Genome-Wide Binding Map of the HIV-1 Tat Protein to

the Human Genome

Céline Marban1,2., Trent Su2,3., Roberto Ferrari2, Bing Li2, Dimitrios Vatakis4, Matteo Pellegrini5,6,
Jerome A. Zack4,6, Olivier Rohr1,7*, Siavash K. Kurdistani2,6,8*
1 Institut de Virologie, Université de Strasbourg, Strasbourg, France, 2 Department of Biological Chemistry, David Geffen School of Medicine, University of California Los
Angeles, Los Angeles, California, United States of America, 3 Division of Oral Biology and Medicine, School of Dentistry, University of California Los Angeles, Los Angeles,
California, United States of America, 4 Division of Hematology and Oncology, Department of Medicine, David Geffen School of Medicine, University of California Los
Angeles, Los Angeles, California, United States of America, 5 Department of Molecular, Cellular, and Developmental Biology, University of California Los Angeles, Los
Angeles, California, United States of America, 6 Eli and Edythe Broad Centre of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los
Angeles, California, United States of America, 7 Institut Universitaire de France (IUF), Paris, France, 8 Department of Pathology and Laboratory Medicine, David Geffen
School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America

Abstract
The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level
of transcription of the HIV genome. While Tat’s control of viral transcription is well-studied, much less is known about the
interaction of Tat with the human genome. Here, we report the genome-wide binding map of Tat to the human genome in
Jurkat T cells using chromatin immunoprecipitation combined with next-generation sequencing. Surprisingly, we found
that ,53% of the Tat target regions are within DNA repeat elements, greater than half of which are Alu sequences. The
remaining target regions are located in introns and distal intergenic regions; only ,7% of Tat-bound regions are near
transcription start sites (TSS) at gene promoters. Interestingly, Tat binds to promoters of genes that, in Jurkat cells, are
bound by the ETS1 transcription factor, the CBP histone acetyltransferase and/or are enriched for histone H3 lysine 4 tri-
methylation (H3K4me3) and H3K27me3. Tat binding is associated with genes enriched with functions in T cell biology and
immune response. Our data reveal that Tat’s interaction with the host genome is more extensive than previously thought,
with potentially important implications for the viral life cycle.

Citation: Marban C, Su T, Ferrari R, Li B, Vatakis D, et al. (2011) Genome-Wide Binding Map of the HIV-1 Tat Protein to the Human Genome. PLoS ONE 6(11):
e26894. doi:10.1371/journal.pone.0026894
Editor: John J. Rossi, Beckman Research Institute of the City of Hope, United States of America
Received July 12, 2011; Accepted September 22, 2011; Published November 4, 2011
Copyright: ß 2011 Marban et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Dr. Marban was supported in part by a fellowship from the Agence Nationale de Recherche sur le Sida. This work was also supported by a Howard
Hughes Medical Institute award, and a National Institutes of Health (NIH) Innovator award as well as a California Institute for Regenerative Medicine grant to Dr.
Kurdistani. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: skurdistani@mednet.ucla.edu (SKK); olivier.rohr@unistra.fr (OR)
. These authors contributed equally to this work.

Introduction PP2A subunits [8]. Therefore, Tat may have roles in regulation of
gene expression from the viral as well as the host genome.
After gaining entry into host cell, the HIV-1 genome is reverse- However, a genome-wide map of Tat interaction with the human
transcribed and the proviral DNA is integrated into the host genome is still lacking. Such a binding map may reveal additional
genome. Subsequently, the HIV-1 provirus is transcribed allowing roles for Tat in creating the proper cellular environment for
assembly and release of new viral particles from the infected cell. generating progeny virions.
HIV-1 Tat is essential for efficient viral gene expression and To generate a genome-wide map of Tat binding to the human
replication [1]. By recruiting the general RNA polymerase II genome, we performed chromatin immunoprecipitation combined
elongation factor P-TEFb to Tat response element (TAR) that with next generation sequencing (ChIP-seq) of Tat in Jurkat T cells
forms at the 59 end of nascent viral transcripts, Tat promotes (Jurkat-Tat). We also utilized microarrays to compare global gene
efficient elongation of viral transcription [2]. Moreover, Tat expression changes in Jurkat-Tat versus Jurkat T cells and related
acetylation by cellular histone acetyltransferases (HATs) such as the expression differences to histone acetylation changes. We
p300, CBP and PCAF is crucial for its transactivation activity [3]. found that the bulk of Tat binding sites are outside the immediate
While the role of Tat in viral gene expression has been well promoter regions of genes. Intriguingly, Tat binds preferentially to
studied, much less is known about the interaction of Tat with the specific DNA repetitive elements, especially the Alu repeat
host genome. Previous studies that aimed to define the role of Tat elements. Binding of Tat to the promoter regions did not correlate
at the host gene promoters found that Tat regulates transcription with gene expression. The majority of Tat binding sites at gene
of the interleukin 6 [4], MHC class I [5], ß2 microglobulin [6] and promoters in Jurkat-Tat cells are in close proximity with regions
mannose receptor [7] promoters. Tat also induces host cell bound by the ETS1 transcription factor or CBP in Jurkat T cells.
apoptosis through association with promoters of PTEN and two Our data provide the first comprehensive map of Tat binding to

