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

Virology 405 (2010) 322–333

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Virology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o

Transcription through the HIV-1 nucleosomes: Effects of the PBAF complex in Tat activated transcription Rebecca Easley a, Lawrence Carpio a, Luke Dannenberg a, Soyun Choi a, Dowser Alani a, Rachel Van Duyne a, Irene Guendel a, Zachary Klase b, Emmanuel Agbottah a, Kylene Kehn-Hall c, Fatah Kashanchi a,⁎ a b c

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA NIAID, National Institutes of Health, Bethesda, MD 20892, USA Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, George Mason University, Manassas, VA 20110, USA

a r t i c l e

i n f o

Article history: Received 21 January 2010 Returned to author for revision 22 February 2010 Accepted 3 June 2010 Keywords: HIV-1 Chromatin BAF PBAF FACT Transcription Remodel

a b s t r a c t 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. © 2010 Elsevier Inc. All rights reserved.

Introduction Human immunodeficiency virus (HIV-1) is shown to become latent upon infection in T lymphocyte (T cell) (as well as other cell types) when cell activation has halted and transcription factor levels needed for both the cell and virus have become dramatically reduced or absent altogether. HIV-1 latency is especially important in light of the fact that viral reservoir can reanimate years later and avoid the consequences of immune clearance or antiviral drugs. The HIV-1 promoter contains many critical DNA binding sites including two NF-κB sites, three Sp1-binding sites, and a TATA box. The activity of the LTR depends on the viral activator Tat which is believed to function at the level of both initiation and elongation (Bohan et al., 1992; Feinberg et al., 1991; Kato et al., 1992; Laspia et al., 1989; Marciniak et al., 1990; Marciniak and Sharp, 1991). It is additionally involved in the ⁎ Corresponding author. National Center for Biodefense and Infectious Diseases, George Mason University, Discovery Hall, Room 306,10900 University Blvd. MS 1H8, Manassas, VA 20110, USA. E-mail addresses: [email protected] (R. Easley), [email protected] (L. Carpio), [email protected] (L. Dannenberg), [email protected] (S. Choi), [email protected] (D. Alani), [email protected] (R. Van Duyne), [email protected] (I. Guendel), [email protected] (Z. Klase), [email protected] (E. Agbottah), [email protected] (K. Kehn-Hall), [email protected] (F. Kashanchi). 0042-6822/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2010.06.009

stabilization of the TFIID/TFIIA complex on the HIV-1 TATA box (Kashanchi et al., 1992) and recruitment of a functional TATA-binding protein (TBP) (Chiang and Roeder, 1995; Dal Monte et al., 1997; GarciaMartinez et al., 1997; Kashanchi et al., 1996; Roebuck et al., 1997; Veschambre et al., 1995). Furthermore, van Opijnen et al. (2004) have proposed that the HIV-1 promoter contains a CATA box instead of a TATA box in order to achieve optimal transcription and replication. Tat binds to and interacts with a number of transcriptional cofactors including Sp1, cyclin E/cdk2, TFIIH, Tip 60, Pol II and the activators CBP/p300 and p/CAF. Transcriptional activity from the HIV-1 LTR is restricted in vivo partially due to the higher order chromatin structure of the HIV-1 proviral DNA. The 5′ LTR contains four distinct DNase I hypersensitive sites (DHS) (Van Lint et al., 1996; Verdin, 1991; Verdin et al., 1993). It has been largely assumed that DHS in native chromatin are free of histones and allow unrestricted access to DNA-binding proteins (McGhee et al., 1981). Furthermore, independent of the viral integration site, five nucleosomes (nuc-0 to nuc-4) are precisely positioned within the 5′ LTR. In the transcriptionally silent provirus, these nucleosomes define two large nucleosome-free regions spanning nucleotides (nt) −255 to −3 and nt +141 to +265; nuc-1 (which encompasses the transcription start site) is located between these two regions. The first nucleosome-free region contains many promoter/enhancer elements which are already occupied by transcription factors (Demarchi et al., 1993). When cells are activated with tumor necrosis factor alpha

