Histone H3 lysine 9 trimethylation and HP1γ favor inclusion of alternative exons
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articles
Histone H3 lysine 9 trimethylation and HP1γ favor
inclusion of alternative exons
Violaine Saint-André1–3, Eric Batsché1–3, Christophe Rachez1–3 & Christian Muchardt1–3
Pre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally. It is therefore conceivable that chromatin-borne
information participates in alternative splicing. Here we find that elevated levels of trimethylation of histone H3 on Lys9
(H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene the chromodomain
© 2011 Nature America, Inc. All rights reserved.
protein HP1g, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism
involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1g on the variant region of the CD44
gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone
modifications impacting alternative splicing. They further implicate HP1g as a possible bridging molecule between the chromatin
and the maturating mRNA, with a general impact on splicing decisions.
Alternative splicing is a regulated process by which pre-mRNAs are by regulating the charges of the tails and thereby modifying their
differentially spliced, leading to expression of several mRNAs from contacts with neighboring nucleosomes. Histone modifications also
a single gene. It provides a mechanism for producing a wide variety function as flags, recruiting chromatin factors. For instance, histone
of proteins from a small number of transcription units. It is now esti- acetylation is detected by bromodomain-containing proteins, whereas
mated that about 90% of human pre-mRNAs are alternatively spliced, methylation of lysines can create a binding site for proteins contain-
such that the product of a gene will frequently vary from one tissue to ing chromodomains13,14. These proteins in turn affect the level of
the other, dictated by the balance of splicing enhancer and repressor chromatin condensation and DNA accessibility15.
activities that are present1,2. Several reports suggest that such histone modifications may
Transcription factors recruited to promoter sequences can also influence the regulation of alternative splicing. In particular, treatment
affect the outcome of alternative splicing3. This is understandable of cells with HDAC inhibitors, which favor acetylation of histones,
because splicing of pre-mRNA is initiated cotranscriptionally (see, results in increased exon skipping on several genes that have been
for example, refs. 4,5). Consistent with this, RNA polymerase II monitored (see, for example, ref. 16). Furthermore, trimethylation
(RNAP II) participates in the recruitment of splicing factors to of histone H3 on Lys4 (H3K4me3; see related abbreviations below
nascent transcripts6, and on some genes Ser2 phosphorylation of the reflecting different positions of lysine and di- or trimethylation)
polymerase C-terminal domain (CTD) heptamer repeat is required leads to the recruitment of the CHD1 ATPase with consequences
for the processing of the pre-mRNA by the splicing machinery7. on pre-mRNA splicing efficiencies17. An enrichment of H3K36
The involvement of the human SWI/SNF chromatin remodeling trimethylation has also been detected inside exons12,18,19. This modi-
complex in the inclusion of alternative exons has revealed that splicing fication facilitates recruitment of the polypyrimidine tract binding
decisions are also governed by chromatin and its components8. Exons protein involved in repression of specific alternative exons 20. Finally,
are on average 140–150 nucleotides long, a length strikingly similar on the fibronectin gene, local dimethylation of histone H3 on Lys9
to the 147 nucleotides necessary to accommodate a nucleosome, induced by short interfering RNAs (siRNAs) directed against intronic
which is the fundamental building block of chromatin9. In agreement sequences can favor the inclusion of alternative exons21.
with this, genome-wide analyses show that nucleosomes are preferen- It is possible that chromatin factors and histone modifications
tially located on exons and may participate in the definition of splice affect inclusion of alternative exons by modulating the recruitment
sites10–12. These studies also establish a positive correlation between of the spliceosome. For example, depletion of CHD1 leads to reduced
nucleosome occupancy and the degree of exon inclusion, consistent recruitment of the U2 small nuclear ribonuclear protein component17.
with the impact of chromatin on alternative splicing. Likewise, the SWI/SNF complex interacts with, and possibly recruits,
The histones that compose the nucleosomes are substrates for several components of the spliceosome 8,22. In addition, chromatin
post-translational modifications on their lysine- and arginine-rich may affect the RNAP II elongation rate, a parameter that regulates
N-terminal tails. These modifications, including lysine acetylation inclusion of alternative exons23. The rationale here is that the slowing
and serine/threonine phosphorylation, affect chromatin structure of the RNAP II increases the chances for the spliceosome to recognize
1Institut
Pasteur, Département de Biologie du Développement, Unité de Régulation Epigénétique, Paris, France. 2Centre National de la Recherche Scientifique
(CNRS), URA2578, Paris, France. 3INSERM Avenir, Paris, France. Correspondence should be addressed to C.M. (muchardt@pasteur.fr).
Received 25 February 2010; accepted 2 December 2010; published online 27 February 2011; doi:10.1038/nsmb.1995
nature structural & molecular biology VOLUME 18 NUMBER 3 MARCH 2011 337
articles
Figure 1 Histone H3K9 trimethylation (H3K9me3) is enriched on
the CD44 alternative exons. (a,b) ChIP assays with antibodies to H3,
a 6 H3K4me3
Relative enrichment
5 H3K4me2
H3K4me2, H3K4me3 and H3K36me3 (a) and antibodies to H3K9me2
H3K36me3
(versus H3)
and H3K9me3 (b), and chromatin from HeLa cells. The relative 4
IgG
enrichment of each modification along the CD44 gene was measured by 3
qPCR using primer sets targeting the indicated exons and introns and is 2
expressed as a fraction of the signal obtained with antibody to H3. The 1
signal obtained upon immunoprecipitation with nonimmune IgG indicates 0
the level of background (dotted line). Values are means ± s.e.m. of six PP C1 C2 C3 C4 C5 v2 v3v4v5v6 v7 v8 v9 v10 C16 C17 C18 C19
qPCR values from one representative experiment of five independent
CD44 Constant exons (C) Variant exons (v)
experiments. Constant and variant exons are represented as black
and gray boxes, respectively, on the CD44 gene map below the graphs. b 2 H3K9me3
The proximal promoter (PP) corresponds to the transcriptional start site. H3K9me2
Relative enrichment
IgG
(versus H3)
1
splice sites as they appear and thus minimizes the probability of skip-
ping to stronger splice sites farther down the gene. Our earlier data
show that the SWI/SNF-dependent inclusion of CD44 variant exons 0
observed upon activation of protein kinase C (PKC) is accompanied PP C1 C2 C3 C4 C5 v2 v3v4v5v6 v7 v8 v9 v10 C16 C17 C18 C19
by a local accumulation of RNAP II on the regions encoding the vari-
CD44 Constant exons (C) Variant exons (v) 3.5 kb:
ant domain of the CD44 protein8. This suggests that the SWI/SNF
complex affects variant exon inclusion in part by modulating the
© 2011 Nature America, Inc. All rights reserved.