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

Table 1. ChIP-seq alignment results using Bowtie 0.12.7 window. Using the Poisson distribution, we calculated P-values for
(Hg19). the enrichment of ChIPed reads in each window. Significant peaks
were defined as those windows with a P-value,1024 and with two
neighboring windows at the same significance P-value. Based on
DNA Total reads Aligned Reads (%) these criteria, we identified 2074 genomic regions occupied by Tat
in Jurkat-Tat T cells.
Input 42249133 26416727 (62.5)
Using the cis-regulatory element annotation system (CEAS)
ChIPed 44360277 27831745 (62.7) software [11] to determine the Tat binding distribution pattern
doi:10.1371/journal.pone.0026894.t001
across individual chromosomes, we observed that chromosomes 2,
9, 14, 15, 21 and 22 were significantly (P,1024) enriched for Tat
binding (Figure 1A). The majority (82%) of Tat binding occurs
the human genome, revealing an unexpected array of target
within introns and intergenic regions in the genome (Figure 1B).
regions.
Intergenic regions are defined as those regions that are at least
3 kilo bases (kb) away from any known gene. Only ,7% of Tat
Results binding sites are located within the promoter regions. These data
Genome-wide Tat binding locations defined by ChIP-seq suggest that Tat binding to the genome is non-random, with
To determine whether Tat binds to specific regions in the host preferential binding to certain chromosomes and intergenic
genome, we performed ChIP-seq to map Tat binding sites in regions.
Jurkat T cells that stably express Tat under G418 selection [9]. We
first validated Tat expression in Jurkat-Tat cells with Western Tat binding loci are enriched within repeat sequences
blotting (Figure S1). Subsequently, we sequenced both input and To determine whether Tat binding regions are associated with
ChIPed Tat-bound DNA using the Illumina GAIIx Sequencer. specific genomic features, we obtained the coordinates of various
The obtained sequences were aligned to the human genome DNA elements from the UCSC table browser website [12]. To our
(version Hg19) using the Bowtie software [10]. For both input and surprise, we found that 53% of Tat bound regions lie within
ChIPed samples, ,62% of all sequences were uniquely aligned to repeat-masker (rmsk) regions, which record repeat elements found
the human genome (Table 1). We segmented the human genome by RepeatMasker [13,14] (Figure 2A). We systematically deter-
into 100 bp windows and calculated the ChIPed DNA read mined the enrichment of Tat binding in various repeat elements
counts, which were compared to input DNA read counts in each and found that, strikingly, 58 percent of all repeat elements bound

Figure 1. Chromosomal distribution and genomic location of Tat binding sites. (A) Enrichment pattern of Tat-bound regions among
individual chromosomes is shown as a bar chart. Percent of total Tat-binding sites (red bars) and what would be expected by random chance (blue
bars) for each chromosome is shown. The asterisks denotes enrichment P-value,1024. (B) Distribution of all Tat-binding peaks in relation to gene
structure is shown as a pie chart. Intergenic regions are defined as at least 3 kb away from the start and end of any transcript.
doi:10.1371/journal.pone.0026894.g001

