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10.1101/gad.621811Access the most recent version at doi:published online March 29, 2011Genes Dev.

Guillermo Pablo Vicent, A. Silvina Nacht, Jofre Font-Mateu, et al.minute of hormonal gene activationFour enzymes cooperate to displace histone H1 during the first

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Four enzymes cooperate to displacehistone H1 during the first minute

of hormonal gene activationGuillermo Pablo Vicent,1,2 A. Silvina Nacht,1 Jofre Font-Mateu, Giancarlo Castellano, Laura Gaveglia,Cecilia Ballare, and Miguel Beato

Centre de Regulacio Genomica (CRG), Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain

Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes,which is limited by the compaction of DNA in chromatin. Although we have gained insight into how corehistones and their modifications influence this process, the role of linker histones remains unclear. Here we showthat, within the first minute of progesterone action, a complex cooperation between different enzymes acting onchromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First,

activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2[activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 bythe MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylaseKDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which isrequired for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, andkinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histoneH2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) andexpression arrays show that H1 displacement is required for hormone induction of most hormone target genes,some of which are involved in cell proliferation.

[Keywords: chromatin remodeling; histone H1; hormonal gene activation; hormone receptors]

Supplemental material is available for this article.

Received December 23, 2010; revised version accepted February 28, 2011.

In eukaryotic cells, chromatin presents a barrier to pro-teins that must access DNA, but also offers an opportu-nity for modulating gene expression. The basic unit ofchromatin, the nucleosome core particle, consists of anoctameric disc composed of two copies of each of the fourcore histone (H3, H4, H2A, and H2B), around which 147base pairs (bp) of DNA are wrapped in 1.65 left-handedsuperhelical turns. However, the repeating unit of meta-zoan chromatin is the nucleosome, which includes alinker histone of the H1 family and some additional 2030 bp of linker DNA. Histone H1 is localized at the dyad

of the nucleosome and contacts asymmetrically thelinker DNA at the nucleosomal entry and exit sites, lim-iting the mobility of the nucleosome core particle andcontributing to the folding and stabilization of the 30-nmchromatin fiber (Brown 2003; Bustin et al. 2005).

Regulation of eukaryotic gene expression implies mech-anisms that facilitate access of transcription factors to

DNA sequences in chromatin. This is the function ofchromatin remodeling complexes, which either chemi-cally modify the core histones, mainly in their N-termi-nal tails, or use the energy of ATP hydrolysis to weakenthe interaction of histones with DNA. Four differentfamilies of ATP-dependent chromatin remodeling com-plexes have been described, all sharing similar ATPases:the SWI/SNF, the ISWI (imitation switch), the CHD, andthe INO80 (Clapier and Cairns 2009). The chromatinremodelers of the ISWI family contain two to four sub-units and include NURF, CHRAC, and ACF complexes,

initially purified from Drosophila melanogaster, and thehuman WICH or NoRC. Many ISWI family complexes(ACF and CHRAC) optimize nucleosome spacing to pro-mote proper chromatin folding and the repression oftranscription. NURF, the founding member of this family,has been isolated from human cells and has been shownto catalyze energy-dependent nucleosome sliding (Xiaoet al. 2001; Barak et al. 2003), but its function in generegulation is not clear.

Histone-modifying enzymes are also key actors inchromatin remodeling, and there are indications thatthey cooperate with ATP-dependent remodelers. Initial

1These authors contributed equally to this work.2Corresponding author.E-MAIL [email protected]; FAX 34-933-160099.Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.621811.

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studies focused on the role of histone acetylation and, to alesser extent, on histone phosphorylation and histonearginine methylation (Bartsch et al. 1996; Spencer et al.1997; Li et al. 2003; Metivier et al. 2003; Vicent et al.2006b), while lysine methylation has not been studiedextensively (Metzger et al. 2005; Wissmann et al. 2007).

Although H3 trimethylated at Lys 4 (H3K4me3) is asso-ciated with transcription start sites (TSSs) of active genes(Santos-Rosa et al. 2002, 2003; Schneider et al. 2004), therole of this modification during mouse mammary tumorvirus (MMTV) activation has been questioned (Kinyamuand Archer 2007). The largest subunit of NURF, BPTF/NURF301, has been shown to recognize H3K4me3 bymeans of a plant homeodomain (PHD) located close to abromodomain (Li et al. 2006), thus stabilizing the NURFcomplex on chromatin (Wysocka et al. 2005).

