Hu Proteins Regulate Alternative Splicing by Inducing Localized

Hu proteins regulate alternative splicing by PNAS PLUS inducing localized histone hyperacetylation in an RNA-dependent manner

Hua-Lin Zhoua, Melissa N. Hinmana, Victoria A. Barrona, Cuiyu Genga, Guangjin Zhoua, Guangbin Luoa,b, Ruth E. Siegelc, and Hua Loua,b,d,1

aDepartment of Genetics, bCase Comprehensive Cancer Center, cDepartment of Pharmacology, dCenter for RNA Molecular Biology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106

Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved July 5, 2011 (received for review March 1, 2011)

Recent studies have provided strong evidence for a regulatory link protein CHD1 (18). Moreover, the histone mark H3K36me3 among chromatin structure, histone modification, and splicing reg- can affect alternative splicing by recruiting the splicing regu- ulation. However, it is largely unknown how local histone modifi- lator PTB to pre-mRNA via the chromatin-binding protein cation patterns surrounding alternative exons are connected to MRG15 (19). differential alternative splicing outcomes. Here we show that spli- Chromatin structure regulates various aspects of transcription cing regulator Hu proteins can induce local histone hyperacetyla- that are mediated by RNA polymerase II (RNAPII). As an ob- tion by association with their target sequences on the pre-mRNA vious link between chromatin structure and pre-mRNA splicing, surrounding alternative exons of two different genes. In both the transcriptional behaviors of RNAPII, such as pausing and primary and mouse embryonic stem cell-derived neurons, histone transcriptional elongation rate, have been demonstrated to influ- hyperacetylation leads to an increased local transcriptional elonga- ence alternative splicing outcomes (9, 20–22). To understand how tion rate and decreased inclusion of these exons. Furthermore, we the local rate of transcriptional elongation is regulated to impact demonstrate that Hu proteins interact with histone deacetylase 2 alternative splicing outcomes, it is imperative to investigate the