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(TNF-α), phorbol esters (TPA), or histone deacetylase (HDAC) inhibitors (i.e., trapoxin, Trichostatin A (TSA), and sodium butyrate) DHS3 and DHS4 are extended and nuc-1 is specifically remodeled (Coull et al., 2002; Demarchi et al., 1993; Simone, 2006). Chromatin immunoprecipitation (ChIP) assays suggest that histone acetylation surrounding nuc-1 significantly increases following TSA treatment. To overcome the nucleosomal barrier, post translational modifications, such as histone acetyltransferases (HATs), can result in the opening or closing of the chromatin. Additionally chromatin can be remodeled using the energy from ATP hydrolysis to alter interactions between histones and DNA allowing increased access to DNA within a nucleosomal array (Kingston and Narlikar, 1999). The interactions that can occur in both chromatin assembly and remodeling are very complex and many studies have recently been published showing the importance of this in HIV-1 transcriptional activation. A recent paper that focuses on the Tat interactome identified a number of chromatin remodeling complexes that interact with Tat including the SWI/SNF, NURD, and FACT (Gautier et al., 2009). In addition to this study, other labs have further identified proteins that directly interact with Tat and act to promote viral transcription such as the histone chaperone protein Nucleosome Assembly Protein-I (hNAP-1) (Vardabasso et al., 2008). BAF and PBAF are both in the SWI/SNF family of chromatin remodeling complexes which utilize BRG1, or the closely related BRM, as the main catalytic subunit for ATPase activity (Klochendler-Yeivin et al., 2002; Olave et al., 2002; Yan et al., 2005). Chromatin modifying enzymes (i.e., p300/CBP, p/ CAF) can work together with SWI/SNF to facilitate a chromatin structure that is accessible for the basal transcription machinery (Narlikar et al., 2002). BRM (Treand et al., 2006) and BRG have been shown to be involved in Tat-mediated activation of the HIV-1 LTR (Agbottah et al., 2006; Bukrinsky, 2006; Mahmoudi et al., 2006; Treand et al., 2006). Depletion of endogenous host-cell integrase interactor (INI-1; a SWI/SNF component) and BRG1 by RNAi can abolish Tat-mediated activation. Acetylation of Tat at lysines 50 and 51 promote its binding to SWI/SNF (Mahmoudi et al., 2006); however, we have also shown that lysines 41, 50, and 51 are critical for SWI/SNF binding (Agbottah et al., 2006). INI-1 can also synergize with acetylated Tat and p300/CBP at the HIV-1 promoter to activate the LTR (Mahmoudi et al., 2006). Targeting of BRG1 to the HIV-1 promoter by transcription factors, such as ATF-3 and HMGA1, has also been demonstrated (Henderson et al., 2004). BRG1 expression and TSA treatments of C33A cells (BRG1-deficient) demonstrated that histone acetylation plays a role in maintaining a stable association between BRG1 and the chromatin (Henderson et al., 1995). In the current manuscript, we address the question of which SWI/SNF complex(es) is associated with HIV-1 LTR and how it might regulate gene expression by modifying the nucleosome at the transcription start site (nuc-1). Furthermore we show that some of the histones are removed from this region once Tat activated transcription takes place from the HIV-1 LTR. Results Multiple subunits of BRG1 and its associated complexes are needed for Tat activated transcription The evolutionarily conserved SWI/SNF family exists in several subgroups and it is suggested that these complexes have very different functions. This can be seen by gene knockout experiments, in which loss of BRG1, but not BRM, is embryonically lethal. There are many (at least six) complexes that could potentially associate with BRG1 and form complexes such as SWI/SNF (BAF and PBAF), WINAC, NCoR, NUMAC, and mSin3A/HDAC complexes (Fig. 1A). As chromatin remodelers, these complexes can either act as transcriptional activators, repressors, or both. For example, NUMAC acts as a transcriptional activator while the mSin3A/HDAC complex acts as a transcriptional repressor. SWI/SNF complexes also can act as either repressors or activators (Trotter and Archer, 2008). BAF and PBAF differ only in a few subunits where BAF includes Baf250 and PBAF can include Baf200 and Baf180. The BAF

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complex has two forms, A and B where A utilizes BRG1 for its ATPase energy, and B utilizes BRM. The PBAF complex can exist with (A) or without (B) the Baf180 subunit (Yan et al., 2005). To determine which one of these complex(s) was important for HIV-1 transcription, we used seven siRNAs that covered some of the critical and unique subunits of the BRG1 associated complexes. We asked if using siRNAs against specific subunits to each BRG1 containing complex could determine significance of their activity in transient Tat activated transcription. We transfected cells with LTR-CAT with LTRCAT and a CMV-Tat construct in CEM T-lymphocytes and scored for the presence of the CAT enzyme after 48 h. Results of such an experiment are shown in Fig. 1, panel B, where Tat activated transcription showed optimal activity in these cells (lane 2). Using distinct siRNAs, we found that the most significant suppression of Tat activated transcription was with siBaf200 (lane 5) in a 48-h transient transfection assay, although some of the other siBafs including siBaf180 did show some reduced transcription. Because Baf200 is important for PBAF complex formation and Baf250 is important for BAF complex, we decided to further titrate siRNAs against these two subunits in a similar transient transfection assay. Results in panel C show that a titration of siBaf200 effectively inhibits Tat activated transcription and further reinforces the significance of PBAF complex requirement for Tat activated transcription on HIV-1 LTR. Finally, we look at the Tat levels in these transfections (panel D) and by and large, there were minimal alterations in Tat levels after siRNA transfection. Collectively, these results indicate that, at least in a transfection setting in T-cells, Baf200 may be important for Tat activated transcription.

Acetylated Tat binds to SWI/SNF PBAF complex Both BAF and PBAF have similar core subunits including BRG1, actin, Baf47 (INI1), Baf53, Baf57, Baf60, Baf155, and Baf170. The two complexes differ only in a few unique subunits where the BAF complex contains Baf250 and PBAF contains Baf180 and Baf200 (Simone, 2006; Trotter and Archer, 2008; Yan et al., 2005). We and others have previously shown that Tat can be acetylated at multiple lysines residues leading to altered interactions between Tat and TAR, p300/CBP, and p/CAF, as well as activation of viral transcription and replication (Agbottah et al., 2006; Ariumi et al., 2006; Bukrinsky, 2006; Mahmoudi et al., 2006; Treand et al., 2006). We then asked if acetylated Tat could specifically bring down components of the PBAF complex. For this we utilized a HeLa phosphocellulose nuclear fraction (0.75 M) that contained both BAF and PBAF complexes (Yan et al., 2005) at approximately 0.9–1.0 MDa. We performed GST-Tat pull down experiments with these fractions and looked for specific components of either BAF or PBAF complexes. Results in Fig. 2A show that both acetylated Tat 86 or Tat 101 bound efficiently to Baf200 (component of PBAF) and weakly to Baf250 (component of BAF). Actin, a component of both BAF and PBAF complexes was present in acetylated and unmodified Tat pull down experiments. To rule out the possibility that BAF or PBAF complexes may indirectly associate with contaminating DNA, we used ethidium bromide (EtBr), a DNA intercalating agent that dissociates proteins from DNA in these GST-pull down experiments. It is important to note that under these conditions both Baf200 and BRG1 bound with higher affinity to the acetylated Tat, whereas Baf250 bound with less efficiency to the acetylated Tat and had a preference for the unmodified Tat. Finally, we performed another pull down experiment using wild type Tat 86 and a Tat K50/51 mutant. For these Western blots we included 1/5 of the input phosphocellulose nuclear fraction (0.75 M) in the gels prior to the Western blot. We than ran gels, Western blotted with appropriate antibodies and then counted the bands using a phosphoroImager. Results of such an experiment are shown in Fig. 2B, where acetylated GST-Tat 86 pull down (lane 3), but not the 50/51 mutant, showed binding to both Baf200 and BRG1. Collectively, these results imply that acetylated Tat may have more of a preference to bind to PBAF complex.

