Cell Host & Microbe
The Cellular Lysine Methyltransferase Set7/9-KMT7
Binds HIV-1 TAR RNA, Monomethylates the Viral
Transactivator Tat, and Enhances HIV Transcription
Sara Pagans,1,2Steven E. Kauder,1,2Katrin Kaehlcke,1Naoki Sakane,1,3Sebastian Schroeder,1Wilma Dormeyer,4
Raymond C. Trievel,5Eric Verdin,1,2Martina Schnolzer,4and Melanie Ott1,2,*
1Gladstone Institute of Virology and Immunology
2UCSF Department of Medicine
University of California, San Francisco, San Francisco, CA 94158, USA
3Pharmaceutical Frontier Research Laboratory, Japan Tobacco, 1-13-2 Fukuura, Kanagawa-ku, Yokohama, Kanagawa 236-0004, Japan
4Functional Proteome Analysis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
5Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
The Tat protein of HIV-1 plays an essential role in HIV
gene expression by promoting efficient elongation
of viral transcripts. Posttranslational modifications
of Tat fine-tune interactions of Tat with cellular
cofactors and TAR RNA, a stem-loop structure at
the 50ends of viral transcripts. Here, we identify the
lysine methyltransferase Set7/9 (KMT7) as a coacti-
vator of HIV transcription. Set7/9-KMT7 associates
with the HIV promoter in vivo and monomethylates
lysine 51, a highly conserved residue located in the
RNA-binding domain of Tat. Knockdown of Set7/
9-KMT7 suppresses Tat transactivation of the HIV
promoter, but does not affect the transcriptional
activity of methylation-deficient Tat (K51A). Set7/
9-KMT7 binds TAR RNA by itself and in complex
with Tat and the positive transcription elongation
factor P-TEFb. Our findings uncover a positive role
for Set7/9-KMT7 and Tat methylation during early
steps of the Tat transactivation cycle.
tivator Tat is absent, cellular RNA polymerase II assembles and
initiates properly at the HIV promoter, but production of full-
length viral transcripts is blocked at the elongation step (Kao
et al., 1987; Toohey and Jones, 1989). When Tat is produced
tional elongation becomes highly effective, resulting in the
production of full-length viral transcripts necessary to sustain
high-level viral replication throughout infection.
Tat binds to a specific RNA stem-loop structure called TAR
that forms spontaneously at the 50ends of nascent viral tran-
scripts (Barboric and Peterlin, 2005). Tat binding to TAR RNA
involves a highly conserved arginine-rich motif (ARM), located
between aa 49 and 57 in Tat, and the positive transcription elon-
gation factor b (P-TEFb). Tat and the Cyclin T1 component of
P-TEFb bind TAR RNA cooperatively and induce phosphoryla-
tion of the C-terminal domain of RNApolymerase II bythe cyclin-
associated kinase CDK9, a critical step to improve the elonga-
tion competence of the polymerase complex (Herrmann and
Rice, 1993; Kim et al., 2002; Wei et al., 1998; Zhu et al., 1997).
Tat also recruits the SWI/SNF chromatin remodeling complex
to the HIV promoter (Agbottah et al., 2006; Ariumi et al., 2006;
Mahmoudi et al., 2006; Tre ´and et al., 2006) and interacts with
several histone-modifying enzymes, including histone acetyl-
transferases p300/CBP (renamed KAT3B/KAT3A), p300/CBP-
associated factor (PCAF)/human GCN5 (KAT2B/KAT2A), TAF1
(former TAFII250), and Tip60 (KAT5) (Benkirane et al., 1998;
Col et al., 2001; Creaven et al., 1999; Deng et al., 2000; Hottiger
and Nabel, 1998; Weissman et al., 1998). While it was originally
believed that interactions with histone-modifying enzymes serve
to relieve the elongation block imposed by the nucleosomal
organization of the HIV promoter (Verdin et al., 1993), growing
evidence shows that Tat itself is a target. Tat is acetylated by
p300-KAT3B, human GCN5-KAT2A, and PCAF-KAT2B; deace-
tylated by the NAD+-dependent deacetylase SIRT1; ubiquiti-
nated by the E3 ubiquitin ligase Hdm2; and methylated by the
protein arginine methyltransferase PRMT6 and the lysine meth-
yltransferase SETDB1-KMT1E (Boulanger et al., 2005; Bre `s
et al., 2003; Col et al., 2001; Kiernan et al., 1999; Ott et al.,
1999; Pagans et al., 2005; Van Duyne et al., 2008).
Known Tat modifications can either positively (acetylation/
tional activity by regulating interactions of Tat with TAR RNA and
Cyclin T1. Acetylation of Tat also generates a specific interaction
domain with the PCAF bromodomain (Bre `s et al., 2002; Dorr
Brg-1 subunit of the SWI/SNF chromatin-remodeling complex
(Mahmoudi et al., 2006). Tat ubiquitination also enhances Tat
transactivation, although the molecular mechanism associated
to this function is not yet known (Bre `s et al., 2003).
