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Proc Natl Acad Sci U S A. Sep 6, 2011; 108(36): E627-E635.
Published online Aug 1, 2011. doi:  10.1073/pnas.1103344108
PMCID: PMC3169152
PNAS Plus
Biochemistry

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

Abstract

Recent studies have provided strong evidence for a regulatory link among chromatin structure, histone modification, and splicing regulation. However, it is largely unknown how local histone modification patterns surrounding alternative exons are connected to differential alternative splicing outcomes. Here we show that splicing regulator Hu proteins can induce local histone hyperacetylation by association with their target sequences on the pre-mRNA surrounding alternative exons of two different genes. In both primary and mouse embryonic stem cell-derived neurons, histone hyperacetylation leads to an increased local transcriptional elongation rate and decreased inclusion of these exons. Furthermore, we demonstrate that Hu proteins interact with histone deacetylase 2 and inhibit its deacetylation activity. We propose that splicing regulators may actively modulate chromatin structure when recruited to their target RNA sequences cotranscriptionally. This “reaching back” interaction with chromatin provides a means to ensure accurate and efficient regulation of alternative splicing.

Keywords: histone acetylation, neurofibromatosis type 1, Fas

Recent genome-wide transcriptome analysis has demonstrated that more than 95% of human genes undergo alternative splicing to produce multiple proteins from one gene (14). Most of these alternative splicing events lead to coding differences and occur in a cell type- and/or developmental stage-specific manner (3, 5), underscoring the essential role of alternative splicing in gene expression control. In addition to the well-established role of RNA-binding proteins in the regulation of pre-mRNA alternative splicing (6, 7), recent studies have revealed a role for chromatin-associated proteins and the transcription machinery in splicing regulation (810).

A recent study of large human genes demonstrated that pre-mRNA splicing is cotranscriptional and occurs within 5–10 min of synthesis (11). The tight coupling of transcription and splicing predicts cross-talk between chromatin structure and splicing regulation. Indeed, several recent studies have documented a number of interesting links between chromatin features and exon behavior. First, a ChIP analysis indicated that a specific histone modification, trimethylation of lysine 36 of histone H3 (H3K36me3), differentially marks exons (12, 13). Remarkably, this histone mark appears to be associated more significantly with 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, suggesting that nucleosome position helps to distinguish introns from exons (12, 1417). Although these studies provide significant evidence for cross-talk between chromatin and splicing, the nature of the cross-talk remains largely unknown. Several studies support a model in which histone marks function to recruit basal spliceosomal factors or splicing regulators to ensure efficient splicing regulation. For example, the histone mark H3K4me3 was shown to facilitate efficient splicing through recruiting the spliceosomal component U2 snRNP via the H3K4me3 binding protein CHD1 (18). Moreover, the histone mark H3K36me3 can affect alternative splicing by recruiting the splicing regulator PTB to pre-mRNA via the chromatin-binding protein MRG15 (19).

Chromatin structure regulates various aspects of transcription that are mediated by RNA polymerase II (RNAPII). As an obvious link between chromatin structure and pre-mRNA splicing, the transcriptional behaviors of RNAPII, such as pausing and transcriptional elongation rate, have been demonstrated to influence alternative splicing outcomes (9, 2022). To understand how the local rate of transcriptional elongation is regulated to impact alternative splicing outcomes, it is imperative to investigate the mechanisms by which local chromatin structure and histone modification are modulated. To date, very few studies have addressed this question. Given the fact that splicing of pre-mRNA occurs in situ at its chromosomal gene location, it is expected that cross-talk can occur in both directions (10). In this context, it is reasonable to propose that splicing regulators, in most cases RNA-binding proteins, modulate histone modifications in a localized and RNA-dependent manner. However, to date, no examples have been discovered to suggest an active role for splicing regulators in modulating chromatin structure, transcriptional elongation rate, and alternative splicing.

Here we describe experiments that reveal a unique functional connection between the roles of Hu RNA-binding proteins in regulating alternative splicing and histone acetylation. The Hu proteins (HuA/R, HuB, HuC, and HuD) are a family of mammalian RNA-binding proteins. Of the four Hu family members, HuA/R is widely expressed in many cell types, whereas HuB, HuC, and HuD, are expressed specifically in neurons. We have previously demonstrated a role for Hu proteins as splicing regulators (23). To date, at least four splicing targets of Hu proteins have been identified (2327). These studies show that Hu proteins bind to uridine (U)-rich or adenosine/uridine (AU)-rich RNA sequences and interact with spliceosomal factors to regulate exon inclusion negatively or positively.