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

Figure 2. Tat binds mainly to DNA repeat elements. (A) Distribution of Tat-binding peaks in repeat versus non-repeat elements is shown as a
pie chart. (B) Distribution of Tat-binding peaks within repeat elements in Alu versus non-Alu sequences is shown as a pie chart. (C) Percent of Tat-
binding peaks within individual repeat element subtypes is shown for the top 20 enriched elements.
doi:10.1371/journal.pone.0026894.g002

by Tat are Alu repeats (Figure 2B). In fact, the top ten repeat regions of the genome (Figure 3C). In comparison to global Tat
element types bound by Tat all belong to the Alu family of DNA binding patterns, Alu elements bound by Tat are more enriched
repeats (Figure 2C). These data reveal that a large fraction of Tat within introns (45% vs 53%). Essentially none of the Tat-bound
binding regions in the genome are within DNA repeat elements, Alu elements was located within coding exons. Altogether, our
especially the Alu sequences. data reveals that Tat binds specifically to a fraction of Alu elements
within the human genome. These Alu elements may be near or
Tat binding is enriched in the middle to the 39 end of Alu within the introns of genes with functions potentially related to the
elements viral life cycle such as the Alu element shown in Figure 3B.
Since more than one third of Tat-bound genomic regions
contain Alu elements, we sought to determine the binding profile Genomic regions bound by Tat are associated with genes
of Tat to Alu elements. As shown in Figure 3A, Alu elements have enriched in T cell-related functions
an average size of 300 base pairs (bp). We generated an average As the majority of genomic regions bound by Tat are at least
profile of Tat binding centered at the start of all Tat-bound Alu 3 kb away from any TSS, we asked if the nearest genes to Tat
elements. The profile of Tat enrichment over Alu elements was binding sites are enriched for specific functions. To observe long
generated by averaging P-values of Tat binding enrichment in range interactions between Tat binding sites and their target
100-bp windows 61 kb from the start of Alu elements. Tat genes, we used the Genomic Regions Enrichment Annotations
binding on average peaked in the middle of Alu elements with a Tool (GREAT) [15] to determine whether genomic regions bound
skewed enrichment toward the 39 end and downstream regions, up by Tat are located in potential cis-regulatory regions of genes
to 200 bp past the average length of an Alu element (Figure 3A). important for HIV function. GREAT analysis of Tat-bound
Figure 3B shows an example of Tat binding at an Alu element in regions revealed that the genes potentially associated with distal
an intergenic region of chromosome 10 as indicated. We then Tat binding are mostly 5 kb or further away from Tat binding
asked whether Tat-bound Alu-elements are enriched at specific sites. This distribution implies that there may be long range
genomic regions using the CEAS software [11]. The Tat-bound interactions between Tat and its potential target genes (Figure 4A).
Alu elements are primarily located in introns and intergenic In support of this, genes associated with regions bound by Tat are

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

Figure 3. Analysis of Tat binding to Alu elements. (A) Average P-value of Tat-binding profile centered at the start of Alu elements for all Tat-
bound Alu elements is shown. The x axis depicts 61 kb away from start of Tat-bound Alu elements in 100-bp intervals. The y axis denotes 2log10 of
Tat-binding enrichment P-value of ChIPed Tat DNA count over input DNA count. (B) Genome browser representation of Tat enrichment profile
compared to the input DNA at a representative Alu element is shown. The peak height corresponds to read counts. (C) Distribution of Tat-bound Alu
elements with respect to gene structure is shown as a pie chart.
doi:10.1371/journal.pone.0026894.g003

significantly enriched in Mouse Genome Informatics (MGI) (Figure 4C). These data suggest that Tat may exert its effects on
Phenotype ontology terms related to T cell function (Figures 4B the host genes by binding to distant cis-regulatory elements.
and 4C). MGI analyzes the knockout phenotypes of mouse genes
that are homologous to the queried human genes. The mouse Global gene expression changes in Jurkat-Tat cells
homologues of the potentially Tat-regulated genes exhibit To relate global gene expression changes to Tat binding, we
knockout phenotypes such as changes in T cell morphology and used Agilent microarrays to compare global gene expression
reduced number of CD4+ and CD8+ T cells (Figure 4B). Similar between Jurkat and Jurkat-Tat cells. Overall, 475 and 319
gene ontology terms were observed when only genes associated transcripts showed greater than two-fold increase and decrease
with Tat-bound Alu elements were used for MGI analysis in gene expression, respectively. To investigate the functions of the