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Effect of siRNA SWI/SNF PBAF treatment on transcription of integrated HIV-1 promoter We next used the TZM-bl cell line, which contains an integrated HIV-1 LTR-Luc, to ask whether knockdown of BAF or PBAF specific subunits

could affect LTR transcription. Cells were transfected with no plasmid, Tat plasmid alone or Tat plus siRNAs against BRG1, Baf250, Baf180, Baf200 or BRM using Attractene. After 4 days of transfection cells were processed for luciferase activity (Fig. 3A). In general agreement with our previous findings, we observed that knockdown of Baf200, but not Baf250,

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Fig. 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. Lanes 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. Bar 1 (input) shows quantitative results from the Western blot of input (20 μg) of HeLa phosphocellulose nuclear fraction (0.75 M); bar 2 shows acetylated GST-Tat 86 pull down and bar 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.

inhibited Tat transactivation in this system. Next, to be able to distinguish and better reproduce the difference between the BAF and PBAF complexes for Tat activated transcription, we used a Baf180 mutant cell line, HCC1143 (Yan et al., 2005) in transfection assays. Here the mutant cells have undetectable levels of the Baf180 subunit of PBAF complex but still have normal Baf200 levels. We transfected these cells with pNL4-3 viral clone and collected supernatants for the detection of virus after days 2, 3 and 4. We additionally used siRNA against Baf200 to determine if it is required for viral replication in these Baf180 mutant cells. Results of these experiments are shown in Fig. 3B, where HIV RT was present in the supernatant after 2 days and the levels dramatically dropped after 4 days with knockdown of Baf200. These results collectively suggest that efficient complex involved in Tat activated transcription for integrated LTR and virus production may be a PBAF complex. Finally, we performed a similar set of experiments in HLM1 cells where the HIV-1 virus is integrated (one copy) and it contains a triple mutation in the Tat open reading frame. Virus particles are only made in these cells with the addition of exogenously added Tat. Upon

transfection of Tat we observed robust viral particle formation in these cells (Panel C, lane 2). We then transfected five different siRNAs into these cells and found both siBRG1 and siBaf200 (and to some level siBRM) effectively inhibited viral production in these latent cells. Collectively these experiments, which utilized chromatinized LTR DNA, indicate that optimal Tat activated transcription requires PBAF complex. Remodeling of a nucleosome at the transcription start site We next asked if all or some of the components of the nuc-1 were physically removed after Tat activated transcription in vivo. To do this we used latent OM10.1 cells (containing latent wild type virus) where very little transcription and virus production is observed. We first activated the virus with low levels of the TNF-α followed by chromatin immunoprecipitations (ChIPs) of all four core histones. Here the assumption was that TNF-α would start basal transcription leading to formation of the Tat protein and consequentially enhance activated transcription by the Tat. DNA from the ChIP assay was then used for PCR using primers that

Fig. 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 h and subsequently processed for CAT assay. Fifty micrograms of total extracts was 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 (50 μg of total extract) after 48 h. Experiments in panel B and C are average representative of three independent experiments. D) Cells treated with various siRNAs were processed after 48 h 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 50 μg of total extract were used for Western blot.

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Fig. 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 lines (HCC1143) were transfected with pNL4-3 (20 μg) 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 μg) 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.

spanned the nuc-0, nuc-1 and the Envelope (Env) region. As seen in Fig. 4 (panel A) viral activation by TNF-α made very little changes in the composition of the nuc-0 histones (upstream of the transcription start site). We saw no changes in the H3 or H4 levels on the DNA before or after viral induction, and only minor changes for H2A and H2B. However, we observed almost a complete removal of H2A and H2B proteins from the nuc-1 region after transcriptional activation (middle insert). Interestingly, we did not see a change in the downstream nucleosomes including the Env region even after induction of transcription (bottom insert). These results collectively point to a scenario where

activated transcription may in fact modify the nuc-1 histones by removing both histones H2A and H2B thereby “loosening” nucleosomes for Tat activated transcription. This seems plausible since the removal of histones from DNA immediately downstream of transcription start site has been shown to be involved in transcriptional elongation. In fact, removal of histones H2A and H2B has been seen by the Reinberg lab in an elongation complex called the FACT (facilitates chromatin transcription) complex (Orphanides et al., 1998). We next asked if the FACT complex was associating with the HIV-1 LTR at the nuc-1 region. FACT was initially isolated from extracts that