We report a unique mechanism by which Hu proteins increase histone acetylation in regions surrounding alternative exons leading to an increased local elongation rate and decreased inclusion of these exons. Importantly, this regulation occurs through the association of Hu proteins with their target sequences on nascent pre-mRNA molecules. Furthermore, we show that Hu proteins decrease the deacetylation activity of histone deacetylase 2 (HDAC2). We propose that splicing regulators may actively modulate chromatin structure when recruited to their target RNA sequences cotranscriptionally. This “reaching back” interaction with chromatin provides a means to ensure accurate and efficient regulation of alternative splicing, supporting a more dynamic and integrated view of gene expression control.

Results

Hu Proteins Associate with Transcriptionally Active RNAPII in Neuronal Cells.

Our previous studies demonstrated that Hu proteins play an important role in the nucleus as alternative splicing regulators. As splicing occurs in situ at the site of transcription (11), we examined 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 primary 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 AC; sequences of the two 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 other three members are significantly enriched in neurons (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.

Fig. 1.
Splicing pattern of two Hu targets and association of Hu proteins with RNAPII. (A) Schematic diagram of the alternative splicing pathways of Nf1 and Fas pre-mRNA. Black and white boxes represent alternative and constitutive exons, respectively. Hu binding ...

The C-terminal domain (CTD) of the largest subunit of 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 phosphorylation peaks at the promoter region of a gene and Ser2 phosphorylation accumulates during transcriptional elongation (31). To determine if Hu proteins interact with differentially phosphorylated RNAPII, we carried out coimmunoprecipitation (coIP) analyses using protein lysates isolated from primary neurons and three different antibodies against RNAPII. Interactions between endogenous HuR and RNAPII that was either unphosphorylated or phosphorylated at Ser5 or Ser2 were detected in reciprocal coIP assays (Fig. 1 E and F). These results indicate that HuR is associated with both initiating RNAPII and elongating RNAPII. Next, we demonstrated that HuR also interacts with Cdk9, a component of the elongation factor P-TEFb complex that phosphorylates Ser-2 (Fig. 1F). Furthermore, these interactions are RNA-independent indicative of direct protein–protein interaction (Fig. 1G).

To determine if HuR directly interacts with the RNAPII complex, we carried out a GST-pull-down experiment using an immunopurified RNAPII core complex that contains all of the previously defined RNA polymerase II subunits (RPB) (32, 33). As indicated in Fig. 1H, GST-HuR did pull down the large subunit RPB1 of RNAPII. This finding suggests a direct interaction between HuR and the RNAPII core complex.

Next, we carried out a number of coIP experiments to further characterize the interaction between Hu proteins and RNAPII. These experiments were conducted in HeLa cells because of the ease of high-efficiency transfection of these cells. We found that (i) the interactions between HuR and RNAPII also occurred in HeLa cells (Fig. S3 AD), (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 Surrounding 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 exonic 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 observed in ES neurons (Fig. 2C). These observations suggest a faster elongation rate in DNA surrounding the two alternative exons in ES-derived neurons.

Fig. 2.
Hu proteins regulate transcriptional elongation rate. (A) Schematic diagrams of Nf1, Fas, and KIFAP3 genes showing the exons analyzed in the following experiments. The numbers shown above each diagram indicate the distance between two exons in kilobases. ...

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 transcripts. 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. Next, the pre-mRNA accumulation was plotted as a time course (Fig. 2D). The neuron-specific alternative exon kinesin-associated protein 3 (KIFAP3) exon 20, which is differentially included in the two types of cells but is not regulated by Hu proteins (Fig. S5 A and B), was used as a negative control. At time zero, no real-time PCR signal was observed, whereas at later points, KIFAP3 exon 20 accumulated at a similar rate in ES cells and ES-derived neurons. For the Nf1 gene, the pre-mRNA accumulation rate of exons 1, 23, and 39 is similar in the two cell types. However, a 2.5-fold increase in pre-mRNA accumulation surrounding the region of exons 23a and 24 in ES-derived neurons was observed indicating a higher elongation rate in this region (Fig. 2D).

To determine if Hu proteins are responsible for the higher elongation rate in the ES-derived neurons, we established an ES cell line that stably incorporated an HuC expression cassette driven by a doxycycline (Dox)-inducible promoter. As indicated in Fig. S5 C and D, overexpression of HuC was observed in the presence of Dox, which led to significantly decreased inclusion of Nf1 exon 23a. Using the anti-HuR antibody that recognized both HuR and HuC, we estimate that in these cells, the level of the overexpressed Myc-HuC is 3- to 4-fold of the endogenous HuR (Fig. S5D). Next we analyzed pre-mRNA accumulation of Nf1 comparing ES cells that express HuC to those that do not express this protein. We found that the pre-mRNA accumulation surrounding the region of exons 23a and 24 was approximately 1.5- to 2-fold higher in HuC-expressing ES cells than in HuC-non-expressing ES cells, whereas KIFAP3 exon 20 and Nf1 exons 1, 23, and 39 had similar accumulation rates in the two types of cells (Fig. 2E). These results indicate that Hu proteins increase the local transcriptional elongation rate of the Nf1 gene surrounding exon 23a.