Unlike histone acetylation and phosphorylation, thereversibility of histone methylation was debated formany years due to the low turnover rate of methylatedhistones (Byvoet et al. 1972). However, recent studies

have demonstrated that histone methylation can also beremoved enzymatically by histone demethylases that in-clude PADI4 (peptidyl arginine deiminase, type 4), LSD1(lysine-specific demethylase 1), and the JmjC (Jumonji C)domain-containing proteins (Klose and Zhang 2007).Within the seven subfamilies of the JmjC domain-con-taining proteins, the JARID1 family members are highlysimilar in domain architecture and contain JmjN,BRIGHT, JmjC, zf-C5HC2, and PHD domains (Yamaneet al. 2007). RBP2/JARID1A and JARID1B/PLU-1/KDM5Bare the most thoroughly characterized JARID1 familymembers, and both have proven roles in cancer cell pro-liferation (Lu et al. 1999; Benevolenskaya et al. 2005).KDM5B is an H3K4-specific histone demethylase abnor-

mally expressed in breast cancer cell lines and tissues(Lu et al. 1999; Barrett et al. 2002; Yamane et al. 2007).

Steroid hormones regulate gene activity by binding oftheir receptors to promoter and/or enhancer regions oftheir target genes and recruitment of protein kinases,histone-modifying enzymes, and ATP-dependent remod-eling complexes. In addition to these transcriptional ef-fects, steroid hormones also signal via cytoplasmic ki-nases that ultimately impinge on and are needed fortranscriptional regulation. In breast cancer cells, a smallsubpopulation of estrogen receptor (ER) and progesteronereceptor (PR) are palmitoylated and attached to the cellmembrane (Pedram et al. 2007). Upon binding of its li-gand in breast cancer cells, the membrane anchored PR

cross-talks and activated the Src/Ras/Erk signaling path-way, eventually leading to Erk-mediated phosphorylationof PR at S294 and of Msk1 at T581, and to the formationof a ternary complex, pPRpErkpMsk1 (Vicent et al.2006a). The activated ternary complex is targeted to theregulated promoters where Msk1 phosphorylates histoneH3 at S10, leading to displacement of a repressive com-plex containing HP1g (Vicent et al. 2006a). This enablesthe recruitment of the ATP-dependent chromatin remod-eling complex BAF and the histone acetyl transferasePCAF, which acetylates histone H3 at K14, contributingto the anchoring of BAF (Vicent et al. 2009).

Many studies on gene regulation by hormones havebeen performed using the MMTV long terminal repeatregion, which encompasses a promoter including, within140 bp, five degenerated hormone-responsive elements(HREs) and a binding site for nuclear factor 1 (NF1). Inchromatin, the MMTV promoter is organized into posi-

tioned nucleosomes (Richard-Foy and Hager 1987), witha nucleosome located over the five HREs and the NF1-binding site (Truss et al. 1995). On this promoter nucle-osome, the binding site for NF1 is not accessible, and onlytwo of the five HREsthe strong palindromic HRE1 andthe weak half-palindrome HRE4can be bound by hor-mone receptors, while the central HREs are not accessiblefor receptor binding (Pina et al. 1990). Following hormoneinduction in vivo, all HREs andthe binding site forNF1 areoccupied simultaneously on the surface of a nucleosome-like structure, and a functional synergism is observed be-tween PR and NF1 (Truss et al. 1995). In 1984, it wasshown that steroid receptors induce chromatin changes attarget promoters a few minutes after hormone induction

(Zaret and Yamamoto 1984). Since then, the role of SWI/SNF complexes during activation of the MMTV promoterhas been widely studied (Trotter and Archer 2004; Vicentet al. 2004, 2009). We showed that BAF catalyzes ATP-dependent displacement of histones H2A and H2B (Vicentet al. 2004), facilitating access of NF1 to its initially hiddenbinding site (Vicent et al. 2009). On the surface of theresulting histone H3/H4 tetramer, NF1 synergizes withadditional PR complexes by facilitating their binding tothe central HREs: HRE2 and HR3 (Vicent et al. 2010).