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

Figure 4. Functional annotation of Tat binding regions. (A) The bar chart shows the distribution of the distances between Tat binding peaks
and transcriptional start sites (TSS). Note that a Tat peak may be assigned to more than one gene. Functional annotation of (B) Tat-bound regions and
(C) Tat-bound Alu elements, using gene ontology terms generated by Mouse Genome Informatics (MGI) from mouse homologous gene knock out
phenotypes. The x-axis values are 2log10 of binomial raw P-values.
doi:10.1371/journal.pone.0026894.g004

deregulated genes and their relevance to Tat over expression, we associations to correlate Tat binding to gene expression.
performed Gene Ontology (GO) enrichment analysis using Surprisingly, we found no significant correlation. Additionally,
DAVID [16]. Genes overexpressed in Jurkat-Tat cells are we performed ChIP combined with Agilent promoter microarrays
enriched in cell immune response, cell adhesion, and regulation (ChIP-chip) to monitor changes in histone H3 lysine 9 acetylation
of cell death (Figure 5A). However, for the down-regulated genes, (H3K9ac)—a histone modification associated with gene activity—
no significant GO enrichment was found. To determine whether in Jurkat-Tat versus Jurkat cells. We found that changes in
Tat binding is associated with increased or decreased expression of H3K9ac correlated positively with gene expression changes;
its target genes, we used the GREAT assigned gene-peak promoter regions of gene that are up- and down-regulated in

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

Figure 5. Gene expression profile of Jurkat-Tat cells. (A) Gene ontology annotation of genes with more than two-fold change in expression
using DAVID. (B) Relationships of gene expression and H3K9ac changes to Tat-binding are shown. Genes are arranged in descending order based on
their expression in Jurkat-Tat versus Jurkat cells. Each row represents one gene. For H3K9ac ChIP-chip analysis, each column represents a 500-bp
window, spanning 25.5 to +2.5 kb of annotated TSS. Moving average of H3K9ac enrichment in three consecutive 500-bp windows is shown in each
column (right panel).
doi:10.1371/journal.pone.0026894.g005

Jurkat-Tat cells have higher and lower levels of H3K9ac, reads using P-value of 1024. To get more similar numbers of total
respectively, compared to Jurkat cells (Figure 5B). These data significant peaks, P-value of 1022 was used to determine significant
indicate that changes in gene expression are associated with similar reads for RUNX1. Significant peaks found in Jurkat T cells were
changes in histone acetylation but Tat may have more subtle then compared to Tat binding sites in Jurkat-Tat cells. We defined
effects on gene expression that is not detected by our microarray positive co-occupancy of each factor with Tat when there was at
analysis. least one significant peak of binding within 6500 bp of the Tat-
binding sites. Figure 6A shows the fraction of all (2074) Tat
Tat in Jurkat-Tat cells binds to locations occupied by CBP binding sites that are occupied by the indicated factors in Jurkat
and ETS1 cells. Only ,12% of all Tat binding sites coincide with H3K4me3
and/or H3K27me3 and even less so with CBP or ETS1. However,
To determine if Tat binding sites are associated with specific
when the analysis is limited to the Tat binding sites within 61 kb of
chromatin marks, cellular transcription factors or co-factors, we
TSS regions (162 sites), 55% and 42% of Tat peaks are bound by
searched the Gene Expression Omnibus (GEO)[17] for published
ETS1 and CBP, respectively, in Jurkat cells; 35% are co-occupied
ChIP-seq data in Jurkat or Jurkat-Tat cells. We found five datasets
by both factors (Figure 6B). Only 1% of Tat binding sites near TSS
that examined global distributions of ETS1, CBP, RUNX1, H3K4
overlap with RUNX1 binding in Jurkat cells with P-value of 1022
tri-methylation (H3K4me3) and H3K27me3 in Jurkat cells
and 1024. Furthermore, 28% of Tat TSS binding sites are enriched
(GSE23080, GSE17954 [18]). No published dataset in Jurkat-
for H3K4me3 and H3K27me3 in Jurkat cells, including 58% of
Tat cells was found. ETS1 is a transcription factor that is highly
binding sites with both ETS1 and CBP bound near the TSS
expressed in lymphoid lineage cells and is important for regulating
(Figure 6B). Figure 6C shows an example of such genes. The
functions of immune cells [19]. In mouse models, inactivation of
promoter of ZNF143 gene is co-occupied by ETS1 and CBP and
ETS1 leads to T cell apoptosis [20]. ETS1 and Tat were enriched for H3K4me3 and H3K27me3 in Jurkat cells. The same
previously shown to bind at the same region upstream of IL-10 region is bound by Tat in Jurkat-Tat cells. In contrast, no significant
promoter and induce IL-10 transcription [21]. P300 and CBP co-occupancy of Tat in Jurkat-Tat and RUNX1 in Jurkat cells was
HATs acetylate Tat and serve as co-activators of Tat-dependent found near TSS. Although the occupancy of these factors in Jurkat-
HIV-1 gene expression [3]. RUNX1 is a member of Runt-related Tat cells is not known, our analysis raises the possibility that specific
transcription factor (RUNX) family of genes that function in cellular transcription factors may help recruit Tat or denote the
normal hematopoiesis. H3K4me3 and H3K27me3 are histone genomic regions to which Tat binds.
modifications that are generally associated with gene activity and
repression, respectively [22].
Discussion
We downloaded ChIP-seq raw data from GEO [17] and
compared each ChIP channel to its corresponding input DNA We have generated a global map of Tat binding to the human
from the same experimental set. Using the same peak finding genome. Surprisingly, the majority of Tat target regions lie within
algorithm described above with a P-value cutoff of ,1024, we DNA repeat elements. In fact, over 30% of all Tat target regions
defined significant peaks of CBP, ETS1, H3K4me3, H3K27me3 are located at or near Alu elements. Interestingly, Tat increases the
in Jurkat T cells. For RUNX1, we found much less significant transcription of Alu repeat elements by increasing the activity of