Transcriptional Elongation Rate Regulates Inclusion of Nf1 Exon 23a.

In light of the aforementioned results, we hypothesized that Hu proteins specifically regulate Nf1 exon 23a alternative splicing by affecting transcriptional elongation rate. To determine if alternative inclusion of Nf1 exon 23a can be modulated by transcriptional elongation rate, we carried out the following experiment. We cotransfected primary neurons with the NF1 reporter, previously used to study alternative splicing of exon 23a (24) (Fig. 3A), and two RNAPII mutants that are α-amanitin-resistant (34). One of the two mutants, C4, also carries an amino acid substitution that reduces the rate of transcriptional elongation by RNAPII. After transfection, cells were treated with α-amanitin to inhibit endogenous RNAPII to ensure that the NF1 reporter would be transcribed solely by the exogenously introduced RNAPII. As shown in Fig. 3B, transcription by the C4 RNAPII mutant resulted in increased exon 23a inclusion (compare lane 4 to lane 3).

Fig. 3.
Transcriptional elongation rate regulates splicing of Nf1 exon 23a. (A) Schematic diagram of the alternative RNA processing pathways of the Nf1 reporter minigene. (B) Transcription by C4 slow RNAPII increases exon 23a inclusion. Primary neurons were cotransfected ...

Hu Proteins Directly Interact with Histone Deacetylase 2.

In an initially parallel avenue of investigation that unexpectedly converged with our investigation of the roles of Hu proteins on transcriptional elongation, we used a yeast two-hybrid screen with HuC as bait and identified HDAC2 as a potential interaction partner (Fig. 4A). Purified recombinant GST-HuC can pull down in vitro translated HDAC2 protein that is 35S-labeled (Fig. 4B). We also confirmed the interaction between Hu proteins and HDAC2 by coIP using anti-HuR antibody and protein lysate prepared from primary neurons (Fig. 4C), and this interaction is RNA-independent, indicating a direct interaction (Fig. 4D). The interaction occurs between HDAC2 and all of the Hu family members (Fig. S6A). Finally, a biochemical analysis using recombinant GST-Hu and MBP-HDAC2 fusion proteins provided definitive evidence for a direct interaction between all of the Hu family members and HDAC2 (Fig. 4E).

Fig. 4.
Hu proteins interact with and inhibit the activity of 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 ...

Using HuC truncation mutants through both in HeLa cells and in in vitro analysis indicate that the hinge and RRM3 domains of the HuC protein are important for its interaction with HDAC2 (Fig. S6 B and C). Although all of the Hu proteins are capable of interacting with HDAC2, it appears that only the class I HDACs that include HDAC1, HDAC2, HDAC3, and HDAC8 can interact with Hu proteins, as we observed an interaction of HuC with HDAC1 and HDAC2 but not HDAC7 (Fig. S6D).

Hu Proteins Inhibit HDAC2 Activity in Vitro.

Two possible explanations might account for the observed interaction between Hu proteins and histone deacetylases: that the splicing regulators are enzymatic substrates, or that they modulate HDAC activity. HDAC proteins catalyze an enzymatic reaction in which acetyl groups, which are added to lysines by members of acetylase family, are removed. This reversible lysine acetylation is a highly regulated posttranslational modification that occurs on more than 195 proteins in HeLa cells in addition to histones (35). To test if Hu proteins can be acetylated on lysines, HuR or histone H4 proteins were immunoprecipitated from ES cells by anti-HuR or anti-H4 antibodies, respectively. A Western blot using antiacetyl lysine antibody demonstrated that H4, but not HuR, was acetylated. The signal from histone H4 proteins could be competed away efficiently by addition of acetylated BSA (Fig. 4F). This result suggests that the interaction between HDAC2 and Hu proteins does not lead to acetylation of Hu proteins.

To test whether Hu proteins regulate the activity of HDAC2, we conducted an HDAC2 activity assay in vitro using tritium-labeled acetylated histone H4 peptides that were conjugated to biotin as a substrate. HDAC2 activity was measured by calculating the amounts of the released tritium-labeled acetate when HDAC2 was added. Recombinant GST-Hu proteins were added to the reaction together with HDAC2 prepared from HeLa nuclear extract through immunopurification (Fig. 4 GI). Addition of GST-HuC reduced the HDAC2 activity by 2- to 2.5-fold (Fig. 4 G and H). Importantly, the Hu-mediated reduction of HDAC2 activity is time- and dose-dependent (Fig. 4 G and H). When the HDAC protein inhibitor sodium butyrate was added to the reaction, the deacetylation was dramatically reduced, confirming that the observed activity was from histone deacetylase present in the HDAC2 protein preparations. Furthermore, consistent with its role in interaction with the HDAC2 protein, deletion of the hinge domain from the HuC protein diminished the ability of the resulting HuC protein to regulate HDAC2 activity (Fig. 4H). HuR, HuB, and HuD showed similar activity (Fig. 4I). These results demonstrate that Hu proteins inhibit the HDAC2 activity through their interaction with HDAC2.