It is unclear whether, in addition to BAF, other ATP-dependent chromatin remodeling complexes are recruitedto the promoter after activation and are needed for pre-paring the chromatin for transcription. In particular, we

do not know the mechanisms by which constraints im-posed on chromatin dynamics by linker histone bindingare managed during gene activation. Incorporation of his-tone H1 into MMTV minichromosomes inhibits basaltranscription by reducing access of general transcriptionfactors to the promoter, but H1 facilitates synergism be-tween PR and NF1, leading to optimal transactivation(Koop et al. 2003). Following PR binding in Drosophilaextracts, histone H1 is phosphorylated and subsequentlyremoved from the promoter upon transcription initiation(Koop et al. 2003). Moreover, depletion of histone H1from target promoters has been reported during hormonalactivation of transcription in cultured cells (Bresnicket al. 1992), and a role for histone H1 phosphorylation

in modulating MMTV activation has been postulated(Lee and Archer 1998). More generally, histone H1 phos-phorylation by Cdk2 has been associated with hormone-dependent transcriptional activation (Bhattacharjee et al.2001). In MMTV minichromosomes lacking histone H1,dNURF catalyzes the ATP-dependent remodeling neededfor simultaneous and synergistic binding of PR and NF1to the MMTV promoter (Di Croce et al. 1999). However,the role of NURF in physiological gene regulation bysteroid hormones has not been addressed so far.

Here, we identify NURF as an ATP-dependent chroma-tin remodeling complex participating in and required for

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activation of hormone-dependent genes. The NURF com-plex is recruited as early as 1 min after induction and isanchored at the promoter chromatin by the H3K4me3signal produced by the MLL2 and MLL3 components ofthe ASCOM (ASC-2 [activating signal cointegrator-2]complex) complex. The increase of the H3K4me3 signal

is also due to localized displacement of the demethylaseKDM5B from target chromatin. In vivo, NURF remodel-ing allows Cdk2 loading at the promoter and, subse-quently, histone H1 phosphorylation and displacement.These steps precede and are required for recruitment ofBAF to the promoter. Thus, we identify four additionalenzymatic activities (NURF, ASCOM, KDM5B, andCdk2) involved in the very initial chromatin remodelingevents required for progesterone gene activation.

Results

NURF is required for hormonal induction and is

recruited by activated PR to the MMTV promoterTo assess whether SNF2h-containing complexes are in-volved in hormonal gene induction, T47D-MTVL cellscarrying a single copy of the MMTV-luc transgene in-tegrated in their genome (Truss et al. 1995) were trans-fected with specific siRNAs. In cells transfected withcontrol siRNA, fivefold and eightfold increases inMMTV-luc transcription were observed after 1 h and 6h of hormone treatment, respectively (Fig. 1A). Trans-fection of siRNAs against BPTF, a specific subunit ofNURF, compromised the induction by 56% and 50%,respectively. In contrast, transfection with siRNAs againstACF1, a subunit present in hACF and in hCHRAC com-plexes (Corona and Tamkun 2004), failed to reduce the

induction of the MMTV promoter, demonstrating an im-portant role of NURF on MMTV promoter activation (Fig.1A). Progestin induction of three endogenous progester-one target genes (DUSP1, 11b-HSD2, and FOS) was alsoimpaired by the BPTF siRNAs to a level similar orsuperior to that observed for MMTV-luc (Fig. 1B). How-ever, BPTF siRNAs did not inhibit progestin inductionof the EGFR gene (Fig. 1B). We conclude that optimalinduction of several, but not all, progesterone target genesdepends on the physiological levels of NURF.

To find out whether NURF is recruited to the MMTVpromoter, we performed chromatin immunoprecipitation(ChIP) experiments at different time points after hormoneaddition (0, 1, 2, 5, and 30 min) using antibodies specific

for the BPTF subunit (Barak et al. 2003). We found thatBPTF is recruited as early as 1 min after hormone additionand remains bound to the promoter, although to a lesserextent, after 2 min (Fig. 1C). Five minutes after hormoneaddition, no BPTF is detected in the promoter, but itappears again at 30 min, suggesting a rapid and transientrecruitment of NURF, followed by a second slower waveof NURF recruitment (Fig. 1C). PR is also recruited to theMMTV promoter 1 min after hormone addition, but itsbinding increased with time until 30 min (Fig. 1C). Si-multaneously with the recruitment of PR to the MMTVpromoter 1 min after hormone treatment, we observed

recruitment of the other components of human NURF,such as SNF2H, RbAp 46/48, and the recently identifiedHMG2L1 (Fig. 1D; Vermeulen et al. 2010). In contrast, wedid not observe recruitment of RSF-1, a subunit present inthe RSF complex (LeRoy et al. 1998; data not shown). Wealso performed ChIP with a HMG2L1 antibody in cells

transfected with control siRNA and siRNAs againsteither BPTF or SNF2H in order to test whether HMG2L1recruitment depends on NURF activity. We found thatdepletion of any of the two NURF subunits compromisedHMG2L1 recruitment (data not shown). Our resultsimply that, in the absence of BPTF or SNF2H, the com-plex either is not formed or, if formed, has lost theHMG2L1 chromatin anchoring activity.