RNAP II elongation rate. amplitude (Fig. 1b). Conversely, H3K27me3, another chromatin modi-
The human CD44 gene consists of two series of constant exons fication associated with transcriptional repression in mammals, was
(C1–C5 and C16–C19) that frame a large cluster of nine variant sparsely distributed within the gene, with no significant differences
exons (v2–v10). By this spatial organization, the gene is amena- between constant and variant exons (Supplementary Fig. 1b).
ble for detecting changes in chromatin structure between constant
and variant exons. Therefore, we have used CD44 to carefully map H3K9 methylation favors inclusion of CD44 variant exons
a series of histone modifications potentially involved in regulation We next investigated whether the H3K9me3 mark detected on CD44
of alternative splicing, including H3K4me2, H3K4me3, H3K36me3, was involved in regulation of the alternative splicing of this gene.
H3K9me2 and H3K9me3. Notably, H3K9me3 is enriched inside the To this end, we used siRNAs to transiently deplete HeLa cells from
variant region, and the presence of this mark can be correlated with several histone lysine methyltransferases (HKMTs) known to modify
an increased inclusion of the alternative exons in the mature CD44 H3K9, including SUV39H1, SUV39H2, EHMT1 (also known as
mRNA. Furthermore, we find that upon activation of PKC, HP1γ, a GLP) and EHMT2 (also known as G9A) (Supplementary Fig. 2a).
chromodomain protein with specificity for histone H3 methylated Depletion of SUV39H1 or SUV39H2 had only minor effects on
on K9, accumulates in the CD44 variant region where it is found amounts of H3K9me3 inside the CD44 gene. In contrast, upon deple-
associated with both the chromatin and the nascent pre-mRNA, and tion of EHMT1 or EHMT2, accumulation of H3K9me3 was substan-
colocalized with splicing factors. Genome-wide analysis further shows tially affected, with a reduction by approximately a factor of 2 of the
that HP1γ has a role in the regulation of alternative splicing on a large methylation peak on v9 and v10 (Supplementary Fig. 2b). In these
number of genes. Thus, our data suggest that the chromatin of vari- experiments, amounts of H3K27me3 on the CD44 gene remained
ant exons carries information conveyed from the nucleosomes to the unaffected (data not shown).
splicing factors, by proteins binding both histones and RNA. ChIP assays further confirmed the presence of EHMT1 or EHMT2
on the CD44 gene, with a notable accumulation on the variant exons
RESULTS v9 and v10. On these exons, depletion of the HKMTs resulted in their
Increased histone H3K9 trimethylation on CD44 variant exons decreased accumulation, evidence for the specificity of the ChIP data
We have probed the coding region of the CD44 gene for differences in (Supplementary Fig. 2c,d). ChIP assays also showed that depletion of
histone modifications between constant and variant exons. Chromatin EHMT1 or EHMT2 reduced accumulation of RNAP II on the CD44
immunoprecipitation (ChIP) walking in HeLa cells detecting histone gene. The effect was stronger on v9 and v10 than on C2 or C5, suggest-
H3 showed a preferential positioning of nucleosomes on exons versus ing that the depletion of the HKMTs increased the elongation rate of
introns but no substantial difference in this positioning between con- the polymerase inside the variant region (Supplementary Fig. 2e).
stant and variant exons (Supplementary Fig. 1a). We detected the We next probed the impact of the HKMTs on exon inclusion.
expected elevated levels of H3K4me2 and H3K4me3 at the promoter, Depletion of EHMT1 and EHMT2 reduced inclusion of variant exon
and of H3K36me3 inside the coding region of the gene (Fig. 1a). couples v8v9 and v9v10 in the mature CD44 transcript by a factor of
H3K36me3 showed a minor enrichment on variant versus constant 2 or more, whereas depletion of SUV39H1 and SUV39H2 had only
exons, and on exons versus their surrounding introns, in the vari- marginal effects on splicing (Supplementary Fig. 2f). We conclude
ant region of the gene. These observations are consistent with earlier from these experiments that methylation of histone H3 in the variant
reports18,20. H3K9me3 was enriched on the promoter of CD44. Notably, region of CD44 involves the histone methyltransferases EHMT1 and
this modification also accumulated on the cluster of variant exons EHMT2, and that high levels of H3K9me3 favor RNAP II accumula-
inside the gene (Fig. 1b). Peaks of H3K9me3 on the variant region of tion and inclusion of CD44 variant exons.
CD44 were also visible in the data from a genome-wide study carried
out on human T cells, illustrating that it is not a phenomenon specific HP1g is involved in efficient inclusion of CD44 variant exons
to HeLa cells24. Expression of H3K9me2 was also higher on the vari- H3K9 methylation creates a binding site for the chromodomain of
ant compared to the constant exons of CD44, although with reduced HP1 proteins. In mammalian cells, this family of proteins includes
338 VOLUME 18 NUMBER 3 MARCH 2011 nature structural & molecular biology
articles
a b 1.5
siGAPDH siHP1β c1.5 siGAPDH siHP1β d e HeLa S3 HeLa S3-HP1γ
siHP1α siHP1γ siHP1α siHP1γ 2
si DH
si DH
si DH
Splicing fold effect
si α
si β
γ
AP
AP
AP
Relative expression
P1
P1
P1
HeLa S3
Splicing fold effect
H
H
H
G
G
G
si
1.0 1.0 Ctl HP1γ
H3
H3 1
0.5 0.5 HP1γ-HA
HP1α HP1β HP1γ HP1γ
0 0 0
RLP0 CD44 C4C5 v2v3 v4v5 v8v9 v9v10 C16C17 C4C5 v2v3 v4v5 v8v9 v9v10 C16C17
C2C3
Figure 2 HP1γ regulates alternative splicing of the CD44 transcript. (a) HeLa cells were transfected with siHP1α, siHP1β, siHP1γ or siGAPDH as a
control. Western blots on nuclear extracts were carried out with antibodies to HP1α (anti-HP1α), anti-HP1β or anti-HP1γ (lower panels) or anti-H3
(H3 used as a loading control, upper panels). Blots are representative of the experimental replicates. (b,c) RNAs from transfected cells were quantified
by RT-qPCR. (b) Expression level of the CD44 C2C3 constant exons relative to that of RLP0 (set to 1) for each transfection condition. (c) Exon inclusion
is measured as the ratio between the indicated CD44 exon couples and the constant exon couple C2C3 and expressed relatively to the values obtained
upon transfection of siGAPDH (set to 1). Values are means ± s.e.m. of six qPCR values from three experimental replicates. (d) Western blots on nuclear
extracts from HeLa S3 and HeLa S3-overexpressing HP1γ-HA (HeLa S3-HP1γ) cells were carried out with anti-HP1γ or anti-H3 (H3 used as a loading
control). Blots are representatives of the replicates. (e) RNAs from the corresponding HeLa S3 or HeLa S3-HP1γ cells were quantified by RT-qPCR. Exon
inclusion is measured as the ratio between the indicated CD44 exon couples and the constant exon couple C2C3, and expressed relatively to the values
obtained with the HeLa S3 cell line (set to 1). Values are means ± s.e.m. from four experimental replicates.