Hu Proteins Promote Local Histone Acetylation.

HDAC2 catalyzes the deacetylation of histone proteins. To determine if Nf1 exon 23a splicing could be modulated by a change in the histone acetylation pattern, we performed ChIP with antibodies recognizing pan-acetylated H3 or H4 histones using ES cells and ES-derived neurons and analyzed the histone acetylation pattern by real-time PCR using primers distributed along the Nf1 gene (Fig. 2A). We also analyzed the alternative exon extra domain I (EDI) of fibronectin and exon 20 of KIFAP for comparison. The signals obtained for Nf1, fibronectin, and KIFAP were normalized to the β-actin signal and a ratio was calculated in which, for any given 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 as exons 23 and 29. In contrast, the levels of pan-acetylated H3 and H4 were increased by approximately twofold between exons 23a and 28 in ES-derived neurons.

Fig. 5.
Hu proteins regulate local histone acetylation levels. (AD) Mapping of pan-histone H3 (light gray bars) or H4 (black bars) acetylation at the indicated positions of Nf1 (A and B) or Fas (C and D) shown in Fig. 2A by ChIP followed by real-time ...

To determine if Hu proteins can regulate the levels of H3 and H4 acetylation, we carried out similar experiments using HuC-expressing and HuC-non-expressing ES cells described earlier (see Fig. 2). We found that H3 and H4 acetylation was increased between exons 23a and 28 in HuC-expressing cells, whereas the acetylation level remained the same at the fibronectin EDI exon, KIFAP3 exon 20, as well as the Nf1 gene at the promoter and at exons 23, 29, and 39 in the two types of ES cells (Fig. 5B). Another Hu-regulated alternative exon, exon 6 of the Fas gene, 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 with the corresponding genomic DNA. Thus, we investigated if HuR antibody could immunoprecipitate exon 23a of Nf1 through ChIP in primary neurons. A ChIP assay demonstrated that HuR proteins are associated with the Nf1 promoter as well as the region surrounding exons 23a and 24 of the Nf1 gene (Fig. 6A). Hu proteins are expected to associate with the promoter region as they interact with the transcription machinery. As Hu proteins regulate splicing through binding to AU-rich elements on pre-mRNA (24), we predicted that they would strongly associate with exon 23a through the interaction with their cognate binding sites in introns surrounding exon 23a. To test this hypothesis, we investigated whether the association between HuR and exon 23a is RNA-dependent. RNase treatment before immunoprecipitation significantly reduced the accumulation of HuR (Fig. 6B), suggesting that the AU-rich elements in introns surrounding exon 23a of pre-mRNA direct local histone modification mediated by the Hu-HDAC2 complex.

Fig. 6.
The role of pre-mRNA targets in Hu-mediated chromatin modification. (A) ChIP analysis indicating HuR accumulation on the Nf1 gene in primary neurons. Anti-T cell intracytoplasmic antigen 1-related protein antibody was used as a negative control (24). ...

To provide further evidence for this argument, we compared the histone acetylation patterns of the wild-type NF1 reporter to that of a mutant reporter in which the AU-rich sequences upstream of exon 23a were disrupted. This mutation disrupted one of the major Hu protein binding sites and led to increased inclusion of exon 23a in neurons from 8 to 30% (Fig. 6C) as well as in neuron-like cells shown in our previous studies (24). As predicted, significantly higher levels of H3 and H4 acetylation was observed in the nucleosomes formed on the wild-type reporter than the mutant reporter surrounding exon 23a (Fig. 6D), which correlates well with the exon 23a inclusion result. These results strongly suggest that Hu-induced histone hyperacetylation depends on their binding sites on the pre-mRNA.