Set7/9-KMT7, a lysine monomethyltransferase, was originally
identified as an enzyme that modifies K4 in histone H3 (Nishioka
et al., 2002; Wang et al., 2001), but was later found to methylate
nonhistone proteins. Its known substrates include the TAF10
component of the TFIID complex, the tumor suppressor p53,
234 Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc.
and estrogen receptor a, all of which are positively influenced
by Set7/9-KMT7-mediated modifications (Chuikov et al., 2004;
Ivanov et al., 2007; Kouskouti et al., 2004; Kurash et al., 2008;
Subramanian et al., 2008). However, methylation of DNA methyl-
transferase 1 (DNMT1) was found to destabilize and inactivate
this factor (Este `ve et al., 2009; Wang et al., 2009). The p65
subunit of NF-kB is also methylated by Set7/9-KMT7, although
different methylation sites and different functions associated to
Set7/9-KMT7-mediated methylation of p65 have been reported
(Ea and Baltimore, 2009; Yang et al., 2009). Notably, Set7/
9-KMT7 acts as an important coactivator of NF-kB-dependent
gene expression in monocytes and pancreatic b cells and
plays a critical role in the regulation of p65 expression during
hyperglycemia (Brasacchio et al., 2009; Deering et al., 2009;
El-Osta et al., 2008; Li et al., 2008). PCAF-KAT2B was recently
identified as a substrate of Set7/9-KMT7, but the function of
PCAF-KAT2B methylation remains unknown (Masatsugu and
The emerging importance of Set7/9-KMT7 as a central regu-
prompted us to investigate whether the enzyme also plays a
role in HIV transcription. Our data identify Tat as a substrate of
Set7/9-KMT7 and demonstrate that monomethylation by Set7/
9-KMT7 defines a positive step in the Tat transactivation cycle.
Set7/9-KMT7 Monomethylates Tat at K51
To test whether Tat is methylated by Set7/9-KMT7, full-length
synthetic Tat protein (aa 1–72) was incubated with recombinant
Set7/9-KMT7 enzyme and radiolabeled S-adenosyl-L-methio-
nine (SAM). Reactions were resolved by gel electrophoresis
and developed by autoradiography. Tat was methylated in
response to increasing amounts of Set7/9-KMT7 (Figure 1A).
No spontaneous methylation was observed with SAM alone.
substrate of Set7/9-KMT7. In contrast, methylation by Set7/
9-KMT7 was not observed with recombinant IkBa (Figure 1A).
The lysine methyltransferase G9a did not methylate Tat, but
methylated histones as reported (Figure 1B) (Tachibana et al.,
Figure 1. In Vitro Methylation of Tat by Set7/9-KMT7
(A) Synthetic Tat (72 aa), histones, or recombinant GST-IkBa proteins were incubated with3H-radiolabeled S-adenosyl-L-methionine (SAM) and increasing
amounts of recombinant Set7/9-KMT7 (0, 0.5, 1, or 2 mg). Tat and histone H3 methylation were visualized by autoradiography (top panels).
(B) Radioactive in vitro methylation reactions with synthetic Tat or histones and recombinant G9a enzyme (0, 1, or 2 mg).
(C) In vitro methylation assays performed with short Tat peptides, recombinant Set7/9-KMT7, and3H-SAM. Peptides were separated on Tris-Tricine gels and
visualized by autoradiography.
(D) In vitro methylation assays of ARM peptides (aa 45–58), containing either two lysines (WT) or alanine substitutions at positions K50 and K51.
(E) MALDI TOF mass spectrometry of nonradioactive methylation reactions performed with the WT, K50A, or K51A ARM peptides. Peptides were incubated with
Set7/9-KMT7 and SAM, SAM alone, or only the reaction buffer. (See also Figure S1.)
Cell Host & Microbe
Monomethylation of Tat at K51
Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc. 235
2001). These results show that Tat is a specific in vitro substrate
To map the site of methylation in Tat, we generated short
synthetic Tatpeptides andsubjected themtoin vitro methylation
assays. Methylation by Set7/9-KMT7 was observed with one
peptide (aa 45–58), corresponding to the Tat ARM (Figure 1C).
Analysis by MALDI TOF mass spectrometry showed that the
Tat ARM is monomethylated by Set7/9-KMT7: the molecular
mass of the peptide increased by 14 Da, corresponding to the
addition of a single methyl group (Figures 1E and S1A). Mass
spectrometry further revealed that recombinant Set7/9-KMT7
methylates the Tat ARM without addition of exogenous SAM,
possibly by utilizing SAM copurified from E. coli (Figure S1B).
The Tat ARM contains two lysines, K50 and K51. Both resi-
dues are strictly conserved among HIV-1 isolates. To determine
which lysine is methylated by Set7/9-KMT7, we generated
ARM peptides containing alanine substitutions at positions
K50, K51, or both. Methylation by Set7/9-KMT7 was abrogated
when K51 or both lysines were mutated while mutation of K50
alone had no effect, indicating that K51 is a target of Set7/
9-KMT7 in the Tat ARM (Figures 1D and 1E).
Tat Is Monomethylated at K51 In Vivo
We then examined Tat methylation in cells. Rabbits were immu-
nized with synthetic peptides corresponding to the monomethy-
lated Tat ARM. The resulting antiserum (a-meARM) specifically
recognized ARM peptides carrying a monomethyl group at
K51, while no cross-reactivities with unmodified, di-, or trimethy-
lated ARM peptides were observed (Figure 2A). The antiserum
also recognized full-length synthetic Tat (aa 1–72) after in vitro
methylation by Set7/9-KMT7, but did not react with the unmodi-
fied protein (Figure 2B). ARM peptide and Tat levels in these
reactions were visualized by immunoblotting with streptavidin-
horseradish peroxidase conjugate (SA-HRP) that recognized
the biotin label attached to the N terminus of the peptides
(Figures 2A and 2B).