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Fig. 4. Remodeling of the nucleosome on the HIV-1 promoter. A) ChIP assays of four core histones were performed from TNF-α-induced OM10.1 cells. ChIP assays were performed using H2A, H2B, H3 and H4 antibodies (10 μg ab/reaction). OM10.1 cells (a total of 5 × 107) were induced with TNF-α (10 ng/ml) for 2 h and subsequently plated for 24 h. 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 μg) and with CMV-Tat (3 μg) for 48 h. Samples were processed for ChIP assay using antibodies (10 μg/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) 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.

supported transcription on chromatinized templates (Orphanides et al., 1998). In humans FACT consists of the proteins Spt16 and the HMG-box containing SSRP1 (structure specific recognition protein-1) which are both highly conserved. FACT is known to contain histone chaperone activity, suggesting that it functions to re-establish disassembled nucleosomes (Belotserkovskaya et al., 2003; Orphanides et al., 1999). In yeast, inactivation of FACT results in transcriptional initiation from cryptic initiation sites within the coding region of genes, implying that

FACT is required for maintaining chromatin structure in vivo (Reinberg and Sims, 2006). To asses if HIV-1 DNA contained FACT at any of the nucleosomal sites in the promoter, we preformed ChIP assays under the same conditions as Panel A using antibodies against H2A, FACT, and Pol II proteins. We observed the absence of FACT (Spt16 subunit) at the nuc-0 region upstream of the transcription start site (Panel B, top). However, FACT was present at low levels at the nuc-1 site in the absence of TNF-α. Upon induction of viral transcription with TNF-α, FACT was more

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apparent on the nuc-1 as well as the Env region (compare lanes 3 and 4). Addition of TNF-α increased the level of Pol II occupancy on both nuc-1 and Env region consistent with increased transcription. We next performed a similar set of experiments in HLM-1 (Tat mutant) cells. We transfected these cells with Tat to induce viral replication and performed a ChIP assay using antibodies for H2A, FACT, and Pol II. Recovered DNA was processed for qPCR. Enriched DNA was quantified using an ABI Prism 7100 and normalized to control empty vector HLM-1 cells. Results in panel C show that H2A is mostly removed from the nuc1, but not from nuc-0 or Env regions. Additionally, FACT is recruited to the promoter region around nuc-1 when Tat is added to the system. Collectively, these results indicate that FACT may be present on the promoter, and the increased levels of FACT seen upon viral activation or addition of Tat may explain the observed removal of H2A/H2B histones from the nuc-1 region. Presence of PBAF complex on the HIV-1 LTR and envelope region in infected PBMC cells To further validate our results in PBMC infection settings, we infected freshly activated PBMCs with dual tropic 89.6 HIV-1. Cells were initially activated with PHA+ IL-2 for 4 days prior to infection with the virus. Subsequently after infection PBMC samples were collected at days 3, 6, and 9 and processed for ChIP. We utilized antibodies against the large subunit of Pol II (for active transcription through HIV-1 DNA), Baf200

(for presence of PBAF complex), Baf250 (for possible presence of BAF complex), co-activator p300, and cdk9 which is required for Tat activated transcription in vivo. Results are shown in Fig. 5A where ChIP-derived DNA was PCR amplified using LTR (nuc-1) or Env primers. Interestingly, Pol II, cdk9, and Baf200 were present on the LTR and Env regions from days 3–9, although less Baf200 was present on the Env region at day 9. This indicates that Pol II, cdk9 and Baf200 complexes are involved in transcriptional initiation and elongation. However, Baf250 and p300 were mostly present on the LTR on days 3 and 6 but not on day 9. No detectable Baf250 or p300 were present on the Env region, which re-emphasizes the notion that both of the protein complexes may be important for transcription initiation or early proximal elongation, but not for distal elongation. Exogenous viral RT levels gradually increased from day 3 and peaked at day 9. Results from the Baf250 were surprising since our previous LTR transient (Fig. 1B and C) or integrated LTR (Fig. 3A) reporter assays showed no apparent need for the Baf250 to obtain activated transcription. However, in an actual infection setting (viral clone in PBMCs) Baf250 was observed on the LTR but not the Env region. Although these results indicate the complexities of various assays used here (LTR vs. full length virus), the presence of the SWI/SNF PBAF complex on the LTR and Env regions clearly indicated its involvement in transcriptional elongation on HIV-1 DNA. To further validate the presence of the PBAF complex on the HIV-1 LTR region in activated PBMCs, chromatin association of Pol II, Baf200, Baf250, p300 and cdk9 was quantitated using ChIP- qPCR. The qPCR in

Fig. 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 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.

