Calcium-Mediated Histone Modifications Regulate Alternative Splicing in Cardiomyocytes
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Calcium-mediated histone modifications regulate PNAS PLUS alternative splicing in cardiomyocytes Alok Sharmaa, Hieu Nguyena, Cuiyu Genga, Melissa N. Hinmana, Guangbin Luoa,b, and Hua Loua,b,c,1 aDepartment of Genetics and Genome Sciences, bCase Comprehensive Cancer Center, and cCenter for RNA Molecular Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Edited* by Alberto R. Kornblihtt, University of Buenos Aires, Buenos Aires, Argentina, and approved October 15, 2014 (received for review May 14, 2014) In cardiomyocytes, calcium is known to control gene expression at RNA–RBP interactions are caused by calcium-induced phos- the level of transcription, whereas its role in regulating alternative phorylation and/or cellular localization changes of RBPs. For splicing has not been explored. Here we report that, in mouse example, in response to higher calcium levels, the splicing regulators primary or embryonic stem cell-derived cardiomyocytes, increased heterogeneous ribonucleoprotein A1 (hnRNPA1) and RNA bind- calcium levels induce robust and reversible skipping of several ing protein, fox-1 homolog (Rbfox1) were shown to have increased alternative exons from endogenously expressed genes. Interest- nuclear localization, whereas transformer-2 protein homolog beta 1 ingly, we demonstrate a calcium-mediated splicing regulatory (Tra2β1) was shown to translocate to the cytoplasm (7, 16, 17). The mechanism that depends on changes of histone modifications. altered nuclear levels of these proteins lead to changes in alternative Specifically, the regulation occurs through changes in calcium- splicing of the downstream target genes that are regulated by these responsive kinase activities that lead to alterations in histone splicing factors. modifications and subsequent changes in the transcriptional By contrast, a recent study of neural cell adhesion molecule elongation rate and exon skipping. We demonstrate that in- (NCAM) alternative splicing implicated an RBP-independent, creased intracellular calcium levels lead to histone hyperacetyla- epigenetic regulatory mechanism that is induced by calcium (11). tion along the body of the genes containing calcium-responsive This study indicated that, in neurons, depolarization-induced alternative exons by disrupting the histone deacetylase-to-histone skipping of NCAM exon 18 is correlated with a localized H3K9 acetyltransferase balance in the nucleus. Consequently, the RNA hyperacetylation and H3K36 trimethylation (11). These results, polymerase II elongation rate increases significantly on those BIOCHEMISTRY in combination with pre-mRNA accumulation data, imply that genes, resulting in skipping of the alternative exons. These studies the local transcriptional elongation rate of RNA polymerase II reveal a mechanism by which calcium-level changes in cardiomyo- (RNAPII) on NCAM near exon 18 was increased in depolarized cytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns. cells (11). As such, this study supports a signaling-responsive mechanism that is consistent with the kinetic coupling model of alternative splicing | histone hyperacetylation | transcription and splicing, where an increased transcriptional transcriptional elongation rate | calcium | cardiomyocytes elongation rate leads to skipping of alternative exons because they are generally surrounded by suboptimal splicing signals and thus need more time to be recognized (18). Although the pro- lternative splicing is a robust mechanism that regulates the posed mechanism is very intriguing, it is based on a single ex- functional output of a genome. More than 90% of human A ample, and it is not clear whether the proposed mechanism can protein-coding genes undergo alternative splicing, which signif- be generalized to other genes and cell types. icantly increases proteomic complexity of the human genome (1, Calcium and calcium-dependent signaling also play impor- 2). The precise spatial–temporal regulation of alternative splic- tant roles in regulating gene expression in cardiomyocytes, and ing plays a crucial role in controlling gene expression. Decades of studies have yielded a wealth of knowledge on how tissue- and developmental stage-specific alternative splicing is regulated (3). Significance However, signaling-controlled alternative splicing in response to environmental cues has attracted far less attention, and regula- Calcium is an important intracellular second messenger that tory mechanistic insights have only begun to emerge in recent regulates many biological processes. Many extracellular envi- years (4). ronmental cues lead to cellular calcium-level changes, which Calcium is an important