Discussion

The current study reveals a unique role for pre-mRNA splicing regulators that integrates chromatin structure, histone modifications, transcriptional elongation rate, and alternative splicing. Although recent studies have established a link between chromatin modification and splicing, they have focused on how histone marks regulate RNA splicing (8, 36). Here we show that splicing 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 these exons to slow down the local transcriptional elongation rate and affect alternative splicing (38). Another study showed that introduction of siRNAs targeting a region surrounding an alternative exon induced formation of a heterochromatic complex involving HP1 and reduced the transcriptional elongation rate, which in turn affected alternative splice-site usage (39). Last, depolarization of a neuronal cell line was found to induce localized 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 transcription (41). The rapid turnover of histone acetylation is very important for nucleosome dynamics during transcriptional elongation (4244). When pre-mRNA is transcribed by RNAPII, acetylation of the nucleosomes in front of the elongation machinery by histone acetyltransferases (HATs) is required (45, 46). The passage of RNAPII causes displacement of histones, which are subsequently redeposited onto the DNA behind RNAPII. These newly deposited nucleosomes are hyperacetylated, but only transiently. In order to keep the normal chromatin configuration, histone deacetylase complexes remove the acetyl marks (47, 48). This dynamic process provides a regulatory step to establish local chromatin acetylation status.

Recently, multiple studies have demonstrated that histone acetylation modification at alternative exons is connected to differential splicing outcomes (34, 39, 49). Here we provide evidence to suggest that splicing regulator Hu proteins may block removal of acetyl marks during the dynamic process of nucleosome repositioning through inhibiting HDAC2 activity. As a result, the histone hyperacetylation status of a specific region sustained by Hu proteins increases transcriptional elongation. We propose that the pioneer round of transcription allows initial chromatin modulation to occur, establishing a local histone hyperacetylation status to be encountered by the later elongating RNAPII. This activity will induce RNAPII to transcribe the region of hyperacetylated chromatin in a faster mode (Fig. 7). The tissue- or developmental stage-specific expression of Hu proteins can turn on or off histone hyperacetylation at a specific region to regulate alternative splicing. Our data suggest that Hu proteins are recruited to the transcribing gene through a direct interaction with a component of the RNAPII complex (Fig. 1H). Furthermore, when the Hu target sites on pre-mRNA emerge from RNAPII, Hu proteins are transferred from the RNAPII complex to RNA. Thus, the local concentration of Hu proteins is increased, which leads to the regulated HDAC2 activity.

Fig. 7.
A model for RNA-directed modification of histone acetylation by Hu proteins.

For future studies, it will be interesting to investigate potential changes of specific histone marks induced by binding of Hu proteins to their targets. For example, depolarization induces localized increase of H3K9ac as well as H3K36 trimethylation level surrounding the NCAM exon 18 (34). It will also be intriguing to examine how neuron activity such as depolarization affects alternative splicing of NF1 exon 23 in the context of histone modifications. The comparison of our results to NCAM exon will provide more insights into histone modification-mediated alternative splicing change.

Hu protein-mediated histone modification at specific exons requires the presence of AU-rich elements on the pre-mRNA (Fig. 6). When the Hu binding element (AU-rich sequence) is present such as in Nf1 and Fas alternative exons (24, 26), the deacetylation of histones surrounding the alternative exon is reduced. Importantly, this regulation of histone acetylation as well as of the transcriptional elongation rate does not occur surrounding exons that are not targets of Hu proteins such as KIFAP3 exon 20 and the EDI exon of the fibronectin gene (Figs. 2 and and5).5). The results of these experiments strongly suggest that association of Hu proteins with AU-rich elements is necessary for histone modification at specific exons. Consistently, HuR ChIP data indicate that association of HuR proteins with Hu targets is dependent on RNA (Fig. 6 A and B). Moreover, disruption of a major Hu binding site upstream of exon 23a abolished Hu-mediated histone hyperacetylation surrounding exon 23a (Fig. 6 C and D). Therefore, the AU-rich elements on pre-mRNA direct local histone acetylation mediated by Hu proteins.

How does splicing activity affect the RNAPII elongation behavior in general? Recent studies provided some interesting insights. Two studies indicated that splicing activity causes RNAPII to pause at the 3′-end of intron-containing genes in yeast (50, 51). In mammalian cells, a fluorescence recovery after photobleaching-based RNAPII elongation kinetics analysis demonstrated that the basal level of splicing activity of multiple intron-containing model genes does not affect transcriptional elongation rate (52). In this context, our results suggest that at specific chromosomal regions, splicing regulators such as Hu proteins may regulate the local transcriptional elongation rate upon binding to their target sequence.

Splicing is regulated at many different levels in a tissue- or developmental stage-specific manner (7, 53). At the most fundamental level, regulation includes splice-site recognition by the spliceosome, which is modulated by many splicing regulators (54). The fact that splicing of pre-mRNA occurs in situ cotranscriptionally at its chromosome locus implicates higher or more integrated levels of additional regulatory mechanisms that involve chromatin structure, histone modification, and transcription behaviors (55). Here we show that Hu proteins can regulate alternative splicing at both the RNA and chromatin levels. Previously, multiple studies demonstrated that Hu proteins regulate splicing by modulating basal splicing factor binding (23, 24, 26). 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 (5658). 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 regulated in this manner. We propose that this integrated regulatory mechanism serves to ensure accuracy and efficiency of splicing regulation, as local changes in transcriptional elongation mediated by splicing factors can reinforce splicing choices that are also regulated by the same splicing factors.