Next, we examined 293 cells transfected with expression
vectors for Tat. We expressed WT or mutant Tat proteins in
which K51 had been changed to alanine (K51A). Tat proteins
were isolated from cell lysates via their FLAG epitope tags and
examined by western blotting with a-meARM antibodies. Tat
methylation was detected in samples expressing WT but not
mutant Tat, demonstrating that Tat is methylated at K51 in cells
(Figure 2C). Expression of WT and mutant Tat proteins was
equivalent, as confirmed by western blotting with a-FLAG anti-
bodies (Figure 2C).
When we coexpressed Set7/9-KMT7 with Tat, Tat methylation
increased, indicating that Set7/9-KMT7 can methylate Tat
in cells (Figure 2D). Recognition of Tat was blocked when
a-meARM antibodies were preincubated with monomethylated
ARM peptides before western blotting. No effect was observed
after incubation with unmodified ARM peptides, excluding
cross-reactivity of theantiserum with unmodified Tat (Figure2D).
To examine whether Tat is also monomethylated in CD4+
T cells, a natural host cell of HIV-1, we used the Jurkat-derived
A2 cell line. A2 cells harbor a latent minimal HIV-1 genome
Figure 2. Tat Is Monomethylated at K51 in Cells
(A) Dot blot analysis of biotinylated ARM peptides, either unmodified, mono-, di-, or trimethylated at K51 with a-meARM antibodies or SA-HRP.
(B)WesternblotanalysisofinvitromethylationreactionswithbiotinylatedsyntheticTat(12.5,25,50, and100ng),recombinant Set7/9-KMT7,andnonradioactive
SAM with a-meARM antibodies or SA-HRP.
(C) Immunoprecipitations of WT or mutant K51A Tat/FLAG in 293 cells, followed by western blotting with a-meARM or a-FLAG antibodies.
(D) Immunoprecipitation/western blot analysis of Tat/FLAG coexpressed with Set7/9-KMT7 in 293 cells. The a-meARM antibodies were preincubated with milk,
a 10 3 molar excess of K51-monomethylated ARM peptide, or a 10 3 molar excess of nonmodified ARM peptide.
(E) Immunoprecipitation of Tat/FLAG in Jurkat A2 cells treated with TNF-a followed by western blotting with a-meARM or a-FLAG antibodies.
Cell Host & Microbe
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236 Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc.
that, upon stimulation with phorbol 12-myristate-13-acetate
(PMA) or TNF-a, expresses Tat and enhanced green fluorescent
protein (GFP) from the HIV long terminal repeat (LTR) (Jordan
et al., 2003). Methylated Tat was detected in TNF-a-stimulated
A2 cell lysates after immunoprecipitation with a-FLAG anti-
bodies, demonstrating that Tat is methylated under conditions
that mimic physiological conditions of infection (Figure 2E).
Set7/9-KMT7 Activates HIV Transcription
To investigate the biological role of Set7/9-KMT7 during HIV
transcription, we transfected HeLa cells with an HIV LTR
luciferase construct and expression vectors for Tat and Set7/
9-KMT7. WT but not catalytically inactive Set7/9-KMT7
(H297A) synergized with Tat in the transactivation of the HIV
LTR (Figure 3A). Set7/9-KMT7 overexpression also activated
HIV LTR activity by ?2-fold in the absence of Tat. In parallel,
we performed reporter assays with a luciferase construct con-
taining the elongation factor 1 a (EF-1a) promoter, which was
driving Tat expression in these experiments (Figure 3A). No
effect of Set7/9-KMT7 overexpression was observed on the
transcriptional activity of the EF-1a promoter, excluding that
Set7/9-KMT7 activated Tat expression in these experiments.
We then introduced siRNA oligonucleotides specific for Set7/
9-KMT7 into HeLa cells to downregulate endogenous Set7/
9-KMT7 expression before transfection of the HIV LTR lucife-
rase reporter and Tat. Set7/9-KMT7 expression was efficiently
Figure 3. Set7/9-KMT7 Activates Tat Transactivation through K51 Methylation
vector and the HIV LTR luciferasereporter (200 ng). In parallel, cotransfections were performed withthe EF-1a-RL reporter (20 ng) and WT or catalytically inactive
Set7/9-KMT7 (H297A; 150 ng). Luciferase or Renilla values were analyzed 24 hr after transfections. The average of three independent experiments (mean ±SEM)
is shown; * corresponds to a p value <0.05 and ** to a p value <0.01 compared to cells transfected with vector control.
(B)siRNA-mediatedknockdown ofSet7/9-KMT7 inHeLacells.CellswerecotransfectedwiththeHIVLTRluciferaseconstruct(200ng)andincreasingamountsof
expression vectors for WT or mutant K51A Tat/FLAG (0, 10, 50, 250 ng) 48 hr after siRNA transfection. Measurements of luciferase activity and western blotting
were performed 24 hr after plasmid transfections. Luciferase values represent the average (mean ±SEM) of three experiments; * corresponds to a p value <0.05
and ** to a p value <0.01 compared to control cells expressing WT Tat.
(C) Transcriptional activity of TatK51A and TatK51R mutants in Set7/9-KMT7 knockdown cells. Experiment was performed as in (B) with 250 ng of WT or mutant
Tat/FLAG. Luciferase values represent the average (mean ±SEM) of three experiments. * corresponds to a p value <0.05 compared to WT Tat-transfected cells.
Cell Host & Microbe
Monomethylation of Tat at K51
Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc. 237
downregulated 72 hr after siRNA transfections (Figure 3B). At
this time, Tat transactivation of the HIV LTR was ?3-fold
reduced, confirming that cellular Set7/9-KMT7 expression is
necessary for full Tat transactivation (Figure 3B).