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Panel B is from PBMC cells 3 days post infection, since the majority of Baf200 was initially observed on the Env region at that time. Lane 1 shows inputs for both LTR and Env region and lane 2 is the IgG control (similar to lanes 2 and 3 in panel A, respectively). Lanes 3–7 show qPCR results from Pol II, Baf200, Baf250, p300 and cdk9 ChIPs, respectively. Results for Baf200 (lane 4) and Baf250 (lane 5) clearly show that these complexes are differentially loaded onto the LTR vs. Env regions. Interestingly, the Pol II loading on LTR and Env regions (lane 3) also shows differential loading where more Pol II loading is seen at the Env region which may indicate that there could possibly be re-initiation of Pol II with activated transcription. To further validate these results, we performed qPCR of the Baf200 and Baf250 ChIPs from the Env region of infected samples at days 3, 6, and 9. Results in Panel C show that Baf200 was present on the Env region at all 3 days (lane 2) and Baf250 was completely absent on the Env region (lane 3). It is also important to note that we have not completely exhausted the possibility that Baf250 may be available on the Env region using various other antibodies or possible protein modifications masking the epitope. Collectively, these current results suggest that PBAF complex is involved in activated transcription and BAF complex may only be present on the LTR for some other function, i.e., splicing. The possible effect of BAF SWI/SNF complex on HIV-1 splicing Our results above indicated that SWI/SNF Baf250 was present on the promoter only, which may have functions other than transcription. Along these lines, several subunits of the SWI/SNF complex including BRG1, Baf155 and INI1 co purify with the nuclear-receptor co-repressor complex (NCoR), which associates with the splicing factor SF3a120 and the spliceosome-associated protein SAP130 (Underhill et al., 2000). The PRP4 kinase can phosphorylate and interact with BRG1 and the U5 small nuclear ribonucleoprotein particle (snRNP) subunit PRP6 (Dellaire et al., 2002). One group has shown that human BRM favors inclusion of variant exons in the mRNA of several SWI/SNF target genes subject to alternative splicing, independent of its chromatin remodeling activity (Batsche et al., 2006). In addition, BRM could be immunoprecipitated with U1 and U5 snRNAs and with PRP6 and Sam68 proteins, suggesting its role in the recruitment of the splicing machinery. BRM enters the coding region of the CD44 gene together with the Pol II causing the accumulation of the Pol II on variant exons of this gene. This accumulation correlates with a change in C-terminal domain (CTD) phosphorylation of the Pol II associated with BRM. The modified pattern of CTD phosphorylation signifies a halt in elongation by the Pol II and suggested that BRM may favor inclusion of alternative exons by kinetically facilitating spliceosome recruitment to the splice sites of the variant exons. In HIV-1 infection constitutive and alternative mRNA splicing occurs to remove upstream Gag and Pol coding sequences from the Env mRNA and to generate the essential regulatory proteins, Tat and Rev, as well as several other proteins (Vif, Vpr and Nef) needed for successful replication in vivo. The pattern of splicing varies in accordance with the phase of infection in HIV-1 cells. While sub-genomic species (doubly and singly spliced) are abundant during early stages of infection, the genomic transcripts (unspliced) appear at the later stage of infection. The splicing of HIV-1 RNA is extremely complex because of the presence of four 5′-splice donor sites D1 to D4 and eight 3′ splice acceptor sites (A1, A2, A3, A4a, b, c, A5 and A7). A comprehensive study examining the steady-state levels of viral mRNA during viral infection determined the utilization of different splice sites for the production of the 2 and 4 kb transcripts. A4 and A7 sites were found to be essential for the production of Tat, A4a, b or c and A7 was used to produce Rev. Nef and Env required A5 and Vpr required A2 sites (Purcell and Martin, 1993). To show whether the presence of SWI/SNF (BAF or PBAF) leads to regulation of splicing, we used an HIV-1 vector with differential splicing patterns. In the plasmid pNLEnv, the Env gene is expressed from an unspliced mRNA, whereas Nef is made from a spliced mRNA (Fig. 6A). We transfected 293T cells with pNLEnv along with low concentrations of Tat

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alone or Tat and various siRNAs against BAF and PBAF. The production of Nef and Env proteins were determined by Western blotting. Results in Fig. 6B show that siBaf200 (knock down of the PBAF complex) increased the presence of doubly spliced Nef, further indicating that splicing can be detected under these conditions (Fig. 6B, lane 4). However, when using siBaf250 (knock down of BAF complex), we observed increased levels of Env protein. Similar levels of actin were seen in siRNA treated cells (bottom panel). This correlated with similar RNA patterns when using RT/PCR from these 293T transfected cells (panel C). Finally, we observed a similar pattern of RNA levels for both Nef and Env regions when using HLM-1 cells treated with Tat and the two siBaf subunits (panel D). Collectively, these results indicate that knock down of PBAF complex increases synthesis of doubly spliced messages and consequentially the presence of the BAF complex on the HIV-1 LTR (Fig. 5) may be important for the regulation of splicing on the HIV-1 LTR. Discussion In order to free the DNA around the HIV-1 LTR, chromatin can be opened or closed through either covalent modifications (such as HATs) or using the energy of ATP hydrolysis (such as SWI/SNF). Tat has been shown to interact with the HATs p300 and p/CAF, and this association could be critical for downstream chromatin remodeling or progressive transcriptional elongation. The proviral DNA can additionally be remodeled using ATPase chromatin remodelers such as BRG1 or BRM. The SWI/SNF complex (including the BAF and PBAF complexes) is known to play a role in Tat transactivation, though there has been no evidence to determine if BAF or PBAF is responsible for chromatin remodeling. Using siRNAs against unique components of the various complexes we found that the PBAF complex was required for Tat activated transcription in TZM-bl cells. We also observed the effects of acetylated versus unacetylated Tat on BAF (Baf250) and PBAF (Baf200) and found that Baf200 bound efficiently to acetylated Tat while Baf250 had very little binding in vitro. Using siRNA against either BAFs, we found that knockdown of Baf200, but not Baf250, suppressed Tat activated transcription in CEM cells and transfection of Baf180 mutant cells with pNL4-3 and siBaf200 showed minimal amount of virus production. These results collectively indicated that PBAF, specifically the Baf200 complex, is important for Tat binding and transactivation. Since SWI/SNF is involved in chromatin remodeling we focused our efforts on the nucleosomes immediately surrounding the transcription start site. We observed a drop in H2A and H2B core histone levels mostly in the nuc-1, and not on the Env region, further emphasizing a site specific organization of the chromatin at the transcription start site. This finding suggests that activated transcription could modify nuc-1 histones for Tat transactivation. Removal of H2A and H2B has additionally been seen at the elongation site by the FACT complex, and we hypothesized that this could also occur in HIV-1 transcription. We observed the presence of the FACT complex at the promoter as well as the Env region when the chromatinized virus was activated. Is it important to note that FACT dependent transcription was haulted when histone polypeptides were covalently cross-linked within nucleosomes, indicating that a complex (such as an octamer) is disassembled during activated transcription (Orphanides et al., 1999). This suggests that FACT could possess nucleosome disassembly activity and is supported by the interaction of Spt16 with H2A/H2B dimers (Belotserkovskaya et al., 2003; Orphanides et al., 1999) as well as histone H3/H4's interaction with SSRP1 (Belotserkovskaya et al., 2003). Transcription is enhanced in vitro by the loss of H2A/H2B dimer which is an effect of FACT-mediated transcription through the nucleosome (Gonzalez and Palacian, 1989), additionally, the specific loss of H2A/H2B dimers is seen during active transcription that is mediated by Pol II in vivo (Kireeva et al., 2002). Interestingly, several independent studies have shown that in higher organisms FACT physically associates with CHD1 and in yeast H3K4me3 correlates with active genes at the 5′-region and crests near the transcriptional