intracellular second messenger that impact on the output of gene expression. In cardiomyocytes, regulates many biological processes. Although most studies have calcium is known to control gene expression at the level of focused on how calcium regulates gene expression at the tran- transcription, whereas its role in regulating alternative splicing scriptional level (5, 6), a small number of studies have also ex- has not been explored. Our studies demonstrate that in these plored how changes in intracellular calcium levels can lead to cells a network of alternatively spliced exons exists, which alternative splicing pattern alterations (7–12). For example, in responds to the altered calcium levels by changing their splic- neuronal cells, a splicing-sensitive exon array identified more ing patterns. Our studies further elucidate an epigenetic reg- ulatory mechanism, triggered by calcium signaling pathways, than 5,000 genes that change their splicing pattern in response to that leads to histone hyperacetylation along gene bodies, elevated calcium levels (13). However, because only a handful of which increases the transcriptional elongation rate of RNA studies have investigated the underlying mechanisms, it remains polymerase II and impacts alternative splicing. largely unknown how calcium-induced alternative splicing changes are regulated. These studies have revealed two distinct Author contributions: A.S. and H.L. designed research; A.S., H.N., C.G., and M.N.H. per- mechanisms by which calcium-responsive alternative exons can formed research; G.L. contributed new reagents/analytic tools; A.S. analyzed data; and be regulated: one RNA-binding protein (RBP)–dependent and A.S. and H.L. wrote the paper. the other RBP-independent (14, 15). The authors declare no conflict of interest. Several studies using depolarized neurons have revealed RBP- *This Direct Submission article had a prearranged editor. dependent mechanisms that influence alternative exon inclusion, 1To whom correspondence should be addressed. Email: [email protected]. which involve calcium-induced interactions between short RNA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sequences and RBP splicing regulators (7, 12). Changes of these 1073/pnas.1408964111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1408964111 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 abnormal calcium levels can affect gene expression at the level chloride (NaCl) was used as a negative control. Semiquantitative of transcription (19). Specifically, a number of studies have reverse transcriptase–PCR (RT-PCR) performed with primers demonstrated that elevated intracellular calcium levels activate annealing to the exons surrounding Nf1 exon 23a using total the calcium/calmodulin-dependent protein kinase (CaMK) or RNA extracted from these cells indicates that exon inclusion the protein kinase C/D (PKC/PKD) (20–25). Consequently, the decreased drastically from 69 to 15% with increasing amounts of activated kinases lead to increased transcription of a number of KCl (Fig. 1 A and B). In a time course experiment, we observed cardiac-specific transcription factors through a cascade of events that inclusion of Nf1 exon 23a decreases from 72 to 9% within including changes in histone modifications (20–25). No studies to 24 h of treatment with 100 mM KCl (Fig. 1C). We observed the date have explored whether calcium signaling can also affect pre- splicing pattern change starting after 6 h of the KCl treatment, mRNA processing in the cardiovascular system. which is expected because the Nf1 mRNA half-life is 6 h (Fig. Here we establish that in cardiomyocytes there is a calcium- S1A). Remarkably, when we replaced KCl-containing culture controlled network of alternative splicing events. Using car- media with regular media, the splicing pattern change was diomyocytes isolated from neonatal mice, we provide a number completely reversed (Fig. 1D), indicating that the splicing pattern of examples where elevated intracellular calcium levels lead to of Nf1 exon 23a is highly dynamic in responding to environmental a significant yet reversible increase in skipping of alternative stimuli. Note that the cells appeared normal throughout the dura- exons. Interestingly, our analysis suggests that calcium-mediated tion of the experiments. In fact, the rhythmic beating of the primary splicing regulation likely occurs through an RBP-independent cardiomyocytes, which ceased during KCl treatment, was restored after KCl was removed. Also, the level of neurofibromin, the pro- mechanism. This mechanism links calcium-responsive kinase tein product of Nf1, remained constant in untreated and treated activities and alternative splicing through controlling histone cardiomyocytes (Fig. S1B). modifications and transcriptional