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

Figure 6. Tat binding peaks in Jurkat-Tat cells are associated with specific cellular factors and chromatin marks in Jurkat cells. (A)
Percent of all Tat peaks in Jurkat-Tat cells that are bound by CBP, ETS1, RUNX1, H3K4me3 and/or H3K27me3 in Jurkat cells is shown. (B) Percent of Tat
peaks at TSS in Jurkat-Tat cells that are bound by CBP, ETS1, RUNX1, H3K4me3 and H3K27me3 in Jurkat cells is shown. (C) Shown is the Tat
enrichment profile at the ZNF143 gene promoter in Jurkat-Tat cells compared to CBP, ETS1, RUNX1, H3K4me3, H3K27me3 enrichment at the same
genomic location in Jurkat cells. The peak heights (y-axis) correspond to read counts. The x-axis represents the genomic coordinates.
doi:10.1371/journal.pone.0026894.g006

cellular transcription factor TFIIIC in Jurkat cells [23]. Since Alu as IL-6 [4] and ß2 microglobulin [6], showed some Tat binding
elements antagonize the interferon-induced protein kinase R above background but did not pass our criteria for statistical
(PKR) activation [24] and PKR is known to repress protein significance. Also, previous studies used different cell culture systems
synthesis when cells are under stress, Tat binding at Alu elements to determine Tat binding in vivo. These differences may also lead to
may be important to enable efficient viral replication in the host identification of different sets of target genes.
cell. In addition, these Alu elements may affect regulation of genes Since Tat is not known to bind DNA directly, the mechanism
with functions related to HIV-1 biology. by which Tat binds to specific regions of the genome may partly
In relation to gene regulation, Tat binding sites are distal to involve interactions with host cellular factors. By comparing our
genes with functions in T cell biology as determined by knockout Tat binding data to the published datasets in Jurkat cells, we found
models in mice. These data suggest that Tat may exert its effects that Tat binds to gene promoters that were bound by ETS1 and
on its target genes through distant regulatory elements. If so, then CBP, but not with RUNX1, in non-Tat expressing Jurkat cells. A
Tat binding, in and of itself, may highlight previously unknown cis- similar positive relationship was found between Tat binding and
regulatory elements within the genome. We did not find a two histone methylation marks, H3K4me3 and H3K27me3.
significant correlation between Tat binding and gene expression. These data raise the possibility that Tat may distinguish its target
The effects of Tat on gene expression may be too subtle for the regions through specific host transcription co-factors or chromatin
microarrays to detect. Alternatively, Tat may affect the regulation marks. However, our data does not include or exclude ETS1, CBP
of its target genes through effects on mRNA structure that is not or the histone modifications as the mediators of Tat recruitment.
evident by expression analysis. Binding analyses of specific Tat mutants that disrupt its interaction
We found only a few genes with Tat binding to the vicinity of with specific cellular factors are required to determine potential
their TSS. This is partly due to the stringent cutoff used in this study mechanisms of Tat recruitment. It is also possible that Tat binds to
to maintain a False Discovery Rate (FDR) under 5%. For instance, the genome through an RNA component. Nonetheless, our results
gene promoters previously shown to be bound by Tat binding such demonstrate that in addition to known roles for Tat in enhancing