Although it has been demonstrated that serine/arginine-rich proteins link splicing and transcription and that splicing factor SC35 can regulate the elongation rate through its association with RNAPII (56, 59), no splicing regulators have been found to be engaged in RNA target sequence-directed regulation of transcriptional elongation rate. Thus, our findings will have major implications for understanding how alternative splicing is regulated in the context of chromatin and transcription. The bidirectional regulation of transcription and splicing described in this study strongly supports the previously described “gene expression machine” view (6062), as well as providing significant insights into a unique mechanism to connect different steps of gene expression.

Methods

Cell Culture and Generation of Cell Lines.

Mouse cerebellar neurons were cultured using a modification of a previously described procedure (63). In brief, cerebella were removed from 6- to 8-d-old mice. The dissociated cerebellar cells were plated onto tissue culture plates coated with 0.1 mg/mL poly-L-lysine (Sigma). The cells were maintained in defined medium composed of neurobasal media supplemented with B-27, 2 mM glutamine, 25 mM KCl, and 0.3 g/mL glucose. Beginning on the second day of culture, cells were treated with 5 μM cytosine arabinoside (AraC), a mitosis inhibitor. Fifty percent of media was replaced with fresh media every 3 d. In transfection experiments, 5 × 106 freshly isolated neurons were used for each transfection with Nucleofector II program C13 and mouse neuron Nucleofector kit (Lonza). The neurons were then cultured in DMEM supplemented with 10% FBS for 1 d before switching to defined media supplemented with AraC. The neurons were collected for analysis after 6 d in culture. The Institutional Animal Care and Use Committee at Case Western Reserve University approved these mouse experiments and confirmed that all experiments conform to the relevant regulatory standards.

Mouse ES cell differentiation was carried out using R1 cells with a previously described procedure (64). To obtain the stable HuC-expressing mouse ES cell line, the Tet-on system (Clontech) was used. In this experiment, 5 × 106 mouse ES cells were transfected with the pTet-on vector using the mouse ES 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 with pTRE2-HuC. The colony ES-HuC-4 that has low background HuC expression without doxycycline (−Dox) and high HuC expression with doxycycline (+Dox) was selected. Next, pTRE2-HuC and pGK-puro were cotransfected into clone ES-HuC-4, and cells were selected for 2 wk with 5 μg/mL of Puromycin and 200 μg/mL of G418. Colonies were picked and cultured for 3 d with 2 μg/mL of doxycycline. Induction of HuC was detected by Western blot assay using anti-Myc antibody.

ChIP and Real-Time PCR.

In the ChIP experiment, 5 × 107 cells were fixed with 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 of the H5, anti-acetyl-Histone H3 (Millipore), or anti-acetyl-Histone H4 (Millipore) antibodies. Nonspecific IgG (Sigma) was used as a control. Cross-linking of bound DNA fragments was reversed, and DNA was dissolved in 100 μL of Tris (10 mM)-EDTA (1 mM). Real-time PCR was carried out using SYBR green PCR mix (Qiagen) and the Chromo4 Real Time PCR system (MJ Research). Relative expression levels were determined using a three-point standard curve generated by diluting a control cDNA sample. The relative proportions of coimmunoprecipitated gene fragments were determined on the basis of the threshold cycle (Ct) for each PCR product. The Ct values obtained from immunoprecipitations using specific antibodies were subtracted from the Ct values obtained from that using the control IgG. The resulting values were further normalized to the value obtained with a primer pair amplifying an intergenic region or β-actin. The fold difference between ES neurons and ES cells was calculated as (Ctneuron-gene × CtES-control)/(CtES-gene × Ctneuron-control). For every analyzed gene fragment, each sample was quantified in duplicate and from at least three independent ChIP analyses. In order to investigate if the association between HuR and exon 23a is RNA dependent, sonicated cell lysates were treated with 400 μg/mL of RNase at 37 °C for 30 min before immunoprecipitation as described (65). See Table S1 for PCR primer sequences.

Transcriptional Elongation Analysis.