The transcriptional activity of mutant Tat (K51A) was ?3-fold
reduced as compared to WT Tat, and no further reduction was
observed after knockdown of Set7/9-KMT7 (Figure 3B). In addi-
tion, no effect of the siRNAs was observed when the HIV LTR
reporter was expressed in the absence of Tat, supporting the
model that endogenous Set7/9-KMT7 activates HIV transcrip-
tion through K51 methylation in Tat (Figure 3B). Similar results
were obtained with a Tat mutant that contained an arginine at
position 51 (TatK51R), excluding that the transcriptional defect
of the K51 mutant was caused by the change in charge instead
of the lack of methylation (Figure 3C). Of note, the addition of
a methyl group to a lysine residue does not neutralize the posi-
tive charge of this position. We verified by western blotting that
WT and mutant Tat proteins were expressed equally in these
experiments and that no difference in Tat expression was
observed when Set7/9-KMT7 expression was downregulated
(Figures 3B and 3C).
Set7/9-KMT7 Regulates Proviral Gene Expression
during HIV Infection
introduced Set7/9-KMT7 siRNAs into Jurkat A2 cells by Amaxa
nucleofection. To induce Tat and GFP expression, cells were
treated with PMA 3 days after siRNA transfection, when Set7/
9-KMT7 expression was downregulated by 80% (Figure 4A).
The rate of GFP+ cells was decreased by ?50% in Set7/
9-KMT7 knockdown cells, confirming a positive role of Set7/
9-KMT7 in HIV gene expression (Figure 4A).
We also examined the role of Set7/9-KMT7 in primary CD4+
T cells and generated lentiviral vectors expressing different
shRNAs against Set7/9-KMT7 (shSet7/9-1 and shSet7/9-2).
These vectors also express the mCherry protein under the
control of the EF-1a promoter, which allows for the identification
of successfully transduced cells (Grskovic et al., 2007; Ventura
et al., 2004). Both shRNAs efficiently suppressed Set7/9-KMT7
expression after infection of Jurkat cells followed by sorting of
mCherry+ cells (Figure 4B). They also caused a 40% reduction
of Set7/9-KMT7 expression in unsorted, primary CD4+ T cells
in which ?40% of the cells were mCherry+ (Figure S2). These
Figure 4. Set7/9-KMT7 Regulates HIV Gene Expression in the Context of Lentiviral Infection
(A) siRNA-mediated knockdown of Set7/9-KMT7 in latently infected Jurkat A2 cells. Three days after nucleofection of Set7/9-KMT7 or control siRNAs, western
blotting was performed, and cells were stimulated with PMA for 12 hr or were left unstimulated. GFP expression was measured by flow cytometry. The average
(mean ±SEM) of three independent experiments is shown, * corresponds to a p value <0.05 compared to control siRNA-transfected cells.
(B) shRNA-mediated knockdown of Set7/9-KMT7 in CD4+T cells. Primary CD4+ T cells isolated from uninfected blood donors were infected first with lentiviral
from the EF-1a promoter. GFP expression within shRNA-expressing cells (as marked by mCherry expression) is expressed relative to cells expressing nontar-
geting shRNA control. Average (mean ±SD) from triplicate experiments performed with three different donors (HIV GFP) or a single donor (HIV [EF-1a] GFP) is
shown. Western blotting was performed in Jurkat cells transduced with indicated shRNAs and sorted for mCherry expression. (See also Figure S2.)
Cell Host & Microbe
Monomethylation of Tat at K51
238 Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc.
unsorted, infected CD4+ T cells were subsequently infected with
a molecular clone of the viral isolate HIVNL4-3(HIV-R7/E?/GFP).
This virus contains the GFP open reading frame (ORF) in place of
nef, allowing identification of infected cells by flow cytometry,
and a frameshift mutation in the env gene, thereby restricting
infection to a single cycle (Jordan et al., 2003). Knockdown of
Set7/9-KMT7, as marked by mCherry expression, caused
a 40% (shSet7/9-1) and 60% (shSet7/9-2) reduction in GFP
expression as compared to cells expressing a nontargeting
shRNA control (Figure 4B). No effect of the Set7/9-KMT7
shRNAs was observed when cells were infected with an HIV-
based lentiviral vector expressing GFP from the EF-1a promoter
instead of the HIV LTR (Figure 4B). These data confirm that Set7/
9-KMT7 regulates proviral gene expression during HIV infection
in primary CD4+ T cells while other steps of the viral life cycle,
such as reverse transcription, nuclear import, and integration,
In Vivo Recruitment of Set7/9-KMT7 tothe HIV Promoter
To examine whether Set7/9-KMT7 is physically recruited to the
HIV promoter during active gene expression, chromatin was
prepared from Jurkat A2 cells treated with PMA or the solvent
control and was immunoprecipitated with antibodies directed
against endogenous Set7/9-KMT7. Real-time PCR analysis of
the immunoprecipitated material showed that Set7/9-KMT7,
while present at the uninduced HIV LTR at low concentrations,
was 5-fold enriched in response to PMA (Figure 5A). No asso-
ciation of Set7/9-KMT7 with the GFP ORF was observed in unin-
duced cells; a slight signal was detected in response to PMA
(Figure 5A). These data demonstrate that Set7/9-KMT7 asso-
ciates in vivo with the HIV promoter and is specifically enriched
during active transcription.
Since the enhancement of Set7/9-KMT7 recruitment to the
HIV LTR coincides with Tat expression, we tested whether Tat
and Set7/9-KMT7 interact. Tat expression was induced in A2
cells after treatment with TNF-a, and cellular lysates were
subjected to immunoprecipitation with Set7/9-KMT7 antibodies.
