter
A) ChIP assays of 4 core histones were performed from TNF-α-induced OM10.1 cells. ChIP assays were performed using H2A, H2B, H3 and H4 antibodies (10 ug ab/reaction). OM10.1 cells (a total of 5 × 107) were induced with TNF-α (10 ng/ml) for 2 hours and subsequently plated for 24hrs. Specific DNA sequences in the immunoprecipitates were detected by PCR, using primers specific for HIV-1 LTR nuc-0 (-415 to -255), HIV-1 LTR nuc-1 (+10 to +165) and Env (+8990 to +9120). The HIV-1 proviral DNA in OM10.1 cells was derived from clade B LAI strain. B) Similar to panel A (ChIP assay from OM10.1 cells), except using antibodies against H2A, FACT, and Pol II. Antibody against the Spt16 subunit of FACT was used for ChIP assay. C) HLM-1 cells were transfected with control empty vector (3 ug) and with CMV-Tat (3 ug) for 48hrs. Samples were processed for ChIP assay using antibodies (10 ug/reaction) against H2A, FACT, and Pol II. Recovered DNA was processed for qPCR. Enriched DNA was quantified using an ABI Prism 7100 instrument and SYBR green (Applied Biosystems, Foster City, CA, USA) and normalized to control empty vector HLM-1 cells. Results for FACT qPCR were similar in presence or absence of Tat. Values are average of three independent experiments.
Figure 5
Figure 5. Presence of PBAF component on the HIV-1 LTR and Env regions
A) Activated PBMCs (PHA+ IL-2) were infected with a dual tropic HIV-1 strain 89.6 (20,100 RT units). Cells (5 × 106) were collected at days 3, 6 and 9 for ChIP analysis with appropriate antibodies. Specific DNA sequences in the immunoprecipitates were detected by PCR, using primers specific for 89.6 strain HIV-1 LTR nuc-1 (+10 to +165) and Env (+8990 to +9120). Bottom panel shows the presence of Baf200, Baf250 and actin (western blot) in these cells. Also, the RT levels in the supernatant of the cells are shown in bottom right. B) PBMC cells in Day 3 (majority of Baf200 was observed on the Env region at that time) was used for qPCR for both LTR and Env regions, readings were normalized to input. Lane 1 is the inputs for both LTR and Env region and lane 2 is for IgG control. Lanes 3-7 are results from from Pol II, Baf200, Baf250, p300 and cdk9 ChIPs, respectively. C) Further validation of results in panel B using qPCR of the Baf200 and Baf250 ChIPs from the Env region of infected samples at days 3, 6, and 9. Lane 1 represents input (1/10); lane 2 is for Baf200 and lane 3 is for Baf250 ChIPs. Readings were normalized to input.
Figure 6
Figure 6. Effects of SWI/SNF on HIV-1 splicing
A) Schematic of the pNLEnv plasmid. Env gene is expressed from an unspliced mRNA, whereas Nef is made from a spliced mRNA. B) 293T cells were transfected with combinations of low levels of Tat plasmid (1 ug) and siGFP, siBaf250, or siBaf200. Western blot analysis using antibodies against Nef, Env, or Actin were performed after 3 days. Knockdown of Baf200 resulted in increased levels of Nef (spliced) protein whereas knockdown of Baf250 resulted in increased levels of Env (unspliced) protein. Western blot of both Baf200 and 250 are shown below. C) RT/PCR of 293T transfected cells with Tat and siGFP, siBaf250, or siBaf200. One hundred nanogram of total RNA was used for cDNA synthesis and PCR with primers for Nef and Env region. D) HLM-1 cells (7 × 106) were transfected with CMV-Tat (3 ug) and various siRNAs (100 nM). Total RNA was extracted 48 hrs later for RT/PCR using Nef and Env primers.
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