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Fig. 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 μg) 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 nanograms 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 μg) and various siRNAs (100 nM). Total RNA was extracted 48 h later for RT/PCR using Nef and Env primers.

start site in mammalian cells (Adelman et al., 2006; Bernstein et al., 2005; Flanagan et al., 2005; Kelley et al., 1999; Krogan et al., 2002; Lusser et al., 2005; Schneider et al., 2004; Shi et al., 1996; Simic et al., 2003; Sims et al., 2005; Squazzo et al., 2002; Woodage et al., 1997; Yan et al., 2005). It is likely that monoubiquitination of H2B assists in FACT mediated removal of the H2A/H2B dimers during transcription, this is further supported by the physical and functional interactions between FACT, PAF and H2B modification. This mode of targeted histone degradation (possibly through the proteasomal ATPases) may be playing a key role in removal of nuc-1 histones on HIV-1 LTR. Current studies are to define which ubiquitinated molecule(s) on the LTR (i.e., H2B) is associated with protein degradation and which proteins in fact assist Pol II in transcriptional elongation. We used a PBMC infectious setting to further observe the presence of BAF or PBAF on the HIV-1 DNA. To our surprise we observe BAF on the LTR but not Env region. This further indicated that the BAF complex may be important for events other than transcription. When looking at the impact of BAF or PBAF on the regulation of splicing we found that knockdown of PBAF increased the presence of doubly spliced Nef protein while siBAF treatment increased the levels of the Env protein. This may indicate that there are at least two stages for activated transcription in vivo. The first stage may utilize the BAF complex and regulate splicing of messages by stringent recognition of the splice acceptor and donor sites in HIV-1 genome, a scenario similar to the CD44 gene and the accumulation of Pol II on the variant exons (as discussed above). The second stage may utilize PBAF which binds to acetylated Tat and recruits new set of complexes needed for repetitive and speedy Pol II activated transcription for the entire genome.

Conclusions From our findings, it is possible to produce a new model of SWI/SNF's role in HIV-1 Tat transactivation. Initiation of transcription occurs in the presence of unmodified Tat which binds to BAF and p-TEFb allowing nuc-1 to become accessible. This, however, only allows for basal transcription and an increase in splicing at the HIV-1 LTR region. In order for elongation and rapid transcription to occur, Tat becomes acetylated and recruits PBAF complexes. This new complex allows for increased chromatin remodeling followed by rapid transcription and decreased splicing to occur. The decreased splicing is probably due to acetylated Tat binding to p32 which is an inhibitor of the splicing factor ASF/SF-2 (Berro et al., 2006). Future experiments will define the role of these complexes on various HIV-1 clades and whether nuc-1 remodeling and RNA splicing are facilitated by both BAF and PBAF complexes in these viruses.

Materials and methods Cell culture HEK293T (293T) and TZM-bl cell lines were grown in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (FBS) (10%), 2 mM L-glutamine, and antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml) (cDMEM). HCC1143 and CEM, cells were grown in RPMI 1640 supplemented with FBS, L-glutamine, and antibiotics as described above (cRPMI). All cell lines were maintained at 37 °C in 5% CO2.