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 Genome-Wide Map of HIV-1 Tat to the Human Genome

elongation of viral transcription, Tat also binds to the host genome ChIP-seq data analysis
at specific genomic locations with potentially important conse- Sequenced reads were base-called using the standard Illumina
quences for the viral life cycle. software. Bowtie 0.12.7 was used to align the reads to the Human
genome (Hg19) allowing up to two mismatches; reads that aligned
Materials and Methods to more than one location in the genome were discarded. For
unaligned reads, 5 bp from the 59 end and 25 bp from the 39 end
Cell culture of the reads were trimmed and re-aligned to the genome. The total
Jurkat T cells were obtained from ATCC (TIB-15) and cultured number of reads in the input sample was normalized to the ChIP
under standard tissue culture conditions. Jurkat-Tat cells were counts. Genomic regions with significant enrichment of Tat
obtained from the NIH AIDS Research & Reference Reagent chromatin over input chromatin were calculated within 100 bp
Program and maintained in RPMI supplemented with 10% fetal windows that tiled the genome. The P-value for enrichment of
bovine serum, 1% penicillin and streptomycin and 800 mg/ml of ChIP versus input reads was calculated using the cumulative
G418. Poisson distribution.

Western blotting Expression profiling

Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 8.0, Total RNA was isolated from Jurkat and Jurkat-Tat cells using
150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate) the RNeasy Mini kit (Qiagen). cRNAs were generated from
supplemented with protease inhibitors (Roche). Cell lysates were 250 ng of total RNA and labeled with Cy3 (Jurkat) or Cy5 (Jurkat-
subjected to SDS-PAGE and analyzed by Western blot using Tat) using the Low Input Quick Amp Labeling Kit (Agilent)
standard procedures. The antibodies used for Western blotting according to manufacturer’s instructions. Labeled cRNAs were
were as follows: Anti-Tat (Abcam ab43014), Anti-Beta-actin hybridized to the Agilent Human whole-genome array (G2534-
(Abcam ab8224), Anti-H3 (Abcam ab10799). 600110) according to Agilent protocol. Raw intensity data from
resulting gene expression data were normalized by medium
Chromatin immunoprecipitation and ChIP-seq library background-subtracted intensities between Cy5 and Cy3 channels
preparation followed by LOWESS normalization using Matlab. Normalized
Jurkat-Tat cells in exponential growth phase were fixed with 1% log2 ratios of Jurkat-Tat cRNAs (Cy5) over Jurkat cRNAs (Cy3)
formaldehyde (v/v) for 10 min at 37uC. Fixation was stopped by were calculated and results from replicates were averaged. Gene
addition of glycine to a final concentration of 140 mM. Cell were ontology enrichment analysis was performed using The Database
lysed and chromatin was digested with the micrococcal nuclease for Annotation, Visualization and Integrated Discovery (DAVID)
from S. aureus (Roche) for 90 min at 4uC according to the v6.7 [16].
manufacturer’s instructions, re-suspended in lysis buffer and All data have been deposited in the Gene Expression Omnibus
sonicated with Misonix ultrasonic liquid processor. 1% of the (GEO) [17] under accession number GSE30739.
lysate was used as an input control. Lysates were immunoprecip-
itated with 5 mg of anti-Tat antibodies (Abcam ab43014) using the
Supporting Information
standard ChIP protocol. Both purified input and Tat chromatin
samples were used to prepare ChIP-seq libraries according to the Figure S1 Tat is expressed in Jurkat-Tat cells. Western
manufacturer’s instructions (Illumina). Libraries were sequenced blots of the indicated factors in normal Jurkat T cells (Jurkat) and
using Illumina Genome Analyser II to obtain 76 bp-long reads. Jurkat cells stably expressing Tat (Jurkat-Tat).
(TIF)
Chromatin immunoprecipitation and microarray
hybridization Author Contributions
Jurkat-Tat and Jurkat cells in exponential growth phase were Conceived and designed the experiments: CM OR SKK. Performed the
fixed with 1% formaldehyde (v/v) for 10 min at 37uC. Fixation experiments: CM. Analyzed the data: TS CM RF BL. Contributed
was stopped by addition of glycine to a final concentration of reagents/materials/analysis tools: MP DV JAZ. Wrote the paper: TS CM
140 mM. Histone H3 acetyl lysine 9 antibody (Upstate 07-352) SKK.
was used for ChIP. The ChIP-chip and subsequent analysis were
performed essentially as described previously [25].

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PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e26894