Inhibition and reinitiation of transcription were performed as described (11). Bromouridine (BrU) labeling was modified from a previously described procedure (59). Before addition of DRB, ES-derived neurons were plated for 6 d, whereas ES cells were 50% confluent. 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 twice with media to remove DRB and then incubated in fresh medium for indicated time periods. At each time point, total RNA was isolated using TRIzol (Invitrogen). To immunoprecipitate the BrU-labeled pre-mRNA, 15 μg of BrU antibody (Sigma) was preincubated with 20 μL of protein G Dynabeads (Invitrogen) for 4 h at 4 °C with rotation. Next, 250 μg of total RNA was incubated with beads for 3 h at 4 °C in 200 μL of RSB-100 buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 0.4% Triton X-100, 0.2 U/μL RNaseOut and 25 μg/mL tRNA). The beads were washed five times with RSB-100 buffer. The RNA bound to beads was eluted by direct addition of 300 μL RLT buffer (Qiagen RNeasy mini kit) supplemented with 2 μg of tRNA. The RNA was purified with Qiagen RNeasy mini kit and eluted in 30 μL of RNase-free water. Eight microliters of the purified RNAs were used for reverse transcription in a 20-μL reaction using the Superscript III First Strand Kit (Invitrogen) and the cDNA (2 μL per well) was used for quantitative PCR. The Ct values obtained from RT-PCR using the BrU antibody were subtracted from the Ct values obtained from templates with control IgG. The resulting value was further normalized to the value obtained with a primer pair amplifying utrophin exon 2. The normalized pre-mRNA expression values were plotted relative to the expression level of the BrU-labeled control without DRB-treatment, which was set to 1 in all experiments (11). For every analyzed gene fragment, each sample was quantified in duplicate and from at least three independent experiments.

In Vitro HDAC2 Activity Assay.

The histone deacetylation assay was conducted as described by a histone deacetylase assay kit (Millipore). Briefly, biotin-conjugated histone H4 peptide was acetylated with 3H-labeled acetyl coenzyme A (Perkin-Elmer) using recombinant histone acetyltransferase PCAF. Subsequently, the labeled histone H4 peptide was bound to streptavidin–agarose beads. The activity of purified HDAC2 was assessed by following the release of 3H label from the 3H-labeled acetyl histone H4 peptide. HDAC reactions were carried out in a final volume of 200 μL comprising 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 8 μg purified MBP-HDAC2, or 50 μL immunoprecipitated HDAC2 complex, agarose beads carrying 40,000 cpm [3H] acetyl histone H4 peptide, and 0.025–1 μg of GST, GST-HuC, GST-ΔHinge, or 50 μL 1M sodium butyrate. The reactions were stopped at 4–30 h. The beads were spun down and 50 μL of the supernatant was counted in a Beckman LS6500 scintillation counter.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank the following individuals for providing antibodies and plasmids: Diane Hayward, HDAC2 plasmid (University of Otago, Otago, New Zealand); Alberto Kornblihtt, mutant RNAPII plasmids (University of Buenos Aires, Buenos Aires, Argentina); and Sachiyo Kawamoto, pMT-6myc [National Institutes of Health (NIH), Bethesda, MD]. We thank Yves Barde for his advice on neuronal differentiation of ES cells and Mats Ljungman for his advice on the transcriptional elongation assay. We thank Cheng-Ming Chiang (University of Texas Southwestern Medical Center, 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. This work was supported by NIH Grant NS-049103 and DOD Grant NF060083 (to H.L.). R.S. was supported by NIH Grant 5R03NS59648. G.L. was supported by NIH Grant CA112094. H.-L.Z. was supported by postdoctoral fellowships from the American Heart Association (0725346B and 09POST2250749). M.N.H. was supported by a genetics training grant from NIH (T32GM008613) and a National Research Service Award predoctoral fellowship from the National Institute of Neutological Disorders and Stroke (1F31NS064724). V.A.B. was supported by a developmental biology training grant from NIH (T32HD00710432) and a predoctoral fellowship from the American Heart Association (0815373D).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Author Summary on page 14717.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103344108/-/DCSupplemental.

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Proc Natl Acad Sci U S A. Sep 6, 2011; 108(36): 14717-14718.
Published online Aug 1, 2011. doi:  10.1073/pnas.1103344108

Author Summary

AUTHOR SUMMARY

Genome-wide studies carried out in eukaryotic cells have indicated that messenger RNA molecules (mRNA), which harbor protein coding information, are processed into protein coding sequences from their precursors at the same time as the precursors are generated from DNA. The tight coupling of transcription (the process by which a messenger RNA precursor, namely pre-mRNA, is generated) and splicing (the process by which a protein coding RNA sequence is assembled from the precursor) predicts cross-talk between chromatin structure and splicing regulation. A number of recent studies have demonstrated that changes in the patterns of histone modification, a posttranslational modification of the component proteins of chromatin, and chromatin structure may lead to differential splicing outcomes (1). For example, two separate studies showed that recruitment of Heterochromatic Protein 1 (HP1) family members to a chromosomal region that is close to an alternative exon (i.e., the part of a mRNA that is selectively included in the coding sequence), reduces the local transcriptional elongation rate of RNA polymerase II, the enzyme responsible for the transcription of pre-mRNA. This reduction leads to an increased level of alternative exon inclusion (2, 3). Consistent with the known function of HP1 proteins as transcription repressors, heterochromatin marks, including di- and tri-methylation of lysine residues in histones, were found to be enriched in these regions (2, 3). However, several questions remain unanswered. First, what induces the initial histone modification pattern change? Second, although it is clear that information can flow from chromatin to RNA, is the reverse true? Finally, can splicing regulators bound at their RNA targets affect histone modification patterns? The current study provides insights into these issues.