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PBMC purification Human PBMCs from three healthy donors were purified by FicollPaque centrifugation and activated for 72 h with of PHA (10 μg/ml) and recombinant human IL-2 (50 U/ml) in cRPMI medium. Freshly activated PBMCs were infected with dual tropic 89.6 HIV-1 virus at MOI or 10. At various time points, cell culture supernatants were collected for ChIP assay. GST-Tat pulldown GST-Tat (wild-type 86 and 101) were purified over a glutathione column and eluted. Acetylated GST-Tat (2 μg) was incubated with p300 and acetyl-CoA (63), washed, and incubated with 100 μg of HeLa phosphocellulose nuclear fraction (0.75 M) that contained both BAF and PBAF complexes. Samples were incubated overnight at 4 °C in presence of 100 ng/ml of EtBr, washed and bound complexes were run on a 4–20% SDS/PAGE and Western blotted for the presence of Baf200, BRG1, Baf250 and actin using specific antibodies. Chromatin immunoprecipitation assay (ChIP) ChIP assays were performed from cells using antibodies as described below. Cells were seeded to 60% confluency, treated as described in the figure legends, then processed for ChIP beginning with cross-linking proteins to DNA by 1.0% formaldehyde. Chromatin was sonicated five times for 20 s each, generating DNA fragments of about 500–100 base pairs. The sonicated supernatants containing the DNA were diluted with ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8.1, and 167 mM NaCl) to a total volume of 5.5 ml, and precleared by rotating for 1 h at 4 °C with ChIP prepared protein A/G beads (beads washed twice with 1 ml TNE50 + NP-40, resuspend in 650 μl; add 40 μl of ssDNA (10 mg/ml) and 75 μl BSA (10 mg/ml)). No proteases or RNAses were used for the extraction. The extract was centrifuged at 3000 rpm for 10 min at 4 °C and the lysate was transferred to a fresh tube. Supernatant (500 μl) was reserved for input and then 10 μg of each antibodies indicated above were added to the reaction mixture. After overnight rotation at 4 °C, the immune complexes were collected by addition of ChIP prepared protein A/G beads. After extensive washes, the immune complexes were eluted with 1% SDS/NaHCO3 solution for 30 min at room temperature. The eluted complexes were treated with NaCl solution and reverse cross-linked overnight. DNA was extracted using 1:1 phenol/chloroform (500 μl) followed by addition of 1 ml of absolute ethanol and 3 M sodium acetate (50 μl) incubated at −20 °C for at least 20–30 min. The solution was spun for 20 min at 14,000 rpm at 4 °C followed by a 70% ethanol wash and 5 min spin. DNA pellet was resuspended in 1× TE and stored at 4 °C. Afterwards, DNA was purified by PCR purification Kit (BiONEER) and amplified by PCR. For qPCR, enriched DNA was also quantified using an ABI Prism 7100 instrument and SYBR green (Applied Biosystems, Foster City, CA, USA). Primer pairs for quantitative-PCR analysis of chromatin immunoprecipitation amplified sequences flanked the HIV-1 LTR nuc-1 (+10 to + 165) and Env (+8990 to +9120). For qPCR, enriched DNA was also quantified using an ABI Prism 7100 instrument and SYBR green (Applied Biosystems). Primer pairs for quantitativePCR analysis of chromatin immunoprecipitation amplified sequences flanked the HIV-1 LTR nuc-0 (−415 to −255, nuc-1 (+10 to + 165) and Env (+ 8990 to + 9120). Readings were normalized to either Tat deficient samples or input as specified in the figure legend. Antibodies Antibodies used were obtained from Upstate Biotech (H2A, 07146; H2B, 07-371; H3, 04-801; and H4, 05-858) and Santa Cruz (Baf200, sc81050; Baf250, sc48791; SPT16, sc28734; p300, sc584;

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BRG1, sc10768; and Actin, 7210). The Pol II (8WG16 against the unphosphorylated CTD) has previously been used by us for many ChIP assays. Cdk9 antibody was from Biodesign. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Nef (2949) and Env gp160 (1209). CAT assays CEM cells were transfected (electroporation) with HIV-LTR-CAT (5 μg), CMV-Tat (3 μg), and 20 nM (or higher concentrations) of either siBaf200 or siBaf250. After 48 h, cells were lysed and chloramphenicol acetyltransferase (CAT) activity was determined. A standard reaction was performed by adding the cofactor acetyl coenzyme A to a microcentrifuge tube containing cell extract and radiolabeled (14C) chloramphenicol in a final volume of 50 μl and incubating the mixture at 37 °C for 1 h. The reaction mixture was then extracted with ethyl acetate and separated by thin-later chromatography on silica gel plates (Bakerflex silica gel thin-later chromatography plates) in a chloroform–methanol (19:1) solvent. The resolved reaction products were then detected by exposing the plate to a PhosphorImager cassette. Western blots Cell extracts were resolved by SDS PAGE on a 4–20% tris-glycine gel (Invitrogen). Proteins were transferred to Immobilon membranes (Millipore) at 200 mA for 2 h. Membranes were blocked with Dulbecco's phosphate-buffered saline (PBS) 0.1% Tween-20+ 5% BSA. Primary antibody against specified antibodies was incubated with the membrane in PBS+0.1% Tween-20 overnight at 4 °C. Membranes were washed three times with PBS+0.1% Tween-20 and incubated with HRP-conjugated secondary antibody for 1 h. Presence of secondary antibody was detected by SuperSignal West Dura Extended Duration Substrate (Pierce). Luminescence was visualized on a Kodak 1D image station. siRNA Treatment BRG1 (sc29827), BRM (sc29831), Baf200 (sc96225), and Baf250 (sc43628) siRNAs were purchased from Santa Cruz Biotechnology Inc. siGENOME SMART pool for PB1 (Baf180) (M-008692-01-0005) was purchased from Dharmacon. siRNA (at various concentrations) was transfected into cells using Attractene (Qiagen) according to the manufacturer's instructions. Transfections and RT assays HCC1143 cells were seeded in 6 well plates at 400,000 cells/well and then transfected with 20 μg of pNL4-3 either alone or with 30 nM of siBaf200 using Attractene (Qiagen). Supernatants were collected to test for the presence of virus on days 2, 3, and 4. Viral supernatants (10 μl) were incubated in a 96-well plate with reverse transcriptase (RT) reaction mixture containing 1× RT buffer (50 mM Tris–HCl, 1 mM DTT, 5 mM MgCl2, 20 mM KCl), 0.1% Triton, poly(A) (1 U/ml), pd(T) (1 U/ ml), and [3H]TTP. The mixture was incubated overnight at 37 °C, and 10 ml of the reaction mix was spotted on a DEAE Filtermat paper, washed four times with 5% Na2HPO4 and three times with water, and then dried completely. RT activity was measured in a Betaplate counter (Wallac, Gaithersburg, MD). Cells (day 4) were also processed for Western blot analysis using anti-Baf200 and anti-actin antibodies. Competing interests The author(s) declare that they have no competing interests.

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Author's contributions RE and FK designed the experiments, and wrote the manuscript. LC, SC and IG performed the Western blots. ZK assisted in the design of knockdown experiments. LD performed ChIP experiments. KK aided in the preparation of the manuscript. FK oversaw the research and aided in the preparation of the manuscript. Funding This work was supported by grants from the George Washington University REF funds to FK and NIH grant AI043894 and AI074410. Acknowledgments We would like to thank the members of the Kashanchi lab for experiments and assistance with the manuscript. We would also like to thank Dr. John D. Wade (Queensland, Australia) for the Tat peptides. pNLEnv was a kind gift from H. G. Kräusslich (Universität Heidelberg, Germany). References Adelman, K., Wei, W., Ardehali, M.B., Werner, J., Zhu, B., Reinberg, D., Lis, J.T., 2006. Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26 (1), 250–260. Agbottah, E., Deng, L., Dannenberg, L.O., Pumfery, A., Kashanchi, F., 2006. Effect of SWI/ SNF chromatin remodeling complex on HIV-1 Tat activated transcription. Retrovirology 3, 48. Ariumi, Y., Serhan, F., Turelli, P., Telenti, A., Trono, D., 2006. The integrase interactor 1 (INI1) proteins facilitate Tat-mediated human immunodeficiency virus type 1 transcription. Retrovirology 3, 47. Batsche, E., Yaniv, M., Muchardt, C., 2006. The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nat. Struct. Mol. Biol. 13 (1), 22–29. Belotserkovskaya, R., Oh, S., Bondarenko, V.A., Orphanides, G., Studitsky, V.M., Reinberg, D., 2003. FACT facilitates transcription-dependent nucleosome alteration. Science 301 (5636), 1090–1093. Bernstein, B.E., Kamal, M., Lindblad-Toh, K., Bekiranov, S., Bailey, D.K., Huebert, D.J., McMahon, S., Karlsson, E.K., Kulbokas III, E.J., Gingeras, T.R., Schreiber, S.L., Lander, E.S., 2005. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120 (2), 169–181. Berro, R., Kehn, K., de la Fuente, C., Pumfery, A., Adair, R., Wade, J., Colberg-Poley, A.M., Hiscott, J., Kashanchi, F., 2006. Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J. Virol. 80 (7), 3189–3204. Bohan, C.A., Kashanchi, F., Ensoli, B., Buonaguro, L., Boris-Lawrie, K.A., Brady, J.N., 1992. Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro. Gene Expr. 2 (4), 391–407. Bukrinsky, M., 2006. SNFing HIV transcription. Retrovirology 3, 49. Chiang, C.M., Roeder, R.G., 1995. Cloning of an intrinsic human TFIID subunit that interacts with multiple transcriptional activators. Science 267 (5197), 531–536. Coull, J.J., He, G., Melander, C., Rucker, V.C., Dervan, P.B., Margolis, D.M., 2002. Targeted derepression of the human immunodeficiency virus type 1 long terminal repeat by pyrrole-imidazole polyamides. J. Virol. 76 (23), 12349–12354. Dal Monte, P., Landini, M.P., Sinclair, J., Virelizier, J.L., Michelson, S., 1997. TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2. Aids 11 (3), 297–303. Dellaire, G., Makarov, E.M., Cowger, J.J., Longman, D., Sutherland, H.G., Luhrmann, R., Torchia, J., Bickmore, W.A., 2002. Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes. Mol. Cell. Biol. 22 (14), 5141–5156. Demarchi, F., D'Agaro, P., Falaschi, A., Giacca, M., 1993. In vivo footprinting analysis of constitutive and inducible protein–DNA interactions at the long terminal repeat of human immunodeficiency virus type 1. J. Virol. 67 (12), 7450–7460. Feinberg, M.B., Baltimore, D., Frankel, A.D., 1991. The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation. Proc. Natl. Acad. Sci. U. S. A. 88 (9), 4045–4049. Flanagan, J.F., Mi, L.Z., Chruszcz, M., Cymborowski, M., Clines, K.L., Kim, Y., Minor, W., Rastinejad, F., Khorasanizadeh, S., 2005. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438 (7071), 1181–1185. Garcia-Martinez, L.F., Ivanov, D., Gaynor, R.B., 1997. Association of Tat with purified HIV-1 and HIV-2 transcription preinitiation complexes. J. Biol. Chem. 272 (11), 6951–6958. Gautier, V.W., Gu, L., O'Donoghue, N., Pennington, S., Sheehy, N., Hall, W.W., 2009. In vitro nuclear interactome of the HIV-1 Tat protein. Retrovirology 6, 47. Gonzalez, P.J., Palacian, E., 1989. Interaction of RNA polymerase II with structurally altered nucleosomal particles. Transcription is facilitated by loss of one H2A.H2B dimer. J. Biol. Chem. 264 (31), 18457–18462.

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