three members, namely HP1α, HP1β and HP1γ. Of these, only HP1γ (Fig. 3c, ). This led us to explore the distribution of H3K9me3
© 2011 Nature America, Inc. All rights reserved.
has been reported to be on the coding region of active genes25–27. and HP1γ upon activation of the PKC pathway by the phorbol ester
To investigate the possible involvement of HP1 proteins in regula- PMA. This treatment enhances accumulation of RNAP II inside the
tion of CD44 variant exon inclusion, we carried out siRNA-mediated CD44 variant region and stimulates inclusion of the variant exons
depletion of each HP1 isoform in HeLa cells (Fig. 2a; abbreviated as (Fig. 3a,f)8,28. Under these conditions, amounts of H3K9me3 were
siHP1α, etc.). These siRNAs did not affect expression of the CD44 increased throughout the gene, but the profile was unchanged as
gene (Fig. 2b). Inactivation of HP1α and HP1β had little or no effect a peak inside the variant region of the CD44 gene was maintained
on splicing, whereas inactivation of HP1γ resulted in decreased inclu- (Fig. 3b). In contrast, HP1γ distribution was markedly changed by
sion of all the variant exons examined (Fig. 2c). Conversely, stable the PMA, which induces a clear accumulation of the protein inside the
moderate overexpression of HP1γ in HeLa cells (Fig. 2d) resulted in variant region of CD44 (Fig. 3c), largely overlapping with the peak of
increased inclusion of the same variant exons (Fig. 2e). RNAP II (Fig. 3f). The PMA also induces phosphorylation of HP1γ
on its Ser83 (HP1γS83p, Fig. 3g). The profile of HP1γ distribution
HP1g slows RNAP II was essentially reproduced in ChIP experiments with an anti–
The impact of HP1γ on inclusion of CD44 variant exons prompted us phospho-S83 HP1γ (Fig. 3d). Although this antibody was of sub-
to explore the distribution of this protein within the chromatin of the optimal quality, these data suggest that the HP1γ recruited to the
CD44 gene. As shown in Figure 3, ChIP walking revealed that HP1γ CD44 gene upon treatment with PMA is phosphorylated. This is
is present inside the gene but with no obvious sites of accumulation consistent with an earlier study showing that HP1γ is modified by
a 3.5
– PMA
d g Figure 3 HP1γ, RNAP II and U2AF65
Splicing fold effect
3.0 + PMA accumulate on the variant region of CD44
Relative enrichment
2.5 HP1γS38p
PMA – + chromatin upon PKC stimulation. (a) HeLa
3 – PMA
2.0
(versus IgG)
1.5 2
+ PMA HP1γS38p cells were incubated (+PMA) or not (−PMA)
1.0 overnight with PMA. After extraction of
1 HP1γ
0.5 cytoplasmic RNA and DNase treatment, each
0 0
C2 C5 v2 v3 v4 v5 v6 v7 v8 v9 v10C19 C2 v4 v9 C19 1 2 CD44 exon was quantified by RT-qPCR.
Exon inclusion is represented as fold change
e 7 16 ± s.e.m. between stimulated and nonstimulated
CD44
Relative enrichment
6 14 – PMA cells, with the signal from nonstimulated
Constant exons (C) Variant exons (v) 12 U2AF65 + PMA
(versus IgG)
5
b – PMA 4
10
8
samples being set to 1. (b–f) ChIP walking
experiments were carried out with antibodies
+ PMA 3
30 6 to H3K9me3 (b), HP1γ (c), HP1γS83p (d),
16 2
Relative enrichment
14 4
25 H3K9me3 1 2 U2AF65 (e) and RNAP II (8WG16) (f) and
12 IgG
20 0 0 chromatin from HeLa cells treated (black bars
(% H3)
10
PP C2 C5 i18 C19
5
0
v3
v4
v5
v7
v8
v19
i1
and squares) or not (white bars and squares)
v
15 8
10 6
4
f 50 8
for 2 h with PMA. Amounts of H3K9me3 are
5 expressed in percent of H3, and amounts of
Relative enrichment
2 IgG 7 RNAP II
0 0 40 HP1γ, U2AF65, RNAP II and HP1γS83p are
c 6 (8WG16)
(versus IgG)
PP C2 C5 i18 C19
5
0
v2
v3
v4
v5
v6
v7
v8
v19
i1
5
v
7 30
30 expressed relatively to the signal obtained for
4
Relative enrichment
6 25 HP1γ 20 3 ChIP using nonimmune IgG. Values are means
± s.e.m. of three independent experiments.
(versus IgG)
5 20 10 2
1 IgG
4
15 0
(g) HeLa cells were stimulated with PMA for
3 0
10 PP C2 C5 i18 C19 2 h. Western blots on total protein extracts
0
5
v3
v4
v5
v7
v8
v19
i1
v
2
1 5 were carried out with the indicated antibodies.
IgG
0 0 CD44 Constant exons (C) Variant exons (v) PP, proximal promoter.
PP C2 C5 i18 C19
5
0
v3
v4
v5
v7
v8
v19
i1
v
nature structural & molecular biology VOLUME 18 NUMBER 3 MARCH 2011 339
articles
a Input + PMA
b 90 HP1γ
c 16
d siGAPDH
100
Relative enrichment of
H3 siHP1γ
Relative enrichment of
80
Relative enrichment of
Input – PMA
RNA (versus DNA)
14
RNA (versus DNA)
RNA (versus DNA)
70 – PMA – PMA RNA-ChlP with anti-H3
12 + PMA
Relative enrichment
10 60 + PMA
10 0.8
50
40 8
(% input)
1 30 6
0.4
20 4
10 2 lgG
0.1 0 0 0
C2 C4 C5 i18 C19 C2 C5 i18 C19 C2 C5 i18 C19 C2 v5 v8
v4
v5
v6
v19
0
5
6b
v4
v5
v6
v19
i10
5
v4
v5
v6
v19
0
5
v
i1
v
v
i1
i1
CD44 CD44 CD44
Constant exons (C) Variant exons (v) Constant exons (C) Variant exons (v) Constant exons (C) Variant exons (v)
Figure 4 HP1γ bridges chromatin to pre-mRNA. (a) Quantification by qPCR of CD44 pre-mRNA exons retained on chromatin. Non-cross-linked DNase-treated
chromatin from HeLa cells stimulated (+PMA) or not (−PMA) with PMA for 2 h was extracted as described in Online Methods. Graph shows means ± s.e.m.
of chromatin-bound RNA (quantified by RT-qPCR) relative to the DNA in the input (quantified by qPCR). (b,c) Quantification of CD44 pre-mRNA exons
associated with HP1γ (b) and H3 (c) in the non-cross-linked chromatin fraction. RNA-ChIP assays were carried out with the indicated antibodies and chromatin
prepared as in a. Levels of immunoprecipitated RNA are expressed relatively to the levels of corresponding DNA sequences present in the extracted chromatin
fraction. (d) Association of histone H3 with the CD44 pre-mRNA requires HP1γ. RNA-ChIPs were carried out with anti-H3 and cross-linked chromatin from
HeLa cells transfected with the indicated siRNAs and stimulated with PMA for 2 h. The qPCR values are from a representative experiment and are expressed as
a percent of input for each indicated amplicon. For all panels, values are means ± s.e.m. from two independent experiments.
hosphorylation when it is present inside active genes29. Finally, we
p (Supplementary Fig. 5b). We also carefully verified that the signals
noted that the PMA treatment also causes accumulation of the splic- were due to detection of pre-mRNA and not contaminating mature
© 2011 Nature America, Inc. All rights reserved.
ing factor U2AF65 on the variant region of CD44 (Fig. 3e) in a pattern mRNA (Supplementary Fig. 5c,d). We observed that, although the
similar to that of HP1γ and the RNAP II. variant exons are enriched in RNAP II (Fig. 3d), RNA encoded by
We next depleted the HeLa cells of HP1γ using siRNAs. This these exons is less abundant on the chromatin than that encoded by
resulted in decreased accumulation of RNAP II on the coding region constant exons (Fig. 4a, m). In addition, amounts of RNA encoded
of CD44, suggestive of an increased elongation rate of the polymerase by variant exons and neighboring introns increase on the chromatin
(Supplementary Fig. 3d). In these knockdown experiments, the by a factor about 10 upon treatment of the cells with PMA, whereas
transcriptional activity of the CD44 gene was unaffected (as illus- amounts of RNA encoded by constant exons are increased only by
trated by levels of constant exons in Fig. 2c), consistent with the a factor of 2 (Fig. 4a; compare m and ; Supplementary Fig. 5e).
essentially unchanged levels of RNAP II accumulation on its pro- These observations suggest a correlation between chromatin retention
moter region (Supplementary Fig. 3d, left). Depletion of HP1γ also of a pre-mRNA sequence and its presence in the mature message.
reduced recruitment of U2AF65 (Supplementary Fig. 3c). Likewise, They also suggest that proteins other than the RNAP II bridge the
recruitment of the splicing factor PRP8, involved in later stages of chromatin to the pre-mRNA.
the spliceosome assembly30, was also affected by reduced levels To investigate this issue, we used the chromatin fraction described
of HP1γ (Supplementary Fig. 4). Finally, we noted that depletion of above to carry out RNA-ChIP experiments. With antibodies to RNAP II
HP1γ moderately decreased levels of H3K9me3 within the gene (anti-RNAP II), surprisingly little CD44 pre-mRNA was immuno-
(Supplementary Fig. 3a). A similar link between levels of HP1γ and precipitated, suggesting that the interaction between the polymer-
H3K9me3 had already been observed31–33 and may illustrate the ase and the RNA was largely disrupted by the extraction protocol.
implication of HP1 proteins in the recruitment of HKMTs34. Nevertheless, after treatment of the cells with PMA we observed a
Altogether our experiments show that activation of the PKC path- peak of interaction between the RNAP II and the RNA encoded by the
way causes HP1γ and U2AF65 to largely distribute together with the CD44 variant exons (Supplementary Fig. 5g). This is consistent with
RNAP II, with a peak of accumulation appearing inside the CD44 the accumulation of polymerase observed on these exons.
variant region. In addition, depletion of HP1γ reduces accumulation In contrast to the anti–RNAP II, antibody to HP1γ (anti-HP1γ)
of RNAP II and spliceosome components throughout the gene. These immunoprecipitated a large fraction of the input CD44 pre-mRNA.
observations suggest that HP1γ participate in the PMA-induced inclu- The HP1γ-RNA interaction required induction of the cells with PMA
sion of CD44 variant exons by slowing the RNAP II and by favoring and peaked inside the region encoded by the variant exons (Fig. 4b).
recruitment of splicing factors either directly or via the RNAP II. Histone H3 and H3K9me3 were also found associated with the
CD44 pre-mRNA upon PMA treatment (Fig. 4c and Supplementary
HP1g binds CD44 pre-mRNA inside the variant region of CD44 Fig. 5f). But as with the RNAP II, we observed weaker PCR signals
HP1γ is a chromatin protein that binds RNA35. This prompted for H3 than for HP1γ, although the antibody to H3 (anti-H3) is more
us to examine the contacts between the chromatin and the CD44 efficient at recruiting DNA material in ChIP than the antibody to
pre-mRNA. To this end, we extracted native non-cross-linked chro- HP1γ. In addition, siRNA-mediated depletion of HP1γ reduced the
matin from HeLa cells. From this chromatin we collected a fraction interaction of H3 with the CD44 pre-mRNA, indicating that HP1γ is
solubilized by deoxyribonuclease (DNase) that was then mildly soni- in between the transcript and histone H3 (Fig. 4d).
cated so as to fragment larger RNA molecules. This fraction is strongly These observations suggest that HP1γ, through its dual function as
enriched in RNAP II and U2AF65, suggesting that it contains tran- both a chromatin- and an RNA-binding protein, is involved in linking the
scriptionally active chromatin (Supplementary Fig. 5a). Using PCR nascent CD44 transcript to the chromatin template during elongation.
after reverse transcription (RT-PCR) we quantified CD44 message
present in this fraction at different positions throughout the CD44 RNAP II accumulation and variant exon inclusion
gene. Because the RNA was fragmented, this analysis reflected the To identify additional situations connecting amounts of H3K9me3
retention on chromatin of nascent transcripts. In these experiments we and HP1γ with exon inclusion, we characterized the CD44 locus in
verified that DNA was uniformly extracted throughout the CD44 gene SW626 and SKOV3 cells, two cell lines derived from ovarian tumors.
340 VOLUME 18 NUMBER 3 MARCH 2011 nature structural & molecular biologyarticles
Figure 5 Cell type–specific distribution of 2.5 a SW626 d e f
γ
v9v10 / C16C17
H3K9me3 and HP1γ correlates with variant
P1
SKOV3 25
-H
Relative expression
2.0
Splicing fold effect
V3
V3
SK 626
SK 3
exon inclusion and RNAP II accumulation.
V
– PMA
O
20
O
O
SW
SK
+ PMA
(a) Each CD44 exon was quantified by 1.5 HP1α
HP1γ-HA
15
HP1β
RT-qPCR after DNase treatment of SKOV3 1.0
HP1γ
HP1γ 10
5
(white) or SW626 (black) RNAs. Relative 0.5 H3 H3
0
abundance of each exon on the mature SKOV3 SKOV3-
g
0
C2 C5 v2 v3 v4 v5 v6 v7 v8 v9 v10 C19 HP1γ
transcript was measured by using the same
external reference for each amplicon. Values HP1γ SKOV3-HP1γ
Relative enrichment
CD44 6 5 SKOV3
are means ± s.e.m. from three independent
(versus IgG)
5 4 – +
Constant exons (C) Variant exons (v) 4 PMA PMA
experiments. (b,c) ChIP assays with antibodies 3
to H3K9me3, H3 and HP1γ and chromatin 4 b
0.6 H3K9me3 SW626
3
2
1
2
1 lgG
Relative enrichment
SKOV3
from SW626 or SKOV3 cells. The relative 3 0
– +
0
C2 C5 i18 C19
(versus H3)
5
h
0
v4
v5
v8
v19
enrichment on IL8 and GAPDH promoters,
i1
0.4 PP
v
2 RNAPII (N20)
on satellite sequences (hSat) or along the
Relative enrichment
20 6
0.2
CD44 gene was quantified by qPCR using 1
(versus IgG)
15
4
primer sets targeting the exons and introns 0
lgG 10
0
indicated on the map of the gene and expressed hSat C2 C5 v2 v4 v5 v7 v8 v9 v10 C19 5 2
as a fraction of histone H3 in b or relatively 4 c HP1γ
0 – + 0
C2 C5 i18 C19
lgG
5
0
v4
v5
v8
v19
3.0
Relative enrichment
PP
i1
v
SW626
to IgG in c (set to 1 for each cell line). Values 3 SKOV3 i
(versus IgG)
2.5
are means ± s.e.m. of three independent H3K9me3
Relative enrichment
2 2.0 6 3
qPCRs from one representative experiment. 1.5
4 2
(% H3)
(d,e) Western blots with the indicated antibodies
© 2011 Nature America, Inc. All rights reserved.
1
1.0
lgG
and nuclear protein extracts from SW626 or 0 0.5
2 1
lgG
SKOV3 (d) or from SKOV3 and SKOV3 stably 0 – + 0
H
at
G IL8
0
D
hS
PP C2 C5 v4 v9 v10 C19 C2 C5 i18 C19
AP
5
0
v4
v5
v8
v19
PP
i1
overexpressing HA-tagged HP1γ (SKOV3-HP1γ)
v
CD44 CD44
(e). (f) RT-qPCR quantification of RNAs from Constant exons (C) Variant exons (v) Constant exons (C) Variant exons (v)
SKOV3 and SKOV3-HP1γ cells stimulated
(+PMA) or not (−PMA) overnight with PMA. Exon inclusion is measured as the ratio between the variant v9v10 and the C16C17 exon couples. Values are
means ± s.e.m. of three RT-qPCR from one representative experiment. (g–i) ChIP assays with antibodies to RNAP II (N20) (g), HP1γ (h) and H3K9me3
and H3 (i), and chromatin from SKOV3 or SKOV3-HP1γ transfected for 48 h with either siHP1γ or siGAPDH, then stimulated (+PMA) or not (−PMA)
with PMA for 2 h. The relative enrichment along the CD44 gene was measured by qPCR and expressed in percent of H3 (for H3K9me3) or relatively to
values obtained with IgG (for RNAP II and HP1γ). PP, proximal promoter.
Earlier studies have shown differential CD44 variant exon inclusion observed only after stimulation of the cells with PMA. Under these
between these two cell lines36. In our hands, the inclusion ratio of conditions, HP1γ peaked on the variant region in a distribution simi-
CD44 variant to constant exons in SKOV3 is about 1:100, whereas it lar to that observed in HeLa cells (see Fig. 5g, ). In the presence of
is about 1:2 in SW626 cells (Fig. 5a). the exogenous HP1γ, PMA stimulation had a stronger effect both on
ChIP walking along the CD44 gene in the SW626 cells line revealed a inclusion of the variant exon couple v9v10 (Fig. 5f) and on accumu-
H3K9 trimethylation pattern similar to that observed in HeLa cells with lation of the RNAP II (Fig. 5h; compare and ). Overexpression
an increased accumulation of this modification on the variant region of HP1γ also regenerated a peak of H3K9me3 on the variant region
(Fig. 5b, ). This observation further documents that the presence of of CD44 (Fig. 5i, and ), possibly explained by the interaction of
the H3K9me3 mark inside the CD44 gene is not specific to HeLa cells. In HP1 proteins with HKMTs34. These experiments confirm that HP1γ
this cell line we also observed clear HP1γ recruitment inside the coding affects RNAP II elongation rate, inclusion of CD44 variant exons, and
region of the gene, with a moderate enrichment inside the variant region amounts of H3K9me3 inside the CD44 gene.
(Fig. 5c, ). In contrast, we observed neither H3K9me3 nor HP1γ accu-
mulation on the CD44 gene of the SKOV3 cell line (Fig. 5b,c, ). In HP1g affects alternative splicing of multiple genes
this cell line, HP1γ is also poorly recruited to another of its target genes, To address the global impact of HP1γ on alternative splicing, we trans-
IL8, whereas its recruitment on satellite sequences (hSat) is equivalent fected HeLa cells with siRNAs targeting either HP1γ or GAPDH (as a
to that observed in SW626 cells. Consistent with the low abundance of control) in triplicates, and used total RNAs from these cells for hybridi-
HP1γ, we also observed no or little accumulation of RNAP II on the zation of Affymetrix exon arrays. In this approach, variations in exon
coding region of CD44 (see Fig. 5h, ◊). These observations suggest composition of more than a factor of 1.2 in the absence of HP1γ were
that amounts of CD44 variant exon inclusion in different cell lines can observed in 10.5% of the genes analyzed on the arrays and expressed in
be correlated with amounts of H3K9me3 and recruitment of HP1γ to the HeLa cells either in the presence or in the absence of HP1γ (detailed
the gene. We noted, however, that in the SKOV3 cells, PMA treatment in Supplementary Table 1), whereas only 6.3% of the genes were affected
increases inclusion of variant exon couple v9v10, whereas distribution in their expression level. In addition, two-thirds of the genes regulated at
of HP1γ, RNAP II and H3K9me3 remain essentially unaffected the level of splicing were not affected in their expression. We also noted
(see Fig. 5f–i; compare and ). This possibly illustrates earlier data that depletion of HP1γ did not substantially affect expression of any main
showing that proteins other than HP1γ can respond to activation of PKC splicing factors, suggesting that its effect on splicing is direct.
and favor inclusion of CD44 variant exons8,28. To investigate whether the presence of H3K9me3 could be corre-
In the SKOV3 cells the abundance of HP1γ, but not that of HP1α lated with HP1γ-dependent inclusion of variant exons on genes other
and HP1β, is relatively low compared to SW626 cells (Fig. 5d). We than CD44, we carried out ChIP assays on two of the HP1γ splicing
therefore tested the effect of augmented amounts of HP1γ. Upon sta- targets revealed by the microarrays, namely the serine/threonine pro-
ble overexpression, this protein was reliably detected at some posi- tein kinase N2 (PKN2) and transcription initiation factor TFIID sub
tions inside the CD44 gene (see Fig. 5g, ). Clear accumulation was unit 4B (TAF4B). For PKN2, depletion of HP1γ favors the production
nature structural & molecular biology VOLUME 18 NUMBER 3 MARCH 2011 341articles
a c
H3K9me3
Rel. enrichment
0.8
ChIP
(versus H3)
0.4 1.0 Transcripts
inclusion
Relative
0 0.5
Transcripts
1.0
0
inclusion
Relative
0.5 GLS 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 1617 18
0
PKN2 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 22 d 1.0 Transcripts
b
inclusion
Relative
H3K9me3 0.5
Rel. enrichment
1.2
ChIP
(versus H3)
0
0.6
BRCA1 1 4 5 6 7 10 11 12 13 14 15 16 24
0
1.5 Transcripts
e Transcripts siGAPDH
1.0 siHP1γ
inclusion
1.0
Relative
inclusion
Relative
Forward primer
0.5 0.5 Reverse primer
1 Exon
© 2011 Nature America, Inc. All rights reserved.
0 0
1 Downregulated exon
TAF4B 1 2 3 4 5 6 7 8 9 10 11 121314 15 DSN1 1 2 3 4 5 6 7 8 9 10 11
Figure 6 HP1γ-dependent exon inclusion is guided by an H3K9me3 mark on several genes. HeLa cells were transfected with siHP1γ (black) or siGAPDH
(gray) used as control. RNAs from the transfected cells were quantified by RT-qPCR using primer sets designed to amplify exon couples of which one exon
(in black) is identified on GeneChip Human Exon 1.0 ST Arrays as less included upon depletion of HP1γ. (a–e) H3K9me3 ChIP (a,b, top). Panels show
amounts of H3K9me3 expressed as a fraction of H3 estimated by ChIP at the indicated position. Transcripts are shown in a,b, bottom, and c–e. Panels
show the relative RNA amounts at the indicated position. Schematic of the mRNA represents to scale the coding exons of the longest isoform of the
mRNA and the regulated splicing event for the indicated genes. Values were normalized to those for control genes HPRT and RLP0, whose expression
is not affected by HP1γ depletion, and are means ± s.e.m. of three experimental replicates.
of an isoform lacking the kinase domain encoded by exon 13 (Fig. 6a, affected transcriptionally. HP1α and HP1β did not affect CD44
Transcripts). For TAF4B it results in increased amounts of transcripts splicing, but we cannot exclude the possibility that they may regulate
(as shown by exon couples 1-2, 4-5 and 5-6), with reduced inclusion splicing of other genes; for example, HP1β interacts with ASF–SF2
of exons 3 and 8 (Fig. 6b, Transcripts). On both of these genes the (ref. 41), whereas depletion of HP1α compromises the effect of siR-
H3K9me3 mark is enriched on the regions encoding HP1γ-sensitive NAs on exon inclusion on the fibronectin gene21. In this context, we
exons (Fig. 6a,b, H3K9me3 ChIP). note that Drosophila dHP1a may also be involved in splicing, in that it
Other interesting genes validated by quantitative PCR (qPCR) shows a preference for exon-dense regions on highly expressed puffs
included GLS, which is one of the two genes encoding glutaminases, on polytene chromosomes, and interacts with heterogeneous nuclear
a family of proteins involved in the synthesis of the neurotransmitter ribonucleoproteins and many pre-mRNAs42–44.
glutamate. At least two different transcripts arise from this gene. The We find that methylation of histone H3 on lysine 9 (H3K9me3), the
longer isoform (KGA), skipping exon 15, is expressed predominantly in modification bound by HP1 proteins, is a characteristic of the region
brain and kidney, whereas the shorter isoform (GAC), including exon 15, coding for the variant exons of the CD44 gene. Likewise, this modi-
is mainly expressed in cardiac muscle and pancreas37. Knockdown of fication is enriched on DNA encoding HP1-sensitive variant exons
HP1γ reduces inclusion of exon 15 and therefore impedes the synthesis of the PKN2 and TAF4B genes. Thus, H3K9me3 is the first histone
of the GAC isoform (Fig. 6c). Finally, the genes encoding the breast modification found to function as a marker of alternatively spliced
cancer type 1 susceptibility protein BRCA1 and the kinetochore com- exons. We observed the marking of the CD44 variant region in several
plex component DSN1 also contain HP1γ-sensitive exons (Fig. 6d,e). cell types, including the cervical carcinoma–derived HeLa cells and
the SW626 of ovarian origin (this study) and T cells24. Analysis of
DISCUSSION genome-wide H3K9me3 localization data indicates that this modifica-
The results presented here define HP1γ as a regulator of alternative tion is present mainly at satellite, telomeric and active long terminal
splicing. Within the HP1 family of proteins, HP1γ appears as an repeats45. However, other studies have revealed the presence of
exception. Like HP1α and HP1β, it has been described as a repressor H3K9me3 inside the coding region of genes25,46. Possibly, the function
(see, for example, ref. 38). However, it clearly also has a role after the ini- of the H3K9me3 mark depends on neighboring histone modifications.
tiation of transcription. In particular, it is recruited to some inducible Indeed, H3K9me3 is associated with H3K27me3 on transcriptionally
promoters at the time of transcriptional activation, in replacement for silenced regions, whereas it is associated with H3K36me3 on “less
HP1α and HP1β26,27,39. Likewise, it is detected on the coding region of expressed” exons19. This second combination matches well the situ-
several transcribed genes in an RNAP II–dependent manner25,40. Our ation on the CD44 variant exons, where, together with H3K9me3, we
data show that the presence of HP1γ inside the coding region of CD44 find a moderate enrichment of the H3K36me3 mark.
favors inclusion of alternative exons. More generally, we find that Depletion of the EHMT1 or EHMT2 histone methyltransferases
depletion of HP1γ affects alternative splicing of 10.5% of the expressed reduced amounts of H3K9me3 and decreased inclusion of the CD44
genes analyzed on the arrays, which is more than the number of genes variant exons. This shows that these enzymes are involved in setting the
342 VOLUME 18 NUMBER 3 MARCH 2011 nature structural & molecular biologyarticles
Figure 7 Model for the regulation of CD44 alternative splicing by
a H3K9me3 and HP1γ. (a) In the absence of the H3K9me3 mark, HP1γ is
Without H3K9me3
present at low levels on the chromatin, possibly via its interactions with the
RNAP II or the globular domain of histone H3. Under these conditions the
RNAP II elongates at high rates through the variant region of the gene, and
the spliceosome recognizes only the splice sites of constant exons. These
constant exons remain in contact with the chromatin via the spliceosome
and the RNAP II, whereas the variant exons not bound by spliceosomes
keep contact with the chromatin only via the RNAP II. Altogether, this
b results in inclusion of only the constant exons in the mature mRNA.
(b) When levels of H3K9me3 increase inside the CD44 gene and/or when
With H3K9me3
HP1γ becomes more available (possibly released from the heterochromatin
by phosphorylation), HP1γ forms an additional link with chromatin via its
chromodomain. It accumulates on the coding region where it associates
with the pre-mRNA and favors its transient retention on the chromatin.
The generated chromatin structures slow down the RNAP II, which in turn
facilitates recruitment of splicing factors such as U2AF65 and PRP8 on the
pre-mRNA. This leads to increased inclusion of CD44 variant exons.
Constant exons Variant exons Constant exons
mRNA constant exon Spliceosome
Because HP1γ interacts with the CTD of the RNAP II25,26, it is
Nucleosome
mRNA variant exon very tempting to suggest that it functions as the ‘code reader’, detect-
Nucleosome with K9me3
U2AF65 ing the H3K9me3 modification and relaying the information to the
© 2011 Nature America, Inc. All rights reserved.
Intron
HP1γ Slow RNAPII elongating polymerase. However, the H3K9me3 mark may not be the
Constant exon
Variant exon Fast RNAPII only determinant of HP1γ recruitment. In particular, the SWI/SNF
HP1γS83p
complex that accumulates inside the variant region of CD44 (ref. 8)
can also facilitate HP1γ binding to the chromatin of the CD44 gene
H3K9me3 mark inside the CD44 gene. We note, however, that EHMT1 (Supplementary Fig. 6). Earlier we described a similar observation
and EHMT2 are known to dimethylate lysines and are therefore prob- on interferon-responsive genes39. This would explain why the distribu-
ably assisted in H3K9 trimethylation by other HKMTs47. The EHMT1- tions of HP1γ and H3K9me3 are not strictly identical inside the variant
EHMT2 depletion experiments also clearly illustrate that information region of CD44.
carried by the chromatin can be used by the machinery regulating alter- The HP1γ-dependent accumulation of RNAP II on the variant region
native splicing (see model, Fig. 7). The differential accumulation of the of CD44 gives an important insight on the mechanism allowing this
H3K9me3 mark and HP1γ that we observe between the SKOV3 and protein to regulate alternative splicing. RNAP II accumulation reflects
SW626 tumor cell lines, and that correlates with their respective level of a decreased elongation rate of the enzymes, a pivotal parameter for
variant exon inclusion, further suggests that chromatin-borne informa- splicing that favors the use of the suboptimal splice sites present on
tion relevant for splicing is precisely regulated and possibly unstable in alternative exons23. Possibly, HP1γ may also affect splicing by recruiting
diseases. The targeting of histone methyltransferases specifically to the the spliceosome. Indeed, accumulation of HP1γ on the variant region of
variant exons remains to be explored. In yeast, histone methyltransferases CD44 correlates with the presence of U2AF65 and PRP8. Likewise, an
can be targeted to particular chromatin domains by micro-RNAs48. earlier study showed an interaction between HP1γ and the SR protein
A similar machinery may exist in mammals, in that several of the ASF/SF2 (ref. 41). However, HP1γ has never been associated with the
involved yeast proteins have known homologs in higher eukaryotes49. spliceosome in any of the several proteomic studies of this complex
In addition, an earlier study has shown that alternative splicing is (reviewed in ref. 54). We therefore favor a model where HP1γ functions
controlled through siRNA-mediated transcriptional gene silencing21. as a hub, connecting chromatin and pre-mRNA to create structures
Alternatively, the H3K9me3 mark may be set during early rounds of tran- slowing the elongating RNAP II, which in turn facilitates recruitment
scription at a specific phase of development, possibly guided by splicing of the spliceosome (see model, Fig. 7).
factors expressed at that time. The mark may then be maintained in the
adult by complexes containing both HP1 and HKMT proteins34,50,51. Methods
Activation of PKC and the downstream mitogen-activated pro- Methods and any associated references are available in the online
tein kinase ERK results in increased inclusion of alternative CD44 version of the paper at http://www.nature.com/nsmb/.
exons. This effect is mediated in part by Sam68 (approved symbol
KHDRBS1), which upon phosphorylation binds the CD44 mRNA Accession codes. Exon array data can be accessed on GEO (code:
inside exon v5 (ref. 52) and favors its inclusion in part by interacting GSE25282).
with the SWI/SNF complex8. Activated ERK also phosphorylates the
RNAP II on the Ser5 in the CTD repeats53 and may thereby affect Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
the elongation rate of the polymerase. In the present study we find
that activation of PKC also has a marked effect on the accumula-
Acknowledgments
tion of HP1γ on the variant region of CD44. This accumulation can We thank E. Allemand and J. Seeler for critical reading of the manuscript,
be correlated with phosphorylation of HP1γ on Ser83 (HP1γS83p). and D. Auboeuf (INSERM U590, Centre Léon Bérard, France), P de la Grange
HP1γS83p was unaffected in its interaction with the RNAP II (data (Genosplice Technology, France), V. Ogryzko (CNRS, IGR, Université Paris-XI,
not shown). However, an earlier study has shown that HP1γS83p France), L. Fritsch and S. Ait-Si-Ali (Université Paris–Diderot, Paris, France)
for advice and gifts of reagents. V.S.-A. received fellowships from Région Ile-
is strictly euchromatic29. We therefore suggest that PKC activation de-France and L’Association pour la Recherche sur le Cancer. The work was
allows HP1γ retrieval from storage in the pericentromeric heterochro- supported by grants from the Agence National de la Recherche and Cancéropôle
matin, for it to be recruited on transcribed genes. Ile-de-France.
nature structural & molecular biology VOLUME 18 NUMBER 3 MARCH 2011 343articles
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344 VOLUME 18 NUMBER 3 MARCH 2011 nature structural & molecular biologyONLINE METHODS a ntibodies. Saturated magnetic beads coupled to protein A or G or to anti-rabbit
Cells culture. SKOV3 cells (HTB-77; American Type Culture Collection, ATCC) or anti-mouse antibodies (Dynabeads; Invitrogen) were used to recover the com-
stably overexpressing HP1γ were constructed by infection55 with a Flag-HA-His6– plexes. After 2 h of incubation the bound complexes were washed extensively and
tagged HP1γ-containing retroviral vector pOZ-FHHN-HP1γ. (HA, hemagglutinin). nucleic acids were purified. DNA bound to the immunoprecipitated proteins was
HeLa (ATCC), HeLa S3– and HeLa S3 HP1γ–overexpressing cells26, and SKOV3 quantified by qPCR. Values were measured relatively to the input and expressed
(ATCC), SKOV3-HP1γ and SW626 (HTB-78; ATCC) cells were cultured in relatively to the signal obtained for the immunoprecipitation with nonimmune
DMEM (Invitrogen) supplemented with 7% (v/v) FCS and 100 U ml−1 penicillin- IgG (set to1 or as a fraction of H3). For RNA-ChIP (Fig. 4d), quickly after elution,
streptomycin. When indicated, 40 ng ml−1 of phorbol 12-myristate 13-acetate samples were treated with DNase I (10 U; Roche) for 1 h before reverse transcrip-
(PMA) in dimethyl sulfoxide (DMSO) were added on confluent cells. tion and qPCR.
siRNAs and transfection. siRNAs targeting HP1α, HP1β, HP1γ and BRM were Isolation of native non-cross-linked chromatin and immunoprecipitation.
described earlier8,26. The siRNAs targeting SUV39H1, SUV39H2, EHMT1, Non-cross-linked chromatin was isolated as described56, except that micrococcal
EHMT2 and GAPDH were designed by Dharmacon. Target sequences are avail- nuclease digestion was replaced by a combination of TURBO DNase (Ambion)
able upon request. All siRNAs were synthesized as ON-TARGETplus grade from digestion, according to the manufacturer’s conditions, and sonication (three
Dharmacon. siRNAs were transfected with Dharmafect no. 1 (Dharmacon) for cycles of 10 s on a Diagenode Bioruptor). For RNA-ChIP with non-cross-linked
48 h or 72 h according to the manufacturer’s instructions. chromatin, the chromatin from 106 cells was incubated for 2 h with 1 µg of
indicated antibodies or nonimmune IgG as negative control, and processed as
Antibodies. Anti-H3 (Abcam 1791), anti-H3K36me3 (Abcam 9050), anti- described for ChIP (see above).
H3K4me2 (Upstate 07-030), anti-H3K4me3 (Abcam 8580), anti-H3K27me3
(Upstate 07-448), anti-H3K9me2 (Upstate 07-441), anti-H3K9me3 (Abcam Exon microarray. HeLa cells were transfected twice consecutively with siHP1α,
8898), anti-HP1γ (42s2; Upstate 05-690), anti–RNAP II (N20; Santa Cruz 890 siHP1β, siHP1γ or siGAPDH. mRNAs from these cells were hybridized on
and 8WG16, Covance), anti-U2AF65 (MC3; Santa Cruz 53942), and nonim- GeneChip Human Exon 1.0 ST Arrays (Affymetrix), in triplicates, following the
© 2011 Nature America, Inc. All rights reserved.
mune isotype-matched IgG antibodies (Sigma) were used for ChIP and RNA- manufacturer’s instructions. Scanning was performed on an Affymetrix station.
ChIP assays. Anti-HP1α (Euromedex 2G9), anti-HP1β (Euromedex 1A9), Analysis was performed by using GenoSplice technology (http://www.genosplice.
anti-HP1γ (Euromedex 1G6) and anti-H3 (Abcam 1791) were used for western com) EASANA visualization interface. Only genes expressed in the cells for at
blot assays. least one of the two compared conditions and giving a signal of good quality
as described57 were considered for this analysis. Only significant variations
RNA extracts. Total RNA was extracted using commercially available kits (Qiagen (P < 0.05) in exon inclusion above 20% were taken into account.
or Macherey-Nagel) with DNase treatment. Reverse transcription was carried out
with SuperScript III (Invitrogen) and random hexanucleotides for 1 h at 50 °C on Equipment and settings. Quantitative PCR data were processed with Stratagene
1 µg RNA, quantified with a nanodrop (Thermo Scientific). MxPro 4.01. Graphs were generated with Microsoft Excel. Gel images were
acquired in Adobe Photoshop and saturated. Figures were mounted in Microsoft
Quantitative PCR. Real-time qPCR was carried out on a Stratagene Mx3005p PowerPoint. The figures comply with Nature Publishing Group policy concern-
with Brilliant II SYBR Green kits (Stratagene) according to the manufacturer’s ing image integrity.
instructions. Data were computed as described 8. Primer sequences are described
in Supplementary Table 2. 55. Morgenstern, J.P. & Land, H. Advanced mammalian gene transfer: high titre
retroviral vectors with multiple drug selection markers and a complementary helper-
free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).
ChIP and RNA-ChIP. Briefly, cells were fixed in 1% (v/v) formaldehyde for 56. Méndez, J. & Stillman, B. Chromatin association of human origin recognition
10 min at room temperature (20–25 °C). The chromatin preparation was proc- complex, cdc6, and minichromosome maintenance proteins during the cell cycle:
essed as described8. Chromatin was incubated overnight with 1 µg of the spe- assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20,
8602–8612 (2000).
cific antibodies or nonimmune IgG as negative control. 106 cells were used to
57. de la Grange, P., Gratadou, L., Delord, M., Dutertre, M. & Auboeuf, D. Splicing
prepare chromatin for immunoprecipitation with antibodies to histone, HP1γ factor and exon profiling across human tissues. Nucleic Acids Res. 38, 2825–2838
and RNAP II and 3 × 106 to 5 × 106 cells for immunoprecipitation with other (2010).
doi:10.1038/nsmb.1995 nature structural & molecular biologyYou can also read