and inhibit its deacetylation activity. We propose that splicing reg- mechanisms by which local chromatin structure and histone mod- BIOCHEMISTRY ulators may actively modulate chromatin structure when recruited ification are modulated. To date, very few studies have addressed to their target RNA sequences cotranscriptionally. This “reaching this question. Given the fact that splicing of pre-mRNA occurs back” interaction with chromatin provides a means to ensure accu- in situ at its chromosomal gene location, it is expected that rate and efficient regulation of alternative splicing. cross-talk can occur in both directions (10). In this context, it is reasonable to propose that splicing regulators, in most cases histone acetylation ∣ neurofibromatosis type 1 ∣ Fas RNA-binding proteins, modulate histone modifications in a localized and RNA-dependent manner. However, to date, no exam- ecent genome-wide transcriptome analysis has demonstrated ples have been discovered to suggest an active role for splicing Rthat more than 95% of human genes undergo alternative spli- regulators in modulating chromatin structure, transcriptional cing to produce multiple proteins from one gene (1–4). Most of elongation rate, and alternative splicing. these alternative splicing events lead to coding differences and Here we describe experiments that reveal a unique functional occur in a cell type- and/or developmental stage-specific manner connection between the roles of Hu RNA-binding proteins in (3, 5), underscoring the essential role of alternative splicing in regulating alternative splicing and histone acetylation. The Hu gene expression control. In addition to the well-established role proteins (HuA/R, HuB, HuC, and HuD) are a family of mamma- of RNA-binding proteins in the regulation of pre-mRNA alter- lian RNA-binding proteins. Of the four Hu family members, native splicing (6, 7), recent studies have revealed a role for chro- HuA/R is widely expressed in many cell types, whereas HuB, matin-associated proteins and the transcription machinery in HuC, and HuD, are expressed specifically in neurons. We have – splicing regulation (8 10). previously demonstrated a role for Hu proteins as splicing regu- A recent study of large human genes demonstrated that pre- lators (23). To date, at least four splicing targets of Hu proteins – mRNA splicing is cotranscriptional and occurs within 5 10 min of have been identified (23–27). These studies show that Hu pro- synthesis (11). The tight coupling of transcription and splicing teins bind to uridine (U)-rich or adenosine/uridine (AU)-rich predicts cross-talk between chromatin structure and splicing RNA sequences and interact with spliceosomal factors to regu- regulation. Indeed, several recent studies have documented a late exon inclusion negatively or positively. number of interesting links between chromatin features and We report a unique mechanism by which Hu proteins increase exon behavior. First, a ChIP analysis indicated that a specific histone acetylation in regions surrounding alternative exons histone modification, trimethylation of lysine 36 of histone H3 leading to an increased local elongation rate and decreased inclu- (H3K36me3), differentially marks exons (12, 13). Remarkably, sion of these exons. Importantly, this regulation occurs through this histone mark appears to be associated more significantly with the association of Hu proteins with their target sequences on constitutive exons than with alternative exons (13). Second, a genome-wide analysis of nucleosome occupancy showed that nucleosomes are enriched in exons and are depleted in introns, Author contributions: H.-L.Z. and H.L. designed research; H.-L.Z., M.N.H., V.A.B., and C.G. suggesting that nucleosome position helps to distinguish introns performed research; H.-L.Z., G.Z., G.L., and R.E.S. contributed new reagents/analytic tools; from exons (12, 14–17). Although these studies provide signifi- H.-L.Z. analyzed data; and H.-L.Z. and H.L. wrote the paper. cant evidence for cross-talk between chromatin and splicing, the The authors declare no conflict of interest. nature of the cross-talk remains largely unknown. Several studies This article is a PNAS Direct Submission. support a model in which histone marks function to recruit basal Freely available online through the PNAS open access option. spliceosomal factors or splicing regulators to ensure efficient 1To whom correspondence should be addressed. E-mail: [email protected]. splicing regulation. For example, the histone mark H3K4me3 See Author Summary on page 14717. was shown to facilitate efficient splicing through recruiting the This article contains supporting information online at www.pnas.org/lookup/suppl/ spliceosomal component U2 snRNP via the H3K4me3 binding doi:10.1073/pnas.1103344108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103344108 PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ E627–E635 Downloaded by guest on October 7, 2021 nascent pre-mRNA molecules. Furthermore, we show that Hu A proteins decrease the deacetylation activity of histone deacetylase Neuron: 23 24 NF1 23 23a 2 (HDAC2). We propose that splicing regulators may actively 24 ES: 23 23a 24 modulate chromatin structure when recruited to their target Neuron: 5 7 RNA sequences cotranscriptionally. This “reaching back” inter- Fas 5 6 7 action with chromatin provides a means to ensure accurate and ES: 5 6 7 efficient regulation of alternative splicing, supporting a more dynamic and integrated view of gene expression control. B C Results Hu Proteins Associate with Transcriptionally Active RNAPII in Neuronal Cells. Our previous studies demonstrated that Hu proteins play an 23 23a 24 5 6 7 important role in the nucleus as alternative splicing regulators. As splicing occurs in situ at the site of transcription (11), we exam- 23 24 5 7 ined if and how Hu proteins function in the context of coupled transcription and splicing. Given that three of four Hu protein family members are almost exclusively expressed in neurons, we used mouse primary cerebellar neurons and neurons differentiated from mouse ES cells throughout our studies. These pri- D mary neurons and ES cell-derived neurons (ES neurons) have proven to be ideal systems for three reasons. First, two of the previously characterized Hu protein targets, neurofibromatosis type 1 (NF1 for human and Nf1 for mouse) and apoptosis-promoting receptor Fas are endogenously expressed in mouse ES cells and in neurons differentiated from ES cells. Second, both alternative exons, exon 23a of Nf1 and exon 6 of Fas, are regulated differentially in neuronal cells with almost exclusive skipping of the alternative exon in neurons (Fig. 1 A–C; sequences of the two E F exons shown are in Fig. S1). Third, Hu proteins are abundantly expressed in neurons, as shown in Western blot and RT-PCR analysis (Fig. 1D and Fig. S2B). Of the four Hu protein members, HuA/R is expressed in both ES cells and neurons, whereas the RNAPII other three members are significantly enriched in neurons RNAPII (Fig. 1D and Fig. S2B). It should be noted that the commercial anti-HuR antibody has cross-reactivity to HuB, HuC, and HuD (Fig. S2A) and therefore was used to detect expression of these neuron-specific Hu protein members. The C-terminal domain (CTD) of the largest subunit of G H RNAPII, Rbp1, consisting of multiple repeats of a heptamer sequence, provides a crucial link between transcription and splicing (22, 28, 29). The three serines in the heptamer sequence, serine 2 (Ser2), serine 5 (Ser5), and serine 7 (Ser7), are differentially phosphorylated during different stages of transcription (30). Ser5 RNAPII phosphorylation peaks at the promoter region of a gene and Ser2 RNAPII phosphorylation accumulates during transcriptional elongation (31). To determine if Hu proteins interact with differentially phosphorylated RNAPII, we carried out coimmunoprecipitation Fig. 1. Splicing pattern of two Hu targets and association of Hu proteins (coIP) analyses using protein lysates isolated from primary neu- with RNAPII. (A) Schematic diagram of the alternative splicing pathways rons and three different antibodies against RNAPII. Interactions of Nf1 and Fas pre-mRNA. Black and white boxes represent alternative between endogenous HuR and RNAPII that was either unpho- and constitutive exons, respectively. Hu binding sites are 111 nt upstream sphorylated or phosphorylated at Ser5 or Ser2 were detected in and 16 nt downstream of NF1 exon 23a, and 25 nt downstream of the 5′- reciprocal coIP assays (Fig. 1 E and F). These results indicate that end of Fas exon 6. (B and C) Splicing of exon 23a of the endogenous Nf1 HuR is associated with both initiating RNAPII and elongating pre-mRNA (B) and exon 6 of the endogenous Fas pre-mRNA (C) in ES cells, RNAPII. Next, we demonstrated that HuR also interacts with ES-derived neurons, and primary neurons was detected by RT-PCR. (D) Expres- Cdk9, a component of the elongation factor P-TEFb complex that sion of Hu proteins was detected by Western blot analysis. Anti-HuR antibody (Left) and anti-HuB antibody (Right) were used. U1 70 K was used as phosphorylates Ser-2 (Fig. 1F). Furthermore, these interactions β – a loading control. -tubulin III is a neuronal marker. (E) CoIP was carried are RNA-independent indicative of direct protein protein inter- out with nonspecific IgG or antibodies against hypophosphorylated RNAPII action (Fig. 1G). (8WG16), ser-2 phosphorylated RNAPII (H5), or ser-5 phosphorylated RNAPII To determine if HuR directly interacts with the RNAPII (H14) using protein lysate isolated from mouse cerebellar neurons. Input (5% complex, we carried out a GST-pull-down experiment using an of total lysate) and immunoprecipitated proteins were analyzed by Western immunopurified RNAPII core complex that contains all of the blot using anti-HuR antibody. (F) CoIPs were performed with nonspecific IgG previously defined RNA polymerase II subunits (RPB) (32, 33). or an antibody against HuR using primary neuron lysates. Input (5% of total As indicated in Fig. 1H, GST-HuR did pull down the large sub- lysate) and immunoprecipitated proteins were analyzed by Western blot unit RPB1 of RNAPII. This finding suggests a direct interaction using 8WG16, H5, H14, anti-cdk9, or GAPDH antibodies. (G) The immunoprecipitated proteins were treated with RNase A during IP. Hypo RNAPII: hypo- between HuR and the RNAPII core complex. phosphorylated RNAPII. (H) GST pull-down analysis was carried out using Next, we carried out a number of coIP experiments to further immunopurified RNAPII core complex and GST or GST-HuR. Input (2% of characterize the interaction between Hu proteins and RNAPII. total) and pull-down proteins were analyzed by Western blot using 8WG16 These experiments were conducted in HeLa cells because of antibody.

E628 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1103344108 Zhou et al. Downloaded by guest on October 7, 2021 the ease of high-efficiency transfection of these cells. We found PNAS PLUS that (i) the interactions between HuR and RNAPII also occurred in HeLa cells (Fig. S3 A–D), (ii) all members of the Hu protein family, when overexpressed, interacted with RNAPII (Figs. S3E and S4A), and (iii) the RRM3 and hinge domain appear to be important for this interaction and when deleted, reduced interaction between HuC and RNAPII (Fig. S4B). These experiments demonstrate that all of the Hu family members are capable of interacting with RNAPII and suggest that the RRM3 domain and, to a lesser extent, the hinge domain are responsible for this interaction.

Hu Proteins Regulate the Local Transcriptional Elongation Rate Sur- rounding Alternative Exons. The interaction between Hu proteins and transcriptionally active RNAPII prompted us to hypothesize that Hu proteins play a role in modulating the elongation rate surrounding the alternative exons they regulate. To test this possibility, we first performed ChIP to investigate the distribution of RNAPII on Nf1 and Fas using the H5 antibody specific to Ser-2 phospho-CTD RNAPII. Fig. 2A indicates the PCR products analyzed in a real-time PCR assay. Primer pairs were chosen to flank exon–intron junctions with one primer annealing to an exo- nic sequence and the other to an intronic sequence. These experiments revealed a significant reduction of RNAPII between the alternative exon 23a and exon 28 of the Nf1 gene in ES neurons compared to undifferentiated ES cells (Fig. 2B). A strong reduction of RNAPII after alternative exon 6 of the Fas gene is also C observed in ES neurons (Fig. 2 ). These observations suggest a BIOCHEMISTRY faster elongation rate in DNA surrounding the two alternative exons in ES-derived neurons. To obtain a more direct measurement of the transcriptional elongation rate, we analyzed accumulation of nascent Nf1 pre-mRNA at different exons. The Nf1 gene spans 350 kb with a distance from exon 1 to alternative exon 23a covering more than 120 kb, which makes Nf1 an ideal substrate for elongation rate analysis. We used a method modified from a study by Singh and Padgett (11). In this assay a CDK9 inhibitor, DRB, was used to block transcription elongation. After DRB treatment, cells were incubated with BrU, which is incorporated into all of the newly synthesized pre-mRNA tran- scripts. We then precipitated RNA at different time points using anti-BrU antibody and carried out real-time RT-PCR to analyze pre-mRNA accumulation. As the goal of our experiments was to assess the effect of Hu proteins on transcriptional elongation, we compared pre-mRNA accumulation between high Hu-expressing and low Hu-expressing cells, and between ES-derived neurons and ES cells. The newly synthesized RNAs were isolated from cells at different time points after release from DRB, cDNAs were prepared, and real-time PCR was carried out using primer pairs surrounding each exon–intron junction indicated in Fig. 2A. Fig. 2. Hu proteins regulate transcriptional elongation rate. (A) Schematic Next, the pre-mRNA accumulation was plotted as a time course D diagrams of Nf1, Fas, and KIFAP3 genes showing the exons analyzed in the (Fig. 2 ). The neuron-specific alternative exon kinesin-asso- following experiments. The numbers shown above each diagram indicate the ciated protein 3 (KIFAP3) exon 20, which is differentially in- distance between two exons in kilobases. Dashes indicate amplified PCR pro- cluded in the two types of cells but is not regulated by Hu ducts. (B and C) ChIP analysis using H5 antibody and ES cells or ES-derived proteins (Fig. S5 A and B), was used as a negative control. At neurons. The graphs demonstrate accumulation of RNAPII in ES cells (trian- time zero, no real-time PCR signal was observed, whereas at later gles) and ES-derived neurons (squares) at the indicated positions along the points, KIFAP3 exon 20 accumulated at a similar rate in ES Nf1 (B)orFas(C) gene. The RNAPII signal was normalized to a signal obtained cells and ES-derived neurons. For the Nf1 gene, the pre-mRNA from an intergenic region. (D and E) Real-time RT-PCR was used to measure the expression level of the Nf1 pre-mRNA. ES cells and ES-derived neurons accumulation rate of exons 1, 23, and 39 is similar in the two cell were treated with DRB to block transcription. After release from DRB, expres- types. However, a 2.5-fold increase in pre-mRNA accumulation sion of different exons of Nf1 shown in A was analyzed at indicated times and surrounding the region of exons 23a and 24 in ES-derived neu- normalized to the expression level of utrophin exon 2. The graph demon- rons was observed indicating a higher elongation rate in this strates the expression level of various exons relative to the level in untreated region (Fig. 2D). cells, which was set to 1 in ES cells (triangles) and ES-derived neurons (squares) To determine if Hu proteins are responsible for the higher at indicated times in D as well as in ES cells that do not express HuC (triangles) elongation rate in the ES-derived neurons, we established an and in ES cells that express HuC (squares) at indicated time points in E. ES cell line that stably incorporated an HuC expression cassette driven by a doxycycline (Dox)-inducible promoter. As indicated presence of Dox, which led to significantly decreased inclusion of in Fig. S5 C and D, overexpression of HuC was observed in the Nf1 exon 23a. Using the anti-HuR antibody that recognized both

Zhou et al. PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ E629 Downloaded by guest on October 7, 2021 HuR and HuC, we estimate that in these cells, the level of the the HuC protein are important for its interaction with HDAC2 overexpressed Myc-HuC is 3- to 4-fold of the endogenous (Fig. S6 B and C). Although all of the Hu proteins are capable of HuR (Fig. S5D). Next we analyzed pre-mRNA accumulation interacting with HDAC2, it appears that only the class I HDACs of Nf1 comparing ES cells that express HuC to those that do that include HDAC1, HDAC2, HDAC3, and HDAC8 can inter- not express this protein. We found that the pre-mRNA accumu- act with Hu proteins, as we observed an interaction of HuC with lation surrounding the region of exons 23a and 24 was approxi- HDAC1 and HDAC2 but not HDAC7 (Fig. S6D). mately 1.5- to 2-fold higher in HuC-expressing ES cells than in HuC-non-expressing ES cells, whereas KIFAP3 exon 20 and Hu Proteins Inhibit HDAC2 Activity in Vitro. Two possible explana- Nf1 exons 1, 23, and 39 had similar accumulation rates in the tions might account for the observed interaction between Hu two types of cells (Fig. 2E). These results indicate that Hu pro- proteins and histone deacetylases: that the splicing regulators teins increase the local transcriptional elongation rate of the Nf1 are enzymatic substrates, or that they modulate HDAC activity. gene surrounding exon 23a. HDAC proteins catalyze an enzymatic reaction in which acetyl groups, which are added to lysines by members of acetylase Transcriptional Elongation Rate Regulates Inclusion of Nf1 Exon 23a. family, are removed. This reversible lysine acetylation is a highly In light of the aforementioned results, we hypothesized that Hu regulated posttranslational modification that occurs on more proteins specifically regulate Nf1 exon 23a alternative splicing than 195 proteins in HeLa cells in addition to histones (35). by affecting transcriptional elongation rate. To determine if Totest if Hu proteins can be acetylated on lysines, HuR or histone alternative inclusion of Nf1 exon 23a can be modulated by tran- H4 proteins were immunoprecipitated from ES cells by anti-HuR scriptional elongation rate, we carried out the following experi- or anti-H4 antibodies, respectively. A Western blot using antia- ment. We cotransfected primary neurons with the NF1 reporter, cetyl lysine antibody demonstrated that H4, but not HuR, was previously used to study alternative splicing of exon 23a (24) acetylated. The signal from histone H4 proteins could be com- (Fig. 3A), and two RNAPII mutants that are α-amanitin-resistant peted away efficiently by addition of acetylated BSA (Fig. 4F). (34). One of the two mutants, C4, also carries an amino acid sub- This result suggests that the interaction between HDAC2 and stitution that reduces the rate of transcriptional elongation by Hu proteins does not lead to acetylation of Hu proteins. RNAPII. After transfection, cells were treated with α-amanitin To test whether Hu proteins regulate the activity of HDAC2, to inhibit endogenous RNAPII to ensure that the NF1 reporter we conducted an HDAC2 activity assay in vitro using tritium- would be transcribed solely by the exogenously introduced RNA- labeled acetylated histone H4 peptides that were conjugated PII. As shown in Fig. 3B, transcription by the C4 RNAPII mutant to biotin as a substrate. HDAC2 activity was measured by cal- resulted in increased exon 23a inclusion (compare lane 4 to culating the amounts of the released tritium-labeled acetate lane 3). when HDAC2 was added. Recombinant GST-Hu proteins were added to the reaction together with HDAC2 prepared from HeLa Hu Proteins Directly Interact with Histone Deacetylase 2. In an initi- nuclear extract through immunopurification (Fig. 4 G–I). Addi- ally parallel avenue of investigation that unexpectedly converged tion of GST-HuC reduced the HDAC2 activity by 2- to 2.5-fold with our investigation of the roles of Hu proteins on transcrip- (Fig. 4 G and H). Importantly, the Hu-mediated reduction of tional elongation, we used a yeast two-hybrid screen with HuC HDAC2 activity is time- and dose-dependent (Fig. 4 G and H). as bait and identified HDAC2 as a potential interaction partner When the HDAC protein inhibitor sodium butyrate was added to (Fig. 4A). Purified recombinant GST-HuC can pull down in vitro the reaction, the deacetylation was dramatically reduced, con- translated HDAC2 protein that is 35S-labeled (Fig. 4B). We also firming that the observed activity was from histone deacetylase confirmed the interaction between Hu proteins and HDAC2 by present in the HDAC2 protein preparations. Furthermore, con- coIP using anti-HuR antibody and protein lysate prepared from sistent with its role in interaction with the HDAC2 protein, dele- primary neurons (Fig. 4C), and this interaction is RNA-indepen- tion of the hinge domain from the HuC protein diminished the dent, indicating a direct interaction (Fig. 4D). The interaction ability of the resulting HuC protein to regulate HDAC2 activity occurs between HDAC2 and all of the Hu family members (Fig. 4H). HuR, HuB, and HuD showed similar activity (Fig. 4I). (Fig. S6A). Finally, a biochemical analysis using recombinant These results demonstrate that Hu proteins inhibit the HDAC2 GST-Hu and MBP-HDAC2 fusion proteins provided definitive activity through their interaction with HDAC2. evidence for a direct interaction between all of the Hu family members and HDAC2 (Fig. 4E). Hu Proteins Promote Local Histone Acetylation. HDAC2 catalyzes Using HuC truncation mutants through both in HeLa cells and the deacetylation of histone proteins. To determine if Nf1 exon in in vitro analysis indicate that the hinge and RRM3 domains of 23a splicing could be modulated by a change in the histone acetylation pattern, we performed ChIP with antibodies recognizing A B pan-acetylated H3 or H4 histones using ES cells and ES-derived neurons and analyzed the histone acetylation pattern by real- Reporter NF1 time PCR using primers distributed along the Nf1 gene (Fig. 2A).

1 23a 2 3 We also analyzed the alternative exon extra domain I (EDI) of 1 2 23a 3 fibronectin and exon 20 of KIFAP for comparison. The signals 1 2 3 obtained for Nf1, fibronectin, and KIFAP were normalized to the β-actin signal and a ratio was calculated in which, for any given Reporter NF1 primer pair (Fig. 2A), the normalized signal obtained from ES-derived neurons was divided by that from ES cells. As shown in Fig. 5A, no differences were detected in the levels of pan- acetylation of H3 and H4 on the fibronectin gene at EDI exon and KIFAP exon 20, nor on the Nf1 gene at the promoter as well Fig. 3. Transcriptional elongation rate regulates splicing of Nf1 exon 23a. as exons 23 and 29. In contrast, the levels of pan-acetylated H3 (A) Schematic diagram of the alternative RNA processing pathways of and H4 were increased by approximately twofold between exons the Nf1 reporter minigene. (B) Transcription by C4 slow RNAPII increases exon 23a inclusion. Primary neurons were cotransfected with Nf1 reporter and an 23a and 28 in ES-derived neurons. empty plasmid (control) or plasmids expressing WT or C4 α-amanitin-resistant To determine if Hu proteins can regulate the levels of H3 and RNAPII large subunits. Transfected cells were treated with α-amanitin to H4 acetylation, we carried out similar experiments using HuC- inhibit the endogenous RNAPII for 48 h before RNA isolation. expressing and HuC-non-expressing ES cells described earlier

E630 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1103344108 Zhou et al. Downloaded by guest on October 7, 2021 Fig. 4. Hu proteins interact with and inhibit the activity of A B C PNAS PLUS HDAC2. (A) Yeast two-hybrid analysis. Pairwise two-hybrid interactions are indicated by both growth and the activity of the β- galactosidase reporter. BD: DNA-binding domain, AD: activating domain. (B) In vitro binding of 35S-methionine-labeled HDAC2 to recombinant GST-HuC fusion protein. 35S-methionine-labeled luciferase and HuC were used as negative and positive controls, respectively. (C) Interaction of HuR with HDAC2 in primary neurons. HDAC2 was immunoprecipitated by anti-HuR antibody from neuron lysate (Top), whereas HuR was immunoprecipitated by anti-HDAC2 antibody (Bottom). Input (5% of total D lysate) and immunoprecipitated proteins were analyzed by Wes- F tern blot. (D) Immunoprecipitated proteins were treated with RNase A during IP. (E) Interaction of Hu proteins with HDAC2 E in vitro. GST pull-down experiment was carried out using GST, GST-HuR, GST-HuB, GST-HuC, or GST-HuD and MBP-HDAC2. Wes- tern blot analysis was carried out with anti-HDAC2 antibody (Top) or anti-GST antibody (Bottom). (F) HuR is not acetylated. HuR or histone H4 proteins were immunoprecipitated with corresponding antibodies and probed with an antiacetyl lysine antibody by Western blot assay with (+) or without (−) lysine- acetylated BSA (Ac-BSA). (G) Regulation of HDAC2 activity by G H I HuC. Immunoprecipitated HDAC2 protein (8 nM) from HeLa nuclear extract was incubated with [3H]acetylated histone H4 peptide in the presence of 200 nM GST or 75 nM GST-HuC. The HDAC2 activity was measured during a time course. The cpm on the Y axis indicates radioactivity released from the [3H]acetylated histone H4 peptide into the supernatant through the HDAC2 activity. (H) Dose-dependent inhibition of HDAC2 activity by HuC. Increasing amounts of GST-HuC (1.85–75 nM) was added to the HDAC2 activity assay and the HDAC2 activity was

measured after 24 h of incubation. GST (200 nM), GST-ΔHinge BIOCHEMISTRY (85 nM), or sodium butyrate (HDAC inhibitor, 200 nM) was used as controls. The HDAC2 concentration is 8 nM. (I) HDAC2 activity regulated by Hu family members. Eight nanomolars of HDAC2 and 75 nM of GST-Hu protein were used.

(see Fig. 2). We found that H3 and H4 acetylation was increased exon 23a through the interaction with their cognate binding sites between exons 23a and 28 in HuC-expressing cells, whereas the in introns surrounding exon 23a. To test this hypothesis, we inves- acetylation level remained the same at the fibronectin EDI exon, tigated whether the association between HuR and exon 23a is KIFAP3 exon 20, as well as the Nf1 gene at the promoter and RNA-dependent. RNase treatment before immunoprecipitation B at exons 23, 29, and 39 in the two types of ES cells (Fig. 5 ). significantly reduced the accumulation of HuR (Fig. 6B), suggest- Another Hu-regulated alternative exon, exon 6 of the Fas gene, ing that the AU-rich elements in introns surrounding exon 23a along with the downstream exon 7, exhibited a similar increase of histone acetylation (Fig. 5 C and D). Although we cannot rule out the possibility that the observed data resulted from a change of nucleosome positioning, we believe these results, in combination with those of the HDAC2 activity analysis, strongly suggest that Hu proteins can regulate local histone acetylation levels surrounding Hu-regulated alternative exons. We carried out an shRNA knockdown experiment to examine the contribution of individual Hu protein members. In mouse primary neurons, knockdown of HuB, HuC, or HuD individually resulted in a moderate change in splicing as well as in histone H3 and H4 acetylation (Fig. S7). When the three shRNAs were used in combination, significant changes in both splicing and histone acetylation were observed (Fig. S7), indicating that these activities were regulated by the overall level of Hu proteins instead of specific activities of any particular Hu members.

RNA-Dependent Histone Hyperacetylation Surrounding Exon 23a. We reasoned that if Hu proteins regulate local histone acetylation by blocking the activity of HDAC2, they should be closely associated Fig. 5. Hu proteins regulate local histone acetylation levels. (A–D) Mapping with the corresponding genomic DNA. Thus, we investigated if of pan-histone H3 (light gray bars) or H4 (black bars) acetylation at the HuR antibody could immunoprecipitate exon 23a of Nf1 through indicated positions of Nf1 (A and B)orFas(C and D) shown in Fig. 2A by ChIP ChIP in primary neurons. A ChIP assay demonstrated that HuR followed by real-time PCR. The relative proportions of coimmunoprecipi- proteins are associated with the Nf1 promoter as well as the tated gene fragments were normalized to the value obtained from a β-actin region surrounding exons 23a and 24 of the Nf1 gene (Fig. 6A). control region. (A and C) The graphs demonstrate fold differences of histone H3 or H4 acetylation at different exons of Nf1 and Fas between ES-derived Hu proteins are expected to associate with the promoter region as neurons and ES cells. (B and D) The graphs demonstrate fold differences of they interact with the transcription machinery. As Hu proteins histone H3 or H4 acetylation at different exons of Nf1 and Fas between regulate splicing through binding to AU-rich elements on pre- ES cells that express HuC and those that do not express HuC. The fibronectin mRNA (24), we predicted that they would strongly associate with EDI exon and KIFAP3 exon 20 were used as controls.

Zhou et al. PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ E631 Downloaded by guest on October 7, 2021 A B regulators can modulate histone modifying enzyme activity directly, which leads to altered local transcriptional elongation rates and distinct alternative splicing outcomes. It is conceivable that to efficiently and precisely regulate alternative splicing, chromatin modification and transcriptional elongation rate modulation must be directed to a specific region of a gene and experimental support for this idea is emerging. One study indicated that the SWItch/Sucrose NonFermentable chromatin remodeling complex is involved in slowing down transcription of the variable region of CD44 by RNAPII to increase inclusion of the variable exons (37). The variable exons are associated with an elevated level of H3K9 trimethylation, which appears to recruit HP1γ to the chromosomal region containing C these exons to slow down the local transcriptional elongation rate WT-NF1Mut-NF1 and affect alternative splicing (38). Another study showed that introduction of siRNAs targeting a region surrounding an alter- WT-NF1 1 23a 2 3 1 2 23a 3 native exon induced formation of a heterochromatic complex involving HP1 and reduced the transcriptional elongation rate, Mut-NF1 1 X 23a 2 3 1 2 3 which in turn affected alternative splice-site usage (39). Last, de- 8 30 polarization of a neuronal cell line was found to induce localized D epigenetic changes that increased elongation rate and triggered skipping of NCAM exon 18 (34). However, these studies did not provide insights into how local chromatin structure or histone modifications are modulated. Histone modifications are catalyzed by different types of modifying enzymes. Thus, it is conceivable that recruitment of such modifying enzymes to a targeted site of action as well as regulation of localized modifying enzyme activity determine the establishment and maintenance of local histone modification (40). Here we present evidence to support modulation of histone modifying enzyme activity in an RNA-dependent manner by splicing regulators. Histones can be acetylated and deacetylated to regulate gene Fig. 6. The role of pre-mRNA targets in Hu-mediated chromatin modifica- transcription (41). The rapid turnover of histone acetylation is tion. (A) ChIP analysis indicating HuR accumulation on the Nf1 gene in pri- very important for nucleosome dynamics during transcriptional mary neurons. Anti-T cell intracytoplasmic antigen 1-related protein antibody – was used as a negative control (24). (B) HuR accumulation on the Nf1 gene is elongation (42 44). When pre-mRNA is transcribed by RNAPII, RNA-dependent. ChIP analysis in the absence or presence of RNase A. (C) Spli- acetylation of the nucleosomes in front of the elongation machin- cing of wild-type and mutant NF1 reporter in neurons. The AU-rich sequences ery by histone acetyltransferases (HATs) is required (45, 46). The located 110 nucleotides upstream of exon 23a are mutated in the mutant passage of RNAPII causes displacement of histones, which are reporter (24). The reporters were transfected into mouse primary neurons. subsequently redeposited onto the DNA behind RNAPII. These RT-PCR was carried out using total RNA isolated from the transfected cells. newly deposited nucleosomes are hyperacetylated, but only tran- (D) H3 and H4 acetylation levels were analyzed as described in Fig. 5. PCR products analyzed in the ChIP assay are indicated as bars above the reporter siently. In order to keep the normal chromatin configuration, diagrams in Fig. 6C. histone deacetylase complexes remove the acetyl marks (47, 48). This dynamic process provides a regulatory step to establish local of pre-mRNA direct local histone modification mediated by the chromatin acetylation status. Hu-HDAC2 complex. Recently, multiple studies have demonstrated that histone To provide further evidence for this argument, we compared acetylation modification at alternative exons is connected to the histone acetylation patterns of the wild-type NF1 reporter differential splicing outcomes (34, 39, 49). Here we provide evi- to that of a mutant reporter in which the AU-rich sequences dence to suggest that splicing regulator Hu proteins may block upstream of exon 23a were disrupted. This mutation disrupted removal of acetyl marks during the dynamic process of nucleo- one of the major Hu protein binding sites and led to increased some repositioning through inhibiting HDAC2 activity. As a re- inclusion of exon 23a in neurons from 8 to 30% (Fig. 6C) as well sult, the histone hyperacetylation status of a specific region as in neuron-like cells shown in our previous studies (24). As pre- sustained by Hu proteins increases transcriptional elongation. dicted, significantly higher levels of H3 and H4 acetylation was We propose that the pioneer round of transcription allows initial observed in the nucleosomes formed on the wild-type reporter chromatin modulation to occur, establishing a local histone hy- than the mutant reporter surrounding exon 23a (Fig. 6D), which peracetylation status to be encountered by the later elongating correlates well with the exon 23a inclusion result. These results RNAPII. This activity will induce RNAPII to transcribe the re- strongly suggest that Hu-induced histone hyperacetylation de- gion of hyperacetylated chromatin in a faster mode (Fig. 7). The pends on their binding sites on the pre-mRNA. tissue- or developmental stage-specific expression of Hu proteins can turn on or off histone hyperacetylation at a specific region to Discussion regulate alternative splicing. Our data suggest that Hu proteins The current study reveals a unique role for pre-mRNA splicing are recruited to the transcribing gene through a direct interaction regulators that integrates chromatin structure, histone modifica- with a component of the RNAPII complex (Fig. 1H). Further- tions, transcriptional elongation rate, and alternative splicing. more, when the Hu target sites on pre-mRNA emerge from Although recent studies have established a link between chroma- RNAPII, Hu proteins are transferred from the RNAPII complex tin modification and splicing, they have focused on how histone to RNA. Thus, the local concentration of Hu proteins is in- marks regulate RNA splicing (8, 36). Here we show that splicing creased, which leads to the regulated HDAC2 activity.

E632 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1103344108 Zhou et al. Downloaded by guest on October 7, 2021 splicing by modulating basal splicing factor binding (23, 24, 26). PNAS PLUS The current study reveals a previously undescribed mechanism that integrates the role of Hu proteins in chromatin modification, transcriptional elongation, and alternative splicing regulation. Our results demonstrate that Hu proteins associate with both unphosphorylated and phosphorylated RNAPII. These results are consistent with previous studies indicating that HuR is in association with both the spliceosome and the RNAPII complex (56–58). Thus, we propose that Hu proteins are deposited from the transcribing RNAPII complex to AU-rich elements of the pre-mRNA when Hu protein targets emerge from the transcribing RNAPII (Fig. 7). This mechanism may be applied to other Hu-mediated alternative splicing events as we show that at least two splicing events, Nf1 and Fas alternative splicing, can be regu- Fig. 7. A model for RNA-directed modification of histone acetylation by Hu lated in this manner. We propose that this integrated regulatory proteins. mechanism serves to ensure accuracy and efficiency of splicing regulation, as local changes in transcriptional elongation For future studies, it will be interesting to investigate potential mediated by splicing factors can reinforce splicing choices that changes of specific histone marks induced by binding of Hu pro- are also regulated by the same splicing factors. teins to their targets. For example, depolarization induces loca- Although it has been demonstrated that serine/arginine-rich lized increase of H3K9ac as well as H3K36 trimethylation level proteins link splicing and transcription and that splicing factor surrounding the NCAM exon 18 (34). It will also be intriguing to SC35 can regulate the elongation rate through its association with examine how neuron activity such as depolarization affects alter- RNAPII (56, 59), no splicing regulators have been found to be native splicing of NF1 exon 23 in the context of histone modifica- engaged in RNA target sequence-directed regulation of tran- tions. The comparison of our results to NCAM exon will provide scriptional elongation rate. Thus, our findings will have major im- more insights into histone modification-mediated alternative spli- plications for understanding how alternative splicing is regulated cing change. in the context of chromatin and transcription. The bidirectional

Hu protein-mediated histone modification at specific exons regulation of transcription and splicing described in this study BIOCHEMISTRY requires the presence of AU-rich elements on the pre-mRNA strongly supports the previously described “gene expression ma- (Fig. 6). When the Hu binding element (AU-rich sequence) is chine” view (60–62), as well as providing significant insights into a present such as in Nf1 and Fas alternative exons (24, 26), the unique mechanism to connect different steps of gene expression. deacetylation of histones surrounding the alternative exon is reduced. Importantly, this regulation of histone acetylation as well Methods as of the transcriptional elongation rate does not occur surround- Cell Culture and Generation of Cell Lines. Mouse cerebellar neurons were cultured using a modification of a previously described procedure (63). In brief, ing exons that are not targets of Hu proteins such as KIFAP3 exon cerebella were removed from 6- to 8-d-old mice. The dissociated cerebellar 20 and the EDI exon of the fibronectin gene (Figs. 2 and 5). The cells were plated onto tissue culture plates coated with 0.1 mg∕mL poly-L- results of these experiments strongly suggest that association of lysine (Sigma). The cells were maintained in deﬁned medium composed of Hu proteins with AU-rich elements is necessary for histone mod- neurobasal media supplemented with B-27, 2 mM glutamine, 25 mM KCl, ification at specific exons. Consistently, HuR ChIP data indicate and 0.3 g∕mL glucose. Beginning on the second day of culture, cells were that association of HuR proteins with Hu targets is dependent treated with 5 μM cytosine arabinoside (AraC), a mitosis inhibitor. Fifty per- on RNA (Fig. 6 A and B). Moreover, disruption of a major cent of media was replaced with fresh media every 3 d. In transfection ex- 6 Hu binding site upstream of exon 23a abolished Hu-mediated his- periments, 5 × 10 freshly isolated neurons were used for each transfection tone hyperacetylation surrounding exon 23a (Fig. 6 C and D). with Nucleofector II program C13 and mouse neuron Nucleofector kit Therefore, the AU-rich elements on pre-mRNA direct local his- (Lonza). The neurons were then cultured in DMEM supplemented with 10% FBS for 1 d before switching to defined media supplemented with AraC. The tone acetylation mediated by Hu proteins. neurons were collected for analysis after 6 d in culture. The Institutional Ani- How does splicing activity affect the RNAPII elongation mal Care and Use Committee at Case Western Reserve University approved behavior in general? Recent studies provided some interesting these mouse experiments and confirmed that all experiments conform to insights. Two studies indicated that splicing activity causes RNA- the relevant regulatory standards. PII to pause at the 3′-end of intron-containing genes in yeast Mouse ES cell differentiation was carried out using R1 cells with a pre- (50, 51). In mammalian cells, a fluorescence recovery after photo- viously described procedure (64). To obtain the stable HuC-expressing mouse 6 bleaching-based RNAPII elongation kinetics analysis demon- ES cell line, the Tet-on system (Clontech) was used. In this experiment, 5 × 10 strated that the basal level of splicing activity of multiple mouse ES cells were transfected with the pTet-on vector using the mouse ES intron-containing model genes does not affect transcriptional cell Nucleofector kit (Lonza). The transfected cells were selected for 2 wk with 200 μg∕mL of G418. Thirty colonies were picked and transiently transfected elongation rate (52). In this context, our results suggest that at with pTRE2-HuC. The colony ES-HuC-4 that has low background HuC expres- specific chromosomal regions, splicing regulators such as Hu sion without doxycycline (−Dox) and high HuC expression with doxycycline proteins may regulate the local transcriptional elongation rate (+Dox) was selected. Next, pTRE2-HuC and pGK-puro were cotransfected into upon binding to their target sequence. clone ES-HuC-4, and cells were selected for 2 wk with 5 μg∕mL of Puromycin Splicing is regulated at many different levels in a tissue- or and 200 μg∕mL of G418. Colonies were picked and cultured for 3 d with developmental stage-specific manner (7, 53). At the most funda- 2 μg∕mL of doxycycline. Induction of HuC was detected by Western blot assay mental level, regulation includes splice-site recognition by the using anti-Myc antibody.

spliceosome, which is modulated by many splicing regulators 7 (54). The fact that splicing of pre-mRNA occurs in situ cotran- ChIP and Real-Time PCR. In the ChIP experiment, 5 × 10 cells were fixed with ∕ scriptionally at its chromosome locus implicates higher or more 1% (vol vol) formaldehyde for 30 min. followed by a ChIP assay using a kit (Millipore). Immunoprecipitation was carried out overnight at 4 °C with 15 μg integrated levels of additional regulatory mechanisms that of the H5, anti-acetyl-Histone H3 (Millipore), or anti-acetyl-Histone H4 (Milli- involve chromatin structure, histone modification, and transcrip- pore) antibodies. Nonspecific IgG (Sigma) was used as a control. Cross-linking tion behaviors (55). Here we show that Hu proteins can regulate of bound DNA fragments was reversed, and DNA was dissolved in 100 μLof alternative splicing at both the RNA and chromatin levels. Pre- Tris (10 mM)-EDTA (1 mM). Real-time PCR was carried out using SYBR green viously, multiple studies demonstrated that Hu proteins regulate PCR mix (Qiagen) and the Chromo4 Real Time PCR system (MJ Research).

Zhou et al. PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ E633 Downloaded by guest on October 7, 2021 Relative expression levels were determined using a three-point standard to the expression level of the BrU-labeled control without DRB-treatment, curve generated by diluting a control cDNA sample. The relative proportions which was set to 1 in all experiments (11). For every analyzed gene fragment, of coimmunoprecipitated gene fragments were determined on the basis of each sample was quantified in duplicate and from at least three independent the threshold cycle (Ct) for each PCR product. The Ct values obtained from experiments. immunoprecipitations using specific antibodies were subtracted from the Ct values obtained from that using the control IgG. The resulting values In Vitro HDAC2 Activity Assay. The histone deacetylation assay was conducted were further normalized to the value obtained with a primer pair am- as described by a histone deacetylase assay kit (Millipore). Briefly, biotin- β plifying an intergenic region or -actin. The fold difference between ES conjugated histone H4 peptide was acetylated with 3H-labeled acetyl coen- ð neuron-gene × ES-controlÞ∕ð ES-gene× neurons and ES cells was calculated as Ct Ct Ct zyme A (Perkin-Elmer) using recombinant histone acetyltransferase PCAF. Ctneuron-controlÞ. For every analyzed gene fragment, each sample was quanti- Subsequently, the labeled histone H4 peptide was bound to streptavidin– fied in duplicate and from at least three independent ChIP analyses. In order agarose beads. The activity of purified HDAC2 was assessed by following to investigate if the association between HuR and exon 23a is RNA depen- the release of 3H label from the 3H-labeled acetyl histone H4 peptide. HDAC dent, sonicated cell lysates were treated with 400 μg∕mL of RNase at 37 °C for reactions were carried out in a final volume of 200 μL comprising 10 mM 30 min before immunoprecipitation as described (65). See Table S1 for PCR μ primer sequences. Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 8 g purified MBP-HDAC2, or 50 μL immunoprecipitated HDAC2 complex, agarose 3 – μ Transcriptional Elongation Analysis. Inhibition and reinitiation of transcription beads carrying 40,000 cpm [ H] acetyl histone H4 peptide, and 0.025 1 gof Δ μ were performed as described (11). Bromouridine (BrU) labeling was modified GST, GST-HuC, GST- Hinge, or 50 L 1M sodium butyrate. The reactions were – μ from a previously described procedure (59). Before addition of DRB, ES-de- stopped at 4 30 h. The beads were spun down and 50 L of the supernatant rived neurons were plated for 6 d, whereas ES cells were 50% confluent. was counted in a Beckman LS6500 scintillation counter. These cells were treated with 50 μM DRB (sigma) for 7 h and then 2 mM of BrU was added into the medium for another hour. The cells were washed ACKNOWLEDGMENTS. We thank the following individuals for providing twice with media to remove DRB and then incubated in fresh medium for antibodies and plasmids: Diane Hayward, HDAC2 plasmid (University of indicated time periods. At each time point, total RNA was isolated using TRI- Otago, Otago, New Zealand); Alberto Kornblihtt, mutant RNAPII plasmids zol (Invitrogen). To immunoprecipitate the BrU-labeled pre-mRNA, 15 μgof (University of Buenos Aires, Buenos Aires, Argentina); and Sachiyo Kawamo- BrU antibody (Sigma) was preincubated with 20 μL of protein G Dynabeads to, pMT-6myc [National Institutes of Health (NIH), Bethesda, MD]. We thank (Invitrogen) for 4 h at 4 °C with rotation. Next, 250 μg of total RNA was in- Yves Barde for his advice on neuronal differentiation of ES cells and Mats cubated with beads for 3 h at 4 °C in 200 μL of RSB-100 buffer (10 mM Tris-HCl, Ljungman for his advice on the transcriptional elongation assay. We thank pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 0.4% Triton X-100, 0.2 U∕μL RNaseOut Cheng-Ming Chiang (University of Texas Southwestern Medical Center, and 25 μg∕mL tRNA). The beads were washed five times with RSB-100 buffer. Dallas, TX) for providing the immuno-affinity purified RNAPII core complex. We thank Helen Salz and Jo Ann Wise for critical reading of the manuscript. The RNA bound to beads was eluted by direct addition of 300 μL RLT buffer This work was supported by NIH Grant NS-049103 and DOD Grant NF060083 (Qiagen RNeasy mini kit) supplemented with 2 μg of tRNA. The RNA was (to H.L.). R.S. was supported by NIH Grant 5R03NS59648. G.L. was supported purified with Qiagen RNeasy mini kit and eluted in 30 μL of RNase-free water. by NIH Grant CA112094. H.-L.Z. was supported by postdoctoral fellowships Eight microliters of the purified RNAs were used for reverse transcription in a from the American Heart Association (0725346B and 09POST2250749). μ 20- L reaction using the Superscript III First Strand Kit (Invitrogen) and the M.N.H. was supported by a genetics training grant from NIH μ cDNA (2 L per well) was used for quantitative PCR. The Ct values obtained (T32GM008613) and a National Research Service Award predoctoral fellow- from RT-PCR using the BrU antibody were subtracted from the Ct values ship from the National Institute of Neutological Disorders and Stroke obtained from templates with control IgG. The resulting value was further (1F31NS064724). V.A.B. was supported by a developmental biology training normalized to the value obtained with a primer pair amplifying utrophin grant from NIH (T32HD00710432) and a predoctoral fellowship from the exon 2. The normalized pre-mRNA expression values were plotted relative American Heart Association (0815373D).

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