Next, we used coimmunoprecipitation experiments totest whether NURF forms a complex with PR in culturedcells. One minute after hormone addition, we detectedthe presence of PRBPTF complexes (Fig. 1E, lanes 2,3).Therefore, we conclude that the NURF complex is targetedinitially to the MMTV promoter, likely through an inter-

action with PR, and, after 2 min, leaves the promoter.

Trimethylation of histone H3 at Lys 4 bymethyltransferases of the ASCOM complex

is necessary for hormonal induction andNURF anchoring

As the major subunit of NURF, BPTF, contains PHD do-mains that recognize methyl lysines (Wysocka et al. 2006),we wondered about possible roles of histone methyltrans-ferases (HMTs) in hormone induction. Hormone inductionincreased the H3K4me3 mark at the MMTV promoterwithout changes in the levels of either H3K4me2 (H3dimethylated at K4) or H3K4me1 (H3 methylated at K4)

(Fig. 2A). Previous reports have described a 2-MDa com-plex consisting of eight proteins: the methyltransferasesMLL2 or MLL3 (MLL3/4 in rodents), ASH2L, RBP5,WDR5, hDPY-30, UTX, PTIP, and the protein ASC2(Lee et al. 2008). This complex, named ASCOM, belongsto the Set1-like complexes, a conserved family of relatedH3K4 methyltransferases (Goo et al. 2003; Lee et al.2006). ASC-2 is a coactivator of many nuclear receptorsand transcription factors, including retinoic acid receptorand ERs (Mahajan and Samuels 2005). Its binding tonuclear receptors is dependent on two LXXLL boxes lo-calized in the N-terminal and C-terminal domains ofASC-2 (Li et al. 2007). We tested the function of MLL2and/or MLL3 in hormone regulation using siRNAs (Goo

et al. 2003; Lee et al. 2006). When individual siRNAsagainst MLL2 or MLL3 were used, there was no strikingreduction of the MMTV induction, but the combinationof the two siRNAs (MLL2 + 3) decreased induction by50% and 66% after 1 h and 6 h of hormone addition,respectively (Fig. 2B). We also checked MLL1 and MLL4siRNAs alone and in combination; they failed to elicit aninhibitory effect on MMTV hormone induction (data notshown). Moreover, in cells transfected with siRNAagainst ASC-2 and treated with hormone for 1 h or 6 h,we observed a decrease in hormone activation of 40% and47%, respectively (Fig. 2C). Our results indicate that,

NURF, ASCOM, KDM5B, and CDK2 in H1 eviction

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in T47D-MTVL cells, the MLL2 or MLL3 of the ASCOMcomplex are necessary for hormonal activation of the

MMTV.To assess whether the ASCOM complex is recruited to

the MMTV promoter, we performed ChIP experimentsusing a specific antibody against the ASC-2 subunit. Wefound that ASC-2 is recruited as early as 1 min after hor-mone addition (Fig. 2D), coinciding with the H3K4me3signal. Next, we asked whether the increase in H3K4me3observed after hormone addition is generated by theASCOM complex. Control and MLL2 + 3 siRNA-trans-fected T47DMTVL cells were subjected to ChIP assayswith antibodies against H3K4me3. In control cells, hor-mone treatment induced increased H3K4me3, whereas

knockdown of MLL2 + 3 abrogated K4 methylation andmarkedly reduced SNF2H loading on the promoter (Fig.

2E). To test whether H3K4 methylation and NURF bind-ing take place on the same promoter, we performed se-quential ChIP (re-ChIP) experiments in the MMTV andFOS promoters. Hormone treatment induced methyla-tion of H3K4 accompanied by recruitment of NURF tothe corresponding promoters (Fig. 2F, lanes 3,4), indicat-ing that the H3K4 methylation and NURF recruitmentoccur in the same genomic region. In contrast, control re-ChIPs performed with irrelevant IgG as the first antibodyshowed no amplification product (Fig. 2F, lanes 1,2). Nochange was observed when the adjacent nucleosome D inthe MMTV promoter was amplified by PCR (Fig. 2F, lanes

Figure 1. NURF is needed for full activa-tion of progesterone target genes and is

recruited to their PR-binding sites. (A)Cells were transfected with control siRNAor siRNA against either BPTF or ACF1 as

indicated, and the levels of BPTF, ACF1,and tubulin were determined by Westernblotting. (Right panel) BPTF and ACF1

levels showed a marked reduction of 70%and 65%, respectively. (Left panel) Cells

maintained 1 d in serum-free conditionswere incubated with 10 nM R5020 for 1

and 6 h and total RNA was prepared;cDNA was generated and used as a tem-plate for real-time PCR with luciferase

oligonucleotides. The values represent themean and standard deviation from three

experiments performed in duplicate. (*)P-value < 0.05. (B) cDNA was generatedfrom cells transfected with control and

BPTF siRNAs and used as a template forreal-time PCR with gene-specific primers.

The values were normalized with GAPDH.The histograms show the mean 6 SD of

three experiments performed in duplicate.(*) P-value < 0.05; (**) P-value < 0.01. (C)Cells were untreated (0) or treated for 1, 2,

5, and 30 min with 10 nM R5020 andsubjected to ChIP assays with a-PR- and

a-BPTF-specific antibodies. The precipi-tated DNA fragments were subjected to

PCR analysis to test for the presence ofsequences corresponding to the MMTV nu-cleosome B. Input material (1%) is shown

for comparison. A representative of threeindependent experiments is shown. Thenumbers below each panel correspond to

the quantification by real-time PCR. (D)Cells treated with hormone for 1 min were

subjected to ChIP assays witha-PR, a-BPTF,

a-SNF2H, a-RbAp46/48, and a-HMG2L1.

The precipitated DNA fragments were sub-jected to PCR analysis to test for the pres-

ence of sequences corresponding to theMMTV nucleosome B. A representative of

three independent experiments is shown. The numbersbeloweach panel correspond to the quantification by real-time PCR. (E) Cells

were untreated (0) or treated for 1 min with R5020, lysed, and immunoprecipitated with eithera-PR antibody or normal rabbit IgG asa negative control (IgG). The immunoprecipitates (IP) were analyzed by Western blotting with BPTF- and PR-specific antibodies.

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3,4, second row from the top), underlining the localizednature of the H3K4me3 mark and BPTF recruitment. Theaverage sonicated fragment size of the chromatin used forChIPs and re-ChIPs ranged between 200 and 250 bp (Fig.2F, bottom panel).

Pull-down experiments with T47D-MTVL nuclear ex-tracts and biotinylated peptides of the H3 tail containingvarious modifications were used to test whether H3K4trimethylation influences the binding affinity of the NURF

complex for histone tails. We detected binding of thesubunits of the NURF complexnamely, BPTF andSNF2Hto histone tails containing H3K4me3 (Fig. 2G,cf. lanes 1 and 2). Binding of NURF subunits to H3K4me3peptides containing acetylation at K14 and/or phosphor-ylation at S10 showed a similar affinity as peptides withH3K4me3 alone (Fig. 2G, cf. lanes 2 and 5,7). Phosphor-ylation or acetylation, alone or in combination, had aweak effect on binding of BPTF to the unmethylated

Figure 2. Trimethylation on histoneH3K4 by MLL2/3 of the ASCOM complex

is required for full hormonal transactiva-tion and for anchoring of NURF. (A) Con-trol cells or cells treated with R5020 for 1

min were subjected to ChIP assays with

a-H3K4me3, a-H3K4me2, and a-H3K4me1.The precipitated DNA fragments were ana-

lyzed by PCR for the presence of sequencescorresponding to the MMTV nucleosome B.

A representative of three independent ex-periments is shown. The numbers beloweach panel correspond to the quantificationby real-time PCR. (B, left) Cells were trans-fected with the indicated siRNAs and

treated with 10 nM R5020 for 1 h and6 h. RNA was extracted, and cDNA was

generated and used as a template for real-time PCR with luciferase oligonucleotides.The values represent the mean and stan-

dard deviation from three experiments per-formed in duplicate. (Right) Cells were

transfected with the indicated siRNAs,and the mRNA levels for MLL2, MLL3,

and GAPDH were determined by RTPCR. (*) P-value < 0.05. (C, left) Cells weretransfected with siRNAs and treated with

10 nM R5020 as indicated, and the levels ofluciferase were analyzed by RTPCR. The

values represent the mean and standarddeviation from two experiments performed

in duplicate. (Right) Cells were transfectedwith the siRNAs against ASC-2, and thelevels of ASC-2 and tubulin were deter-

mined by Western blotting. (*) P-value

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