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 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 , fox-1 homolog (Rbfox1) were shown to have increased alternative exons from endogenously expressed . 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 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 (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 elongation rate. To determine the cell-type specificity of this phenomenon, we Results conducted similar experiments in several cell types. Whereas cardiomyocytes differentiated from mouse ES cells showed Robust and Dynamic Calcium-Induced Changes of the Alternative similar changes in the splicing pattern of Nf1 exon 23a (Fig. S2 Splicing of Nf1 Exon 23a in Mouse Cardiomyocytes. The Nf1 gene A–C), no changes were observed in undifferentiated mouse ES plays an essential role in heart development and function (26, cells (Fig. S2D). These results suggest that the KCl-induced 27). Nf1 exon 23a is alternatively spliced, and its inclusion inserts – splicing changes of Nf1 exon 23a are specific to cardiomyocytes. 21 amino acids into the GTPase-activating protein (GAP) The consistently observed stronger RT-PCR signal in KCl- related domain of the Nf1 gene product neurofibromin, leading treated samples prompted us to investigate whether the Nf1 to a significant reduction of its Ras-GAP activity in multiple cell mRNA transcript level changed in these cells. Real-time RT- – types (28 30). In mouse cardiomyocytes, the alternative exon PCR analyses indicate that KCl treatment leads to up to a four- Nf1 exon 23a is included in 70% of transcripts (Fig. 1). To in- fold increase of Nf1 transcript level in both primary and ES cell- vestigate whether the splicing pattern of this exon changes in derived cardiomyocytes but not in ES cells (Fig. S1C). The Nf1 response to environmental stimuli, we exposed cardiomyocytes transcript level is significantly reduced in cells upon recovery prepared from postnatal day 1–3 mouse hearts to increasing from KCl treatment (Fig. S1C). These results indicate that the concentrations of extracellular potassium chloride (KCl), which KCl-induced alternative splicing change of Nf1 exon 23a is ac- elevates intracellular calcium levels by depolarizing cells. Sodium companied by increased transcript level. We hypothesized that the altered levels of intracellular cal- cium are responsible for the KCl-induced splicing changes of Nf1 exon 23a, because elevated levels of extracellular KCl are known A 23 23a 24 to lead to calcium influx via L-type voltage-gated calcium 23 23a 24 channels (31–33). We used two established pharmacological 23 24 approaches to examine the role of calcium in splicing regulation. BCD We found that nifedipine, an L-type calcium channel blocker, KCl Time in KCl Time in recovery significantly reduced the effect of KCl on alternative splicing of NaCl 100 25 50 80 100 mM 04 6 812 24 hr 04682 24 hr Nf1 exon 23a (Fig. S3A). A similar result was observed with the use of AP5, an NMDA receptor antagonist, in conjunction with BAPTA-AM, a calcium chelator (Fig. S3B). To further examine

69 70 50 30 15 72 72 50 42 28 9 91928485068Avg%Inc the role of intracellular calcium in stimulus-induced regulation 100 100 100 of Nf1 alternative splicing, we treated primary or ES cell-derived 80 80 80 cardiomyocytes with increasing concentrations of caffeine, which 60 60 60 is known to increase intracellular calcium levels by acting as 40 40 40 a rynodine receptor agonist, promoting release of calcium from

% Inclusion Inclusion % the sarcoplasmic reticulum to the cytoplasm (34). Interestingly, 20 20 20 caffeine treatment led to a reduction of Nf1 exon 23a inclusion 0 0 0 from 68% of the transcripts in the control cells to 3% of the Fig. 1. KCl induces a change in the alternative splicing pattern of Nf1 exon transcripts in cardiomyocytes in a dose-dependent manner (Fig. 23a in primary mouse cardiomyocytes. (A) Schematic diagram indicating the S3 C and D). These results establish that the alternative splicing alternative splicing pattern of Nf1 exon 23a. The arrows show the locations pattern of Nf1 exon 23a changes in response to altered in- of RT-PCR primers. (B–D) Semiquantitative RT-PCR analysis of the alternative tracellular calcium levels. splicing of Nf1 exon 23a in primary mouse neonatal cardiomyocytes treated with the indicated concentrations of KCl or control NaCl. The numbers in- Calcium-Regulated Changes in the Alternative Splicing Pattern of dicate the % inclusion of Nf1 exon 23a relative to total Nf1 transcripts. (B) – Additional Genes. To determine whether the splicing change Dose response of KCl treatment. Cells were treated with increasing con- regulated by intracellular calcium levels in cardiomyocytes occurs centrations of KCl, as indicated, for 24 h. n = 5. (C) Time course analysis of the KCl treatment. Cells were treated with 100 mM KCl and collected for with other alternative exons, we performed semiquantitative RT- RNA isolation at the indicated times. n = 3. (D) Recovery time course analysis. PCR on nine alternative exons known to be differentially spliced Cells were treated with 100 mM KCl for 24 h followed by recovery without during heart development (35). Interestingly, over 50% of these KCl for the indicated times. n = 3. Error bars represent standard deviations. exons (five of nine) showed changes in their splicing patterns after

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1408964111 Sharma et al. Downloaded by guest on September 25, 2021 PNAS PLUS exposure of the primary cardiomyocytes to KCl. These exons in- A B --++-- ++ siCaMKIIδ clude Ktn1 exon 36, Ank2 exon 21, Enah exon 5, and Mef 2A exon -- --++ + +KN93 -+-+-+ -+ siPKD1 11 (Fig. 2). In all cases, we observed increased skipping of the al- --++ --+ +Gö6976 +--- +--- siControl ternative exons after KCl treatment (Fig. 2). Importantly, for all of Na K Na K Na K Na K Na Na Na Na K K K K these exons, the splicing pattern was reverted back to the baseline level in untreated cells when KCl was removed (Fig. 2). Among the alternative exons that did not show a change in splicing is Capzb exon 8 (Fig. 2), which provided an important negative control in 64 19 68 9 68 5 68 52 68 68 67 68 12 13 12 67 Avg % Inc the subsequent mechanistic studies. These results demonstrate 100 100 that calcium regulates alternative splicing of several genes in car- 80 80 diomyocytes. Interestingly, we noted an apparent correlation be- 60 60 tween the splicing pattern change and transcript levels for these 40 40

genes too (Fig. 2). Inclusion % 20 20 0 0 Calcium-Regulated Alternative Splicing Is Mediated by the CaMKIIδ or Fig. 3. Calcium-mediated alternative splicing is controlled by the CaMKIIδ PKD1 Signaling Pathways. Abnormal calcium signaling has been and PKD1 signaling pathways. Semiquantitative RT-PCR analysis was carried shown to cause cardiac remodeling through several calcium sig- out to examine the Nf1 exon 23a splicing pattern in primary cardiomyocytes naling molecules, including PKC/PKD and CaMKII (21, 25). To treated with 100 mM control NaCl or KCl for 24 h in the absence or presence investigate the signaling pathways involved in the calcium-mediated of inhibitors (A) or siRNA (B). (A) In addition to NaCl or KCl, cