Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription
© Agbottah et al; licensee BioMed Central Ltd. 2006
Received: 13 June 2006
Accepted: 07 August 2006
Published: 07 August 2006
Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency virus (AIDS). Following entry into the host cell, the viral RNA is reverse transcribed into DNA and subsequently integrated into the host genome as a chromatin template. The integrated proviral DNA, along with the specific chromatinized environment in which integration takes place allows for the coordinated regulation of viral transcription and replication. While the specific roles of and interplay between viral and host proteins have not been fully elucidated, numerous reports indicate that HIV-1 retains the ability for self-regulation via the pleiotropic effects of its viral proteins. Though viral transcription is fully dependent upon host cellular factors and the state of host activation, recent findings indicate a complex interplay between viral proteins and host transcription regulatory machineries including histone deacetylases (HDACs), histone acetyltransferases (HATs), cyclin dependent kinases (CDKs), and histone methyltransferases (HMTs).
Here, we describe the effect of Tat activated transcription at the G1/S border of the cell cycle and analyze the interaction of modified Tat with the chromatin remodeling complex, SWI/SNF. HIV-1 LTR DNA reconstituted into nucleosomes can be activated in vitro using various Tat expressing extracts. Optimally activated transcription was observed at the G1/S border of the cell cycle both in vitro and in vivo, where chromatin remodeling complex, SWI/SNF, was present on the immobilized LTR DNA. Using a number of in vitro binding as well as in vivo chromatin immunoprecipitation (ChIP) assays, we detected the presence of both BRG1 and acetylated Tat in the same complex. Finally, we demonstrate that activated transcription resulted in partial or complete removal of the nucleosome from the start site of the LTR as evidenced by a restriction enzyme accessibility assay.
We propose a model where unmodified Tat is involved in binding to the CBP/p300 and cdk9/cyclin T1 complexes facilitating transcription initiation. Acetylated Tat dissociates from the TAR RNA structure and recruits bromodomain-binding chromatin modifying complexes such as p/CAF and SWI/SNF to possibly facilitate transcription elongation.
Human immunodeficiency virus (HIV) is the etiological agent of AIDS. The pathogenesis of HIV-induced disease is complex and multifactorial . Following infection, reverse transcriptase complexes synthesize a double stranded DNA molecule that is then incorporated into the host genome. A robust cellular and humoral immune response inhibits viral production within weeks. However, a chronic persistent infection in lymphoid tissue persists throughout the life (median period of 10–20 years) of the infected individual. Several key HIV-1 and cellular proteins have been determined to be necessary for this course of infection, including the trans-activator Tat. Viral clones deficient in Tat do not effectively replicate in vitro or in vivo. Furthermore, infected T cells quiescent at the G0 phase of the cell cycle (lacking cytokine signals) will not produce high titer virus .
The replication rate of integrated HIV-1 is largely controlled at the level of transcription. The HIV-1 LTR, present at both ends of the integrated viral genome, contains cis-acting elements necessary for transcription initiation from the 5' LTR and for polyadenylation of the viral transcripts in the 3' LTR. The core promoter of HIV-1 includes two NF-κB binding sites, three Sp1 binding sites, the TATA box, and the ligand-binding protein 1 (LBP-1)/YY1 site. There is also a repressor complex sequence (RCS) within the initiation site, which contains three binding motifs for LSF. YY1 and LSF cooperate to allow binding of YY1 to the RCS and subsequent recruitment of HDAC1 . In addition to cellular transcription factors, the activity of the HIV-1 promoter is strongly dependent on the viral trans-activator, Tat. Historically, the mechanism of action by Tat has been assigned to be at the level of initiation and elongation [4–9]. The effect of Tat on pre-initiation, initiation, and elongation has been observed through a number of biochemical interactions, including physical binding to Sp1, stabilization of the TFIID/TFIIA complex on the HIV-1 TATA box , recruitment of a functional TATA-binding protein (TBP) or TFIID [11–16], phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) by a number of kinases, including TFIIH [17–19], hSpt5 which functions in transcription elongation as a stabilization factor of RNAPII , and binding of Tat directly to RNAPII [21, 22]. More recently the role of Tat in transcription initiation has been revisited, where Tat enhanced recruitment of TBP has been observed resulting in activated transcription [23, 24].
Tat activates the HIV LTR by binding to TAR and recruiting and activating cellular factors [25–31]. One of the co-factors required for Tat activity is a protein kinase called TAK (Tat-associated kinase) [32, 33] whose activity is stimulated by Tat. Activation of TAK (cdk9/cyclin T1) results in hyper-phosphorylation of the large subunit of the RNAPII CTD and activation of transcription elongation . Cdk9 is analogous to a component of a positive acting elongation factor (P-TEFb) isolated from Drosophila , which acts to stimulate promoter-paused RNAPII to enter into productive elongation [33, 36–39]. A histidine-rich stretch of cyclin T1 binds to the CTD of RNAPII, which is required for the subsequent expression of full-length transcripts from target genes . Cdk9 phosphorylation is required for high-affinity binding of Tat/P-TEFb to TAR [41–43].
HIV-1 proviral DNA is organized into a higher order chromatin structure in vivo, which regulates viral expression by restricting access of the transcriptional machinery to the HIV-1 LTR. The primary chromatin structure of integrated HIV-1 has been characterized in vivo by the DNase-1 digestion method [44–46]. Two chronically infected human cell lines, ACH2 (T-cell line) and U1 (promonocyte cell line), were analyzed by DNase-I digestion and four distinct DNase-I hypersensitive sites (DHS) were identified in the 5' LTR. It has been largely assumed that DHSs in the native chromatin are free of histones and allow unrestricted access to DNA-binding proteins . The first site, DHS1, located at the 5' end of the integrated LTR, was detected only as a minor DHS in U1 cells. DHS2 and DHS3 are located in the U3 region, encompassing nt 223–325 and nt 390–449, respectively. The NF-κB and Sp1 binding sites, as well as the TATA box, are located in DHS2 and DHS3. DHS4 was found immediately downstream of the U5 region (nt 656–720), where a cluster of potential transcription factor binding sites for AP-1, AP-3 like, DBF-1, and Sp1 are positioned. The location of the DHSs was also verified in vitro using the HIV-1 promoter reconstituted into chromatin by DNase-I footprinting analysis [48–50]. 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 nt -255 to -3 and +141 to +265. One nucleosome, nuc-1, is located between these two regions. The first nucleosome-free region in U3 contains many promoter/enhancer elements which are already occupied by transcriptional factors including repressors . When cells are activated with TNF-α and TPA treatment or with the HDAC-specific inhibitors, trapoxin, Trichostatin A (TSA), or sodium butyrate, DHS3 and DHS4 are extended and nuc-1 is specifically remodeled. Using a ChIP assay, histone acetylation surrounding nuc-1 and the RCS was observed to be significantly increased following TSA treatment . The disruption of nuc-1 occurred independent of transcription and was α-amanitin insensitive, suggesting that this chromatin remodeling was a pre-requisite for transcription, rather than a consequence . Therefore, the integrated proviral DNA, along with the specific chromatinized environment in which integration takes place allows for the coordinated regulation of viral transcription and replication. While the specific roles of and interplay between viral and host proteins have not been fully elucidated, numerous reports indicate that HIV-1 retains the ability for self-regulation via the pleiotropic effects of its viral proteins. Though viral transcription is fully dependent upon host cellular factors, recent findings indicate a complex interplay between viral proteins and host transcription and signaling machineries (i.e. HDACs, HATs, and other protein-modifying factors).
In the current manuscript, we describe the effect of Tat activated transcription at the G1/S border of the cell cycle and analyze the interaction of Tat with the chromatin remodeling complex, SWI/SNF. HIV-1 LTR DNA reconstituted into nucleosomes can be activated in vitro using various Tat expressing extracts. More specifically, optimal activated transcription was observed at the G1/S border of the cell cycle both in vitro and in vivo. In an attempt to define protein complexes that are normally involved in HIV-1 transcription, we used an in vitro immobilized nucleosomal DNA assembly system in the presence of the G1/S extracts and pulled-down complexes followed by their identification using MALDI-TOF mass spectrometry. In addition to known factors such as Sp1, TFIIB, and cdk9/cyclin T1, we also specifically detected the presence of acetylated Tat and members of the SWI/SNF chromatin remodeling complex. Using a number of in vitro binding as well as in vivo ChIP assays, we detected the presence of both BRG1 and acetylated Tat in the same complex. Finally, we demonstrate that removal of nuc-1 resulted in accessibility of the HIV-1 LTR DNA to a restriction enzyme and activated transcription.
Effect of Tat activation at the G1/S border
We previously demonstrated, by in vitro transcription analysis, that Tat-dependent transcription takes place in a cell cycle-dependent manner. We utilized cells that were transfected with either a control plasmid (pCEP4) or a hemagglutinin epitope-tagged Tat plasmid (eTat), selected in hygromycin by single-cell dilution, and maintained in hygromycin. Whole-cell extracts were prepared from control and eTat cells for in vitro transcription assays using wild-type and mutant HIV-1 naked templates. Cells at late G1/early S contained 10-fold higher levels of transcriptional activity on the wild-type LTR template (LTR-TAR+) than on the TAR mutant template (LTR-TAR-) .
Presence of HIV-1 transcripts at the G1/S border in infected cells
Presence of chromatin remodeling factors on HIV-1 DNA at the G1/S border
A limited number of proteins bound to the transcriptionally inactive control promoters including histones H4, H2A, H2B, and H3 (Figure 3A, lanes 4–7). Twenty proteins that bound to the transcriptionally active HIV-1 LTR were identified (Figure 3A, lane 10). The proteins identified included: 1) histone H4; 2) Tat; 3) histone H2A; 4) histone H2B; 5) histone H3; 6) TFIIB; 7) cdk2; 8) cdk9; 9) undetermined; 10A) cyclin E; 10B) cyclin E; 11) cyclin T; 12) undetermined; 13) undetermined; 14) undetermined; 15) undetermined; 16) Sp1; 17) RNA helicase A; 18) RNAPII large subunit; 19) BRG1; and 20) DNA-PK. Additionally, we performed control reactions using HXB2 TATA-/TAR+, HXB2 TATA+/TAR-, and AdML promoters (Figure 3B). A number of unique proteins were bound to the wild-type promoter that were not bound on the HXB2 TATA-/TAR+ and TATA+/TAR- promoters (data not shown). Results in Figure 3A confirms published data showing that TFIIB, cdk2, cdk9, cyclin E, cyclin T1, Sp1, RNAPII, and DNA-PK associate with the HIV-1 LTR during basal transcription or Tat activated transcription. These results also showed that cdk9/cyclin T1 and other cyclin/cdk complexes (i.e. cdk2/cyclin E) are involved in transcription of the HIV-1 promoter. Finally, these results indicate that Tat recruits a component of the chromatin remodeling complex, BRG1, to the HIV-1 LTR.
Effect of acetylated Tat on recruitment of a chromatin remodeling complex
The identification of BRG1 as one of the proteins that bound to transcriptionally active HIV-1 LTR suggests that this component of the SWI/SNF complex may be important in Tat activated transcription. To elucidate the role of BRG1 in HIV-1 gene expression, we determined whether there was a direct association with modified Tat. While the detailed molecular mechanisms underlying Tat dissociation from TAR RNA and its trans-activation of transcription on the integrated HIV-1 genome remain elusive, increasing evidence suggests that Tat activity requires association with several multiprotein complexes, which include the cyclin T1/cdk9 complex [33, 36, 43, 63–69] and the HAT transcriptional coactivators, p300/CBP and p300/CBP-associated factor (p/CAF) [60, 70, 71]. The site of acetylation of Tat was mapped to a double-lysine motif in a highly conserved region (49RKKRRQ54) of the basic RNA-binding motif of Tat. Tat acetylation resulted in its dissociation from TAR RNA and promoted the formation of a multiprotein complex comprised of Tat and p/CAF [60, 70]. Furthermore, we previously had shown that immobilized biotin Tat or acetylated Tat at positions 41, 50, and 51 could selectively pull-down distinct functional complexes including transcription-associated proteins, acetyltransferase-associated proteins, and kinase proteins .
Presence of acetylated Tat and BRG1 on HIV-1 nucleosomal DNA in vivo
To assess the functional relevance of Tat acetylation in vivo, we performed a series of ChIP assays from HIV-1 infected cells. We first raised polyclonal antibodies against acetylated Tat using acetylated Tat peptides as antigens. These antibodies recognize Tat acetylation at lysine 41 or 50 and 51. Next, we transfected HLM-1 cells with a wild-type Tat plasmid (eTat). These cells are HIV-1+/Tat-, thus introduction of wild-type Tat allows for full trans-activation and viral progeny formation. When anti-acetyl-Tat antibodies were used for immunoprecipitation, we consistently observed an association of acetylated Tat at regions where nuc-1 is located but not nuc-0 (Figure 5). These data suggest that Tat is acetylated in vivo and is associated with the nuc-1 region of the HIV-1 promoter, which encompasses the transcription start site.
Effect of BRG1 on HIV-1 transcription in vivo
Effect of BRG1 and acetylated Tat on nucleosomes on the HIV LTR
A number of investigators, including the Verdin lab, have performed pioneering experiments to define the nucleosome positions in HIV-1 infected cells. The HIV-1 genome was first analyzed for the presence of DNase-I hypersensitive sites using an indirect end-labeling technique . Three well characterized, chronically infected cell lines, ACH2, 8E5, and U1, were used to define nucleosome boundaries. Five major hypersensitive sites were identified, some of which were common among the three cell lines. They included DNase-I hypersensitive sites at the 5' LTR, namely HS2 (nt 223–325), HS3 (nt 390–449), and HS4 (nt 656–720) . Both HS3 and HS4 are at the 5' and 3' boundaries of nucleosome-1 (nuc-1), respectively. Nuc-1 is located at the transcriptional start site and poses a block to activated transcription . However, when activated transcription occurs, there is a disappearance of the periodic DNA protection pattern following digestion with DNase-I and reveals a profile of digested template essentially identical to naked DNA . Therefore, nuc-1 is disrupted when activated transcription takes place in all three cell lines tested [45, 46, 76–80].
The chromatin structure presents a significant barrier to transcription. Various complexes, including HATs, covalently modify nucleosomal histone proteins through acetylation while ATP-dependent chromatin remodelers alter the chromatin structure via ATP hydrolysis. These modifications and alterations of chromatin structure increase DNA accessibility to transcription factors and activators thus promoting transcription initiation and efficient elongation. A more current view is that activators must first recruit chromatin remodelers in order to create a chromatin environment permissive for pre-inititation complex (PIC) assembly. Recently, it has become evident that other factors, such as the chromatin structure of the gene promoter and the phase of the cell cycle, also govern how chromatin remodelers collaborate with each other to control steps before, during, or after PIC assembly.
Although the precise nucleosome position of the integrated HIV-1 LTR is well characterized, there is little data about how this primary structure is folded into the chromatin fiber or other secondary structures (>30 nm fiber) and how these structures influence HIV-1 latency and transcriptional activation. Few studies have shown that the integration site and its corresponding chromatin environment affect HIV-1 gene expression [81, 82]. Sequence analysis and mapping of HIV-1 integration sites in the cellular genome has provided evidence that chromatin structure plays an important role in subsequent HIV-1 transcription. Analysis of the site of integration during productive HIV-1 infection has shown that integration preferentially occurs in areas rich in retrotransposable Alu elements [83–85] and transcriptionally active genes (69% of 524 integration sites) . These areas are characterized by increased chromatin accessibility and the accumulation of factors important for chromatin remodeling. These data are also consistent with earlier observations that sites which are preferentially accessible to DNase-I are favored for integration [86–89]. However, a small fraction of cells (less than 1%) exhibited post-integration latency, a state of transcriptional silencing.
Many reports in the last several years have linked Tat trans-activation to chromatin remodeling in vitro and in vivo. When transcription is activated by Tat in a LTR integrated cell line, the chromatin associated with sequences immediately downstream of the transcription start site become accessible to nucleases and the nucleosome adjacent to the transcription start site (nuc-1) becomes disrupted . Similarly, transfection of Tat into Jurkat (Tat-) clones containing a integrated single HIV-1 mini-genome activated HIV-1 transcription and resulted in the disruption of nuc-1 to the same extent as TSA treatment . Furthermore, immunoprecipitation of Tat from human cells identified a protein complex with HAT activity, suggesting that HATs are targeted to the HIV-1 promoter by Tat . Interestingly, in vitro transcription from chromatinized templates indicated that Tat trans-activation is synergistic with Sp1 and NF-κB . In vivo, HIV-1 LTR lacking Sp1 and NF-κB sites do not undergo chromatin remodeling and are unresponsive to Tat trans-activation . Several other groups have observed that Tat is able to form a ternary complex with several HAT complexes, including p300/CBP, p/CAF, TAFII250, and Tip60, targeting these HAT proteins to the viral promoter [70, 90–94].
Similar to our findings, two recent reports have shown the involvement of SWI/SNF as a cofactor for Tat activation of the HIV promoter. Tréand et al.  showed that, via its arginine-rich motif, Tat was able to interact with Brm, the enzymatic subunit of the SWI/SNF chromatin-remodeling complex. This interaction was regulated by Tat acetylation at lysine 50. Tat recruited the SWI/SNF complex to the LTR in vivo, leading to the activation of the integrated HIV-1 promoter. Another recent report from the Verdin lab also showed similar results . Knockdown of INI-1 and BRG1, two components of the SWI/SNF chromatin-remodeling complex, suppressed Tat-mediated trans-activation, and cells deficient in INI-1 or BRG1 exhibited defective Tat trans-activation. Tat was found to be in complex with several SWI/SNF subunits, and the complex synergized with p300 to activate the HIV-1 promoter. A recent study from the Trono lab revealed that INI1 uses its repeat domains (Rpt 1 and 2) to bind and subsequently enhance the trans-activation potential of Tat . While INI1 is dispensable for viral transduction, their findings suggest that incoming PIC might recruit INI1 to promote chromatin remodeling at the HIV-1 promoter and enhance transcription.
It is interesting to note that INI-1 (Snf5/BAF47) is also a potent tumor suppressor whose mechanism of action is largely unknown. Tumor suppressor activity of Snf5 depends on its regulation of cell cycle progression; Snf5 inactivation leads to aberrant up-regulation of E2F targets and increased levels of p53 that are accompanied by apoptosis, polyploidy, and growth arrest. Furthermore, conditional mouse models demonstrate that inactivation of p16Ink4a or Rb does not accelerate tumor formation in Snf5 conditional mice, whereas mutation of p53 leads to a dramatic acceleration of tumor formation . Therefore, it would be interesting to further determine if the binding of Tat to the SWI/SNF complex (either through INI-1 or BRG1) could somehow control the expression of cell cycle genes (i.e., G1/S genes) and alter their activity. Current experiments are in progress to address this critical issue and whether G1/S genes such as various cyclins, including the cyclin E and T promoters, are modulated by the Tat/SWI/SNF complex.
We and others have shown that Tat can be acetylated at lysines 28, 50, and 51 leading to altered interactions between Tat and TAR, p300/CBP, and p/CAF, as well as activation of viral transcription and replication. They collectively support the notion that Tat first recruits p300/CBP to the HIV-1 promoter during initiation. Tat acetylated at lysine 50 then recruits p/CAF, which subsequently acetylates Tat at lysine 28. Acetylation of Tat at lysines 50 and/or 51 promotes the dissociation of Tat from TAR RNA and contributes to stimulation of transcription elongation [60, 99]. The acetylated form of Tat is released from TAR, which can now bind to BRG1, a component of the SWI/SNF chromatin remodeling complex. We hypothesize that the interaction of Tat with BRG1 facilitates SWI/SNF to remodel downstream nucleosomes allowing for further transcription of the HIV-1 genome. We propose that the major function of the interactions between Tat and bromodomain proteins is to modify HIV-1 chromatin such that the LTR becomes responsive to Tat and allows ample and rapid activated transcription to occur. This also may set the stage for re-initiation of transcription where pre-made (activated) complexes may be recycled for HIV-1 activated transcription. Future experiments will address these particular issues along with Tat's effect on the remodeling activity of the SWI/SNF complex at the proximal promoter and ORF regions of HIV-1 genome.
Materials and methods
ACH2 and 8E5 cells are both HIV-1-infected lymphocytic cells, with a single integrated wild-type copy (ACH2) and a single integrated copy containing a defective reverse transcriptase (8E5) in CEM (12D7) cells. The CEM T cell (12D7) is the parental cell for both ACH2 and 8E5 cells. U1 is a monocytic clone harboring two copies of the viral genome from parental U973 cells. All cells were cultured at 37°C with up to 1 × 105 cells per ml in RPMI 1640 media containing 10% fetal bovine serum (FBS), 1% streptomycin/penicillin antibiotics, and 1% L-glutamine (Gibco-BRL, Carlsbad, CA). HLM-1 cells (AIDS Research and Reference Reagent Program, Catalog No. 2029) were derived from HeLa-T41 cells integrated with one copy of the HIV-1 genome containing a Tat-defective mutation. The mutation was introduced as a triple termination linker at the first AUG of the Tat gene. HLM-1 cells are completely negative for virus particle production, but can be induced to express one cycle of infectious HIV-1 particles after transfection with Tat cDNA or mitogens such as TNF-α or sodium butyrate. HLM-1 cells were grown in DMEM containing 10% FBS, 100 mg/ml of G418, plus 1% streptomycin/penicillin, and 1% L-glutamine (Gibco BRL). Cells were grown to 75% confluency prior to the transfection. Plasmids were transfected into HLM-1 cells by electroporation or Amexa, washed after 4 h, and re-fed with fresh complete DMEM with 10% FBS for the remainder of the experiment.
Generation of epitope-tagged Tat-expressing cell line
HeLa CD4+ cells were used for transfection with either an epitope-tagged (the influenza epitope at the C-terminus of Tat 1–86) plasmid or the parental vector pCEP4. Following transfection, cells were selected with 200 μg of hygromycin/μl. Hygromycin-resistant lines established from single-cell clones were maintained for up to 12 months with continuous passage and used to make extracts for in vitro transcription analysis .
Five to ten million infected cells in log phase were incubated for 2 h with or without 5 μg/ml TNF-α to induce transcription of latent proviral DNA. Cells were subsequently left untreated or treated with siRNA. After 48 h, cells were cross-linked (1% formaldehyde, 10 min at 37°C) and samples were sonicated to reduce DNA fragments to 200–800 nt lengths for ChIP assays. Specific transcription complexes were immunoprecipitated with appropriate antibodies. DNA sequences in the immunoprecipitates were detected by PCR using primers specific for the HIV-1 LTR (forward primer, 5'-ACTTTTCCGGGGAGGCGCGATC-3'; reverse primer, 5'-GCCACTGCTAGAGATTTCCACACTG-3') or Env region (forward primer, 5'-CCTTG(T)GAGCCAATTCCCATA-3'; reverse primer, 5'-TAACAAATGCTCTCCCTGGTC-3').
Individual protein bands were excised from the silver-stained gel and destained with a solution of 30 mM potassium ferricyanide/100 mM sodium thiosulfate (1:1) (v/v). Trypsin-digested sample solutions were further desalted and concentrated with C18 ZipTips (Millipore, Framingham, MA). Samples were mixed with the same volume of the matrix solution (α-cyano-4-hydroxycynnamic acid in 50% acetonitrile/0.1% [v/v] trifluoroacetic acid). Two microliters of the mixtures were applied to the sample plate and introduced into the mass spectrometer after drying. Mass spectra were recorded in the reflectron mode of a MALDI-TOF mass spectrometer (Voyager-Elite; PerSeptive Biosystems) by summing 200–300 laser shots with an acceleration voltage of 20 kV, 70% grid voltage, 0.05 guide wire voltage, 100 ns delay, and low mass gate at 700 m/z. Proteins were identified using the peptide mass fingerprinting analysis software ProFound . The NCBInr database was used for the searches with several passes of searching with different limitations for each spot. In general, all bands were searched using methionine oxidation and no limitation for pI as criteria. The most optimal match for each spot was considered using higher coverage rate, more matched peptides, and higher score without limitations on the taxonomic category and protein mass. Zero missed cleavage by trypsin and lowest mass tolerance, i.e. ± 50 ppm, were considered for most of the proteins. A few bands were searched with the following parameters to find the best match: two missed cut cleavages, limited to the "mammal" category, and/or a set ± 50% of total molecular mass. We consistently used multiple parameters such as low miss cut, low ppm, and first methionine oxidation in our searches to obtain reliably matched proteins. Identified proteins (i.e., Sp1, TFIIB, cdk9, Tat, and BRG1) were confirmed by Western blot analysis. Unless stated specifically, all data bases and tools used for bioinformatics analysis were from the following public websites: PubMed , ExPASy , BLAST , Pfam , PBIL , COILS , and PIR .
Oligonucleotides were designed and synthesized using the OligoEngine website  and the accession number for BRG1. Five oligonucleotides (see below), which span the 5' end, middle, and 3' end of the BRG1 mRNA, were chosen. The most optimal sequences had a GC content between 30% and 70%. HIV-1 infected cell lines were treated with TNF-α for 2 h. A mixture of the five oligonucleotides was electroporated into the cells, and HIV-1 replication was monitored by p24 Gag enzyme-linked immunosorbent assay (ELISA). The sequences of oligonucleotides used for BRG1 siRNA (wild-type) were as follows: [GenBank:U29175] -1716, GGACAAGCGCCUGGCCUAC; [GenBank:U29175] -2142, GAAGAUUCCAGAUCCAGAC; [GenBank :U29175] -3210, GAUCUGCAACCACCCCUAC; [GenBank:U29175] -4236, GCAGUGGCUCAAGGCCAUC; [GenBank:U29175] -4776, GGAGGAUGACAGUGAAGGC. The sequences used for BRG1 siRNA (mutant) were as follows: [GenBank:U29175] -1716, GGACAAAAAAAUGGCCUAC; [GenBank:U29175] -2142, GAAGAUUCCAAAAAAAGAC; [GenBank:U29175] -3210, GAUCUGCAACCAAAAAUAC; [GenBank:U29175] -4236, GCAGUGGCUCAAAAAAAUC; [GenBank:U29175] -4776, GGAGGAUGAAAAAAAAGGC.
Cell cycle analysis
The eTat, control (pCEP4), CEM, and OM10.1 cells were either blocked with hydroxyurea for 18 h or blocked with hydroxyurea (2 mM final concentration), washed, and released for 1 h, followed by addition of nocodazole (50 ng/ml) for 14 h. Following the block, the cells were washed twice with phosphate-buffered saline (PBS) and released with complete medium. Samples were collected every 3 h, and the cells were used to make whole-cell extracts (5 × 107 cells/time point) for in vitro transcription or Western blot analysis or processed for fluorescence-activated cell sorting (FACS). Single-color flow cytometric analysis of DNA content was performed on various cell lines. The cells were washed with PBS, and approximately 2 × 106 cells were fixed with 500 μl of 70% ethanol. The cell pellets were washed three times with PBS and incubated in 1 ml of PBS containing 150 μg of RNase A (Sigma)/ml and 20 μg/ml of propidium iodide (Sigma, St. Louis, MO) at 37°C for 30 min. The stained cells were analyzed for red fluorescence (FL2) on a FACScan (Becton Dickinson, San Jose, CA), and the distribution of cells in the G1, S, and G2/M phases of the cell cycle was calculated from the resulting DNA histogram with Cell FIT software, based on a rectangular S-phase model.
In vitro transcription was performed with HeLa or CEM whole-cell extracts (25 to 50 μg total) on immobilized HIV-1 LTR chromatin templates which were assembled as described below. The DNA fragments were biotinylated, gel purified, and reconstituted with core histones by step dilution. Briefly, core histones were purified from HeLa cells and mixed with DNA. The biotinylated mononucleosome were prepared by mixing the biotinylated DNA and purified core histones by sequential dilution from 1 to 0.1 M NaCl and subsequently phased. The biotinylated nucleosomal arrays were then incubated at 30°C for 1 h with paramagnetic beads coupled to streptavidin in a binding buffer containing 10 mM HEPES (pH 7.8), 50 mM KCl, 5 mM DTT, 5 mM phenylmethylsulfonyl fluoride, 5% glycerol, 0.25 mg/ml bovine serum albumin (BSA), and 2 mM MgCl2, supplemented with 300 mM KCl. In vitro transcription reactions were incubated for 1 h at 30°C and contained the nucleoside triphosphates ATP, GTP, and CTP at a final concentration of 50 μM and [32P]UTP (20 μCi; 400 Ci/mmol; Amersham, Piscataway, NJ) in buffer D (10 mM HEPES [pH 7.9], 50 mM KCl, 0.5 mM EDTA, 1.5 mM dithiothreitol, 6.25 mM MgCl2, and 8.5% glycerol). Transcription reactions were terminated by the addition of 20 mM Tris-HCl (pH 7.8), 150 mM NaCl, and 0.2% SDS. The quenched reactions were extracted with equal volumes of phenol-chloroform and precipitated with 2.5 volumes of ethanol and 1/10volume of 3 M sodium acetate. Following centrifugation, the RNA pellets were resuspended in 8 μl of formamide denaturation mix containing xylene cyanol and bromophenol blue, heated at 90°C for 3 min, and seperated at 400 V in a 10% polyacrylamide (19:1 acrylamide-bisacrylamide) gel containing 7 M urea (pre-ran at 200 V for 30 min) in 1 × Tris-borate-EDTA. Transcript sizes of 250 nt for AdLuc and 375 nt for HIV-1 transcripts were observed. The gels were analyzed with the Molecular Dynamics PhosphorImager screen and radioactivity was quantitated with ImageQuant.
Immunofluorescence staining by flow cytometry
OM10.1 cells were used to analyze HIV-1 gene expression at various stages of the cell cycle. Following arrest of cells at G0 by serum starvation, cells were washed in Hanks' balanced salt solution and fixed in 70% ice-cold ethanol. After fixation, cells were rehydrated in PBS containing 1% BSA and aliquots containing 0.5 × 106 to 1 × 106 cells were resuspended in 150 μl of PBS-BSA. The abundance of Nef, Env, cyclin E and cyclin A were determined after immunofluorescence staining . Cells were stained with mouse anti-human monoclonal antibodies to cyclin E (1:100; SC, clone HE67), cyclin A (1:50; SC, clone BF683), Nef (1:50; AIDS reagent catalog, EH1), and Env (1:50; AIDS reagent catalog, 48 d) or a nonspecific immunoglobulin (DAKO) overnight. Samples were washed in PBS-BSA and stained with a fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody (1:30; DAKO) for 30 min in the dark.
BAF, BRG1 associated factors
Brahma related gene-1
human immunodeficiency virus type 1
long terminal repeat
The current manuscript is dedicated to our colleague and friend, Dr. Longwen Deng, who recently passed away. Dr. Deng generated most of the data for the current manuscript. This work was supported by grants from The George Washington University REF funds to FK, and Akos Vertes and by NIH grants AI44357, AI43894, and 13969 to FK.
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