Our study reveals a unique role for splicing regulators in alternative splicing regulation that integrates chromatin structure, histone modification, transcriptional elongation rate, and alternative splicing. In our study, we used exon 23a of the neurofibromatosis type 1 (NF1 for human and Nf1 for mouse) gene as a model system to examine these questions. We have shown previously that inclusion of this exon is negatively regulated by Hu proteins, a family of RNA-binding proteins that consists of one ubiquitously expressed member and three neuron-enriched members (4). Using both primary and mouse embryonic stem cell-derived neurons, we first carried out a coimmunoprecipitation analysis to demonstrate that all of the Hu family members are capable of interacting with elongating RNA polymerase II. Next, we showed that Hu proteins, upon interaction with their target sequences on the pre-mRNA surrounding exon 23a, can induce local histone hyperacetylation. The hyperacetylation starts at a chromosomal region corresponding to exon 23a and persists through exon 28. Furthermore, the histone hyperacetylation leads to an increased local transcriptional elongation rate. The increased transcriptional elongation rate results in skipping of exon 23a, consistent with the idea that exons associated with suboptimal splicing signals are preferentially ignored when the transcriptional elongation rate is increased (1).

We next investigated the underlying mechanism of the Hu-mediated hyperacetylation. We found that Hu proteins interact with the enzyme histone deacetylase 2 (HDAC2) and that all four Hu proteins are capable of inhibiting the deacetylation activity of HDAC2. This is an intriguing finding as HDAC2 activity has never been shown to be regulated by an RNA-binding protein.

Hu protein-mediated histone modification requires the presence of Hu binding targets on mRNA precursors. Disruption of a major Hu binding target upstream of exon 23a abolished Hu-mediated histone hyperacetylation surrounding this exon.

Based on these findings, we propose the model depicted in Fig. P1. We believe that Hu proteins travel with the RNA polymerase II transcription complex during elongation through a direct interaction with a component of the complex. During transcriptional elongation, a dynamic cycle of histone acetylation and deacetylation occurs continuously with histone acetyltransferases (HATs) opening up the chromatin in front of the polymerase by acetylating histones and HDACs closing up chromatin after the polymerase passes through by removing acetylation (5). When exon 23a is transcribed, Hu proteins are unloaded from the RNA polymerase II complex to the pre-mRNA through binding to the AU-rich sequences located upstream and downstream of exon 23a (Fig. P1). The increased level of Hu proteins at the chromosomal region corresponding to exons 23a through 28 leads to a reduced efficiency of histone deacetylation by HDAC2. As a result, the level of hisone acetylation is higher in this region, creating a more “open” chromatin structure. Thus, the elongation rate of the subsequent rounds of transcription by the polymerase is expected to be faster within this region.

Fig. P1.
A model of Hu-mediated histone hyperacetylation. During transcriptional elongation along the Nf1 gene, Hu proteins (red oval) travel with RNAPII (purple oval) and are unloaded to pre-mRNA that contains Hu binding sites surrounding exon 23a. The accumulation ...

We propose that this integrated mechanism serves to ensure accuracy and efficiency of splicing regulation, as local changes in transcriptional elongation mediated by splicing factors can reinforce splicing choices that are also regulated by the same splicing factors. Our findings have implications for understanding how alternative splicing is regulated in the context of chromatin and transcription.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See full research article on page E627 of www.pnas.org.

Cite this Author Summary as: PNAS 10.1073/pnas.1103344108.

References

1. Luco RF, Allo M, Schor IE, Kornblihtt AR, Misteli T. Epigenetics in alternative pre-mRNA splicing. Cell. 2011;144:16–26. [PMC free article] [PubMed]
2. Saint-Andre V, Batsche E, Rachez C, Muchardt C. Histone H3 lysine 9 trimethylation and HP1gamma favor inclusion of alternative exons. Nat Struct Mol Biol. 2011;18:337–344. [PubMed]
3. Allo M, et al. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nat Struct Mol Biol. 2009;16:717–724. [PubMed]
4. Zhu H, Hinman MN, Hasman RA, Mehta P, Lou H. Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA. Mol Cell Biol. 2008;28:1240–1251. [PMC free article] [PubMed]
5. Wang Z, et al. Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes. Cell. 2009;138:1019–1031. [PMC free article] [PubMed]

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