Tat was detected in the immunoprecipitated material by western
blotting with FLAG antibodies, demonstrating that Tat binds
endogenous Set7/9-KMT7 expressed in T cells (Figure 5B).
This interaction was independent of the methylation status of
K51, since the K51A mutant of Tat coimmunoprecipitated with
endogenous Set7/9-KMT7 as efficiently as WT Tat in transfected
293 cells (Figure 5C). In vitro binding reactions of recombinant
Set7/9-KMT7 with synthetic Tat protein demonstrated that
Set7/9 and Tat may bind directly (Figure S3).
Set7/9-KMT7 Binds TAR RNA
K51 lies in the RNA-recognition motif (RRM) of Tat and forms cri-
tical interactions with TAR RNA (Anand et al., 2008). To examine
how methylation by Set7/9-KMT7 influences the interaction
between Tat and TAR RNA, we performed gel mobility shift
labeled TAR RNA as a probe. Surprisingly, addition of Set7/
9-KMT7 alone efficiently shifted the TAR RNA probe, pointing
to intrinsic RNA-binding properties of Set7/9-KMT7 (Figure 6A).
Binding of Set7/9-KMT7 to TAR RNA was successfully com-
peted with unlabeled TAR RNA and, like Tat, required the bulge
region of the TAR stem (Figures 6A and S4A). We excluded the
possibility that the shift was caused by a contamination of
the Set7/9-KMT7 preparation with synthetic Tat and repeated
the experiments with several different Set7/9-KMT7 prepara-
tions (data not shown). We also successfully supershifted the
complex with a-Set7/9-KMT7 antibodies, confirming that it
was indeed a complex formed by Set7/9-KMT7 and TAR (Fig-
ure 6B). No shift was observed when a truncated Set7/9-KMT7
(aa 110–366) was incubated with TAR RNA, indicating that the
TAR-binding domain is located in the N terminus of the enzyme,
outside the catalytic Set domain (Figure 6C). Preparation proce-
dures for full-length and N-terminally truncated Set7/9-KMT7
Figure 5. In Vivo Recruitment of Set7/9-
KMT7 to the HIV LTR
(A) Chromatin immunoprecipitation analysis of
Set7/9-KMT7 in Jurkat A2 cells treated with PMA
for 4 hr or left unstimulated. Real-time PCR was
used to quantify the enrichment of indicated
a-Set7/9-KMT7 antibodies. Quantities of immuno-
precipitated DNA were normalized to input DNA
and expressed relative to the b-actin control.
Three independent experiments were performed
PCR reactions of one experiment is shown.
(B) CoIP of Tat/FLAG and endogenous Set7/
9-KMT7 in Jurkat A2 cells treated with TNF-a.
Cellular lysates were immunoprecipitated with
a-Set7/9-KMT7 antibodies followed by western
blotting with a-Set7/9-KMT7 or a-FLAG anti-
(C) CoIP of WT and K51A mutant Tat with endo-
tations were performed with a-Set7/9-KMT7 anti-
bodies or agarose beads alone and analyzed by
western blotting with a-Set7/9-KMT7 or a-FLAG
antibodies. (See also Figure S3.)
Cell Host & Microbe
Monomethylation of Tat at K51
Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc. 239
proteins were identical, and integrity of both proteins was veri-
fied by Coomassie staining (data not shown).
Interestingly, the main complex formed by Set7/9-KMT7 and
TAR RNA showed the same mobility as the Tat-TAR RNA
complex despite different molecular weights of Tat (72 aa) and
Set7/9-KMT7 (366 aa) (compare Figure 6D, lanes 4 and 5).
Combination of Tat and Set7/9-KMT7 did not result in the forma-
tion of a slower migrating complex, although in general, forma-
tion of an additional, slightly slower migrating complex was
observed in the presence of Set7/9-KMT7 independently from
Tat (Figures 6A–6D).
Set7/9-KMT7 Enhances Tat Binding to TAR
We observed a synergistic increase in TAR binding when
a suboptimal Tat concentration was combined with Set7/
9-KMT7 (Figure 6D, compare lanes 7 and 3). This increase in
binding was obvious when a loop mutant TAR RNA probe
was examined. Intact loop sequences of TAR RNA are required
for the formation of the trimolecular complex of Tat-TAR-P-
TEFb (Wei et al., 1998). Tat alone efficiently interacted with
loop mutant TAR RNA as expected (Figure 6D, lanes 17–20).
In contrast, Set7/9-KMT7 did not bind the loop mutant
probe, demonstrating that, unlike Tat, it requires intact bulge
Figure 6. Set7/9-KMT7 Binds TAR RNA and Tat/P-TEFb
(A) Gel shift assays of recombinant Set7/9-KMT7 (0, 50, 150, and 500 ng) and radiolabeled WT or bulge mutant (Dbulge) TAR RNA probes. A 10 3 excess of
nonradiolabeled TAR RNA was included to compete for binding with the radiolabeled probe. (See also Figure S4A.)
(B) Supershift assays with a-Set7/9-KMT7 antibodies (7 mg) or control rabbit IgGs in reactions containing Set7/9-KMT7 (1 mg) and radiolabeled WT TAR RNA
(C) Gel shift assays with full-length or N-terminally truncated Set7/9-KMT7 (0, 0.1, 0.3, 1 mg) and radiolabeled WT TAR RNA probe.
(D) Tat and P-TEFb binding to TAR in the presence of Set7/9-KMT7. Radiolabeled TAR RNA probes (WT or Dloop) were incubated with synthetic Tat protein
(0, 2.5, 10, and 40 ng) in the absence or presence of recombinant Set7/9-KMT7 (500 ng). The same reactions were performed in the presence of recombinant
Cyclin T1/CDK9 (400 ng).
immunoprecipitated with a-Set7/9-KMT7 antibodies followed by western blotting with a-Set7/9-KMT7, a-FLAG, and a-Cyclin T1 antibodies. (See also
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Monomethylation of Tat at K51
240 Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc.
and loop sequences of TAR for efficient binding (Figure 6D,
lane 21). However, Tat binding to TAR RNA was increased
in the presence of Set7/9-KMT7, supporting a model where
Set7/9-KMT7 enhances the RNA-binding properties of Tat
independently of its physical interaction with TAR RNA (Fig-
ure 6D, lanes 22–24).
Set7/9-KMT7 Forms a Complex with Tat and Cyclin T1
Because in vivo Tat binding to TAR occurs in the presence of
P-TEFb, we also performed gel shift experiments with recombi-
nant P-TEFb (Cyclin T1/CDK9). The Cyclin T1 subunit interacts
with the transactivating domain in Tat and the loop region of
TAR and triggers formation of a large TAR ribonucleoprotein
complex (Figure 6D, lanes 10–12). Formation of this large com-
plex was slightly enhanced when Set7/9-KMT7 was added to
the gel shift reactions (Figure 6D, lanes 14–16) and depended
on intact loop sequences in TAR, irrespective of whether Set7/
9-KMT7 was added to the reaction or not (Figure 6D, lanes
26–28 and lanes 30–32).
Addition of P-TEFb did not affect the complex formed by TAR
RNA and Set7/9-KMT7 in the absence of Tat, indicating that any
engagement of Set7/9-KMT7 in the Tat-TAR-P-TEFb complex
depended on Tat (Figure 6D, lane 13). Because of the large
size of the Tat-TAR-P-TEFb complex, it was difficult to assess
whether migration of the complex changed in the presence of
Set7/9-KMT7 or a-Set7/9-KMT7 antibodies (Figure 6D and
data not shown). We therefore performed coimmunoprecipita-
tion (coIP) experiments to examine whether Set7/9-KMT7 phys-
ically engages in complex formation with Tat and Cyclin T1.
Endogenous Set7/9-KMT7 was immunoprecipitated in 293 cells
expressing HA-tagged Cyclin T1. Cyclin T1 only coimmunopre-
strating that Set7/9-KMT7 cooperatively binds Tat and Cyclin T1
(Figure 6E). Accordingly, Set7/9 expressed in murine cells lack-
ing a Cyclin T1 protein that can interact with Tat only synergized
with Tat when human Cyclin T1 was coexpressed (Figure S4B).
Figure 7. Model of Set7/9-KMT7 Recruitment to
the HIV LTR
(A) Set7/9-KMT7 binds TAR bulge and loop sequences in
newly synthesized HIV transcripts.
(B) When Tat is produced, Set7/9-KMT7 engages into
complex formation with Tat and P-TEFb and methylates
K51 in Tat. See text for details.
Collectively, these data support a model where
Set7/9 promotes the recruitment of Tat and
P-TEFb to TAR RNA.
Our data identify Set7/9-KMT7 as a coactivator
of Tat transactivation. Set7/9-KMT7 methylates
K51 in Tat and activates Tat transactivation in
a K51-dependent manner. In infected T cells,
Set7/9-KMT7 is required for full activation of
HIV gene expression and associates with the
latent and activated HIV LTR in vivo. The trans-
criptional activity of methylation-deficient Tat
is impaired and, unlike WT Tat, not affected by siRNA-mediated
knockdown of Set7/9-KMT7. Importantly, knockdown of Set7/
9-KMT7 also inhibits HIV proviral gene expression in primary
CD4+ T cells. Mechanistically, we link the positive role of Set7/
9-KMT7inHIVtranscriptionto anincreaseinTat-TAR interaction
and demonstrate that Set7/9-KMT7 itself can bind TAR RNA and
the Tat-P-TEFb complex.
Previously, a consensus recognition sequence was proposed
for Set7/9-KMT7 substrates based on structural data derived
from substrates that were known at that time (Couture et al.,
2006). This consensus sequence predicts a lysine or arginine
at position ?2 and a serine or threonine at position ?1 (where
0 is the methylated lysine) and applies to many Set7/9-KMT7
substrates, including histone H3, p53, TAF10, estrogen receptor
a, and DNMT1. However, recently identified target lysines in the
p65 subunit of NF-kB or the PCAF-KAT2B acetyltransferase do
not align with this consensus sequence (Masatsugu and Yama-
moto, 2009; Yang et al., 2009). In Tat, position ?2 is occupied by
R49 and is consistent with the proposed consensus sequence.
However, position ?1 in Tat is occupied by K50, which is similar
residues R49, K50, and K51 are all strictly conserved among HIV
isolates, underlining the importance of recognition of K51 by
Set7/9-KMT7 in the HIV life cycle.
The finding that Set7/9-KMT7 has intrinsic TAR RNA-binding
properties was unexpected. However, other lysine methyltrans-
2005). The fission yeast Set1 (KMT2) protein possesses an
N-terminal canonical RNA recognition motif (RRM) that is essen-
tial for its catalytic activity in vivo (Noma and Grewal, 2002).
Although this RRM is not conserved in Set7/9-KMT7, we show
that TAR RNA binding is also mediated via the N terminus of
low levels of Set7/9-KMT7 are continuously recruited to initiated
HIV transcripts via direct interactions with loop and bulge
sequences in TAR (Figure 7A).
Cell Host & Microbe
Monomethylation of Tat at K51
Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc. 241
HIV LTR is enhanced. We show that Set7/9-KMT7 physically
interacts with Tat and also forms a multimolecular complex
with Tat and P-TEFb. We therefore speculate that during the
elongation phase of HIV transcription, Set7/9-KMT7 becomes
enriched at the HIV LTR via these interactions and methylates
K51 in Tat (Figure 7B). Although it remains unclear from the gel
shift experiments whether Tat methylation directly influences
the formation of the Tat-TAR-P-TEFb complex, the finding that
Set7/9-KMT7 enhances Tat-TAR binding independently from
its interaction with TAR RNA supports such a model. In addition,
we verified by western blot analysis that Tat in the gel shift reac-
tions becomes efficiently methylated by Set7/9-KMT7 whether
exogenous SAM is added or not to the reactions (data not
Important evidence that Set7/9-KMT7 activates Tat function
through K51 methylation comes from the experiments with the
Tat K51A and K51R mutants. In our studies, the transcriptional
activity of these mutants was ?3-fold decreased and overall
unresponsive to Set7/9-KMT7 knockdown, supporting the
model that methylation by Set7/9-KMT7 is an important step in
Tat transactivation. A similar decrease in transactivation with
the Tat K51A mutant was previously reported by Kiernan and
colleagues (Kiernan et al., 1999). Moreover, Bennasser and
colleagues demonstrated that mutation of K51 in the context
of an infectious clone of HIV-1 suppressed viral replication
(Bennasser et al., 2005). However, the latter study did not find
that the K51A mutation affected Tat transcriptional activity, but
RNAi machinery (Bennasser et al., 2005). While our study under-
lines the importance of K51 for Tat transactivation, a potential
involvement of K51 methylation in the RNAi suppressor function
of Tat is interesting and requires further studies.
Additional support for a critical role of K51 in Tat transactiva-
tious anemia virus (EIAV) Tat protein (Anand et al., 2008). This
crystal structure predicts that K51 in HIV-1 Tat plays a central
role in the interaction between Tat and TAR RNA. Our data indi-
cate that addition of a methyl group to K51 could strengthen
these interactions. In contrast, it was reported that K51 methyl-
ation by the H3K9 lysine methyltransferase SETDB1-KMT1E in-
hibited the formation of the TAR ribonucleoprotein complex (Van
Duyne et al., 2008). Since SETDB1-KMT1E can function as a di-
and trimethyltransferase, the possibility exists that addition of
a single methyl group to K51 enhances Tat binding to TAR and
P-TEFb, while addition of two or three methyl groups inhibits
complex formation (Wang et al., 2003).
It remains unclear whether recruitment of Set7/9-KMT7 also
serves to methylate histone H3K4 at the HIV LTR, since the
enzyme cannot efficiently methylate histones when assembled
into nucleosomes (Nishioka et al., 2002; Wang et al., 2001).
HIV transcriptional elongation correlates with an increase in
H3K4 trimethylation at the integrated HIV template (Zhou et al.,
2004). Levels of H3K4 di- or trimethylation have been correlated
with the recruitment of Set7/9-KMT7 to cellular promoters,
including the Ins1/2, Slc2a2, MCP-1, and TNF-a genes, in sup-
port of the concept that Set7/9-KMT7 could act as a histone
methyltransferase in vivo (Deering et al., 2009; Li et al., 2008).
Notably, Set7/9-KMT7 itself is not a di- or trimethyltransferase;
structural analysis of Set7/9-KMT7 revealed that the enzyme
functions mainly as a monomethyltransferase (Xiao et al.,
2003). Accordingly, we show that Tat K51 is monomethylated
The finding that Tat is monomethylated at K51 adds another
of multiple histone-modifying enzymes (Hetzer et al., 2005). Tat
residue K50 is the target of the p300-KAT3B and GCN5-KAT2A
acetyltransferase activities, while R52 and R53 are methylated
by PRMT6 (Col et al., 2001; Kiernan et al., 1999; Ott et al.,
1999; Xie et al., 2007). The dynamics and hierarchy of these
modifications are unknown. However, because of their close
proximity within the Tat ARM, the occurrence and function of
one modification is likely coupled to the modification status of
a neighboring residue, as reported for histones (Jenuwein and
Allis, 2001). Since Set7/9-KMT7 is continuously present at the
HIV promoter even before Tat is produced, we predict that Tat
monomethylation at K51 occurs as an early event in the Tat
transactivation cycle and serves to enhance the formation of
the Tat/TAR/P-TEFb complex. Future studies will focus on
dynamics of Tat methylation at the HIV LTR and its interplay
with other Tat modifications.
Cells, Reagents, and Plasmids
Details for cells, reagents, and plasmids are described in the Supplemental
In Vitro Methylation Assays
In vitro methylation reactions with synthetic Tat peptides, Set7/9-KMT7
enzyme, and3H-SAM were performed as described (Nishioka et al., 2002).
Tat peptide reactions performed with nonradioactive SAM were analyzed by
MALDI TOF mass spectrometry. Details for in vitro methylation assays are
described in the Supplemental Experimental Procedures.
Preparation and Use of Polyclonal a-meARM Antibodies
The a-meARM antibodies were generated after immunization of rabbits with
purification of the antiserum. Details for the preparation and use of polyclonal
a-meARM antibodies are described in the Supplemental Experimental
RNAi and Transient Transfection Experiments
HIV LTR luciferase reporter and protein expression vectors were transfected
into HeLa cells with Lipofectamine (Invitrogen; Carlsbad, CA). Cells were har-
vested 24 hr later and processed for luciferase assays (Promega; Madison,
WI). For RNAi experiments, HeLa cells were transfected with pooled Set7/
9-KMT7 and control siRNAs(both Dharmacon; Lafayette, CO) using Oligofect-
amine (Invitrogen) and were retransfected after 48 hr with the HIV LTR luci-
ferase construct, Tat-expressing vectors, and corresponding amounts of the
empty vector. Cells were harvested 24 hr later and processed for luciferase
assays or western blotting. siRNA-nucleofection of Jurkat A2 cells was per-
formed as described (Mahmoudi et al., 2006). Seventy-two hours after nucleo-
fection, cells were treated with PMA (Sigma, St. Louis; 2 ng/ml) or DMSO for
12 hr. GFP expression was analyzed on a Calibur FACScan (Beckton Dickin-
son; Franklin Lakes, NJ). P values (paired t test) were used for statistical ana-
lysis. Set7/9-KMT7 knockdown efficiency in A2 cells was quantified using the
ImageJ software available at http://rsb.info.nih.gov/ij.
Viral Infections in CD4+ T Cells
Activated CD4+ T cells were transduced with lentiviral vectors containing
shRNAs against Set7/9-KMT7 (shSet7/9-1, shSet7/9-2) or the nontargeting
shRNA control. These lentiviral vectors also express the mCherry protein
Cell Host & Microbe
Monomethylation of Tat at K51
242 Cell Host & Microbe 7, 234–244, March 18, 2010 ª2010 Elsevier Inc.
under the control of the EF-1a promoter. Four days after infection, cells were
reinfected with viral particles produced from the molecular clones HIV-R7/
E?/GFP and pHR0-EF-1a/GFP. The percentage of GFP+ mCherry+ cells
was monitored by flow cytometry 3 days after the second infection. Details
for the viral production and infection are described in the Supplemental Exper-
Chromatin Immunoprecipitation Experiments
Chromatin from Jurkat A2 cells treated with PMA or DMSO was immunopre-
cipitated with a-Set7/9 antibodies. The immunoprecipitated material was
quantified by real-time PCR with primers specific for the HIV LTR, GFP, and
b-actin. Details for the chromatin immunoprecipitation experiments are
described in Supplemental Experimental Procedures.
Jurkat A2 cells were stimulated with 10 ng/ml TNF-a (Biosource; Camarillo,
CA) for 18 hr or were left unstimulated. Cells were lysed in IP buffer and immu-
noprecipitated with a-Set7/9-KMT7 antibodies for 2 hr at 4?C. Beads were
extensively washed and analyzed by western blotting with a-Set7/9-KMT7
or monoclonal a-FLAG antibodies. For coIPs in 293 cells, cells were trans-
fected with vectors expressing Tat/FLAG, Tat K51A/FLAG, or HA-Cyclin T1
as indicated using Lipofectamine reagent, and immunoprecipitations were
performed as described above.
RNA Gel Shift Experiments
TAR RNAs (WT, Dbulge, and Dloop) were synthesized in in vitro transcription
reactions with the Riboprobe system (Promega) as previously described
extracted withaphenol:chloroform mixture, and purified overan illustra Micro-
Spin G-50 column (GE Healthcare; Piscataway, NJ). Gel mobility reactions
(16 ml final volume) were carried out in binding buffer (50 mM Tris [pH 7.4],
0.5 mM EGTA, 150 mM NaCl, 2% glycerol, 0.2% Tween 20, 0.5 mM DTT,
90 mM ZnSO4, 0.005% BSA, and 100 mM ATP) and contained 2 3 104cpm
TAR probes/reaction and the indicated concentrations of Tat, recombinant
Set7/9-KMT7 or Set7/9-KMT7 (aa 110–366), and recombinant Cyclin T1/
CDK9 (Millipore; Billerica, MA). Supershift experiments were conducted in
the presence of 7 mg of a-Set7/9-KMT7 antibodies or corresponding amounts
of preimmune rabbit serum. Reactions were incubated for 30 min at 30?C and
separated on a prerun 4% Tris-glycine gel.
Supplemental Information includes Supplemental Experimental Procedures,
Supplemental References, and four figures and can be found with this article
online at doi:10.1016/j.chom.2010.02.005.
We thank Danny Reinberg, Katherine A. Jones, Matthew Spindler, Silke Wis-
sing, Marielle Cavrois, and members of the Verdin, Ott, and Greene labs for
sharing their reagents and expertise; Sarah Elmes from the UCSF Flow
Core for cell sorting; and Robert Houtz, Lynnette A. Dirk, Shiv Grewal, Matt-
hias Geyer, and Warner C. Greene for helpful discussions. We thank John
Carroll, Alisha Wilson, Teresa Roberts, and Chris Goodfellow for graphics;
Gary Howard for editorial assistance; and Veronica Fonseca for administra-
tive assistance. This work was supported by funds from the Gladstone Insti-
tutes, the University of California San Francisco Gladstone Institute of
Virology & Immunology Center for AIDS Research, collaborative research
between the Gladstone Institutes and JT Pharma, the NIH 1R01AI083139,
and grant number 106736-40-RFRL from amfAR, the American Foundation
for AIDS Research.
Received: July 29, 2009
Revised: December 11, 2009
Accepted: February 12, 2010
Published: March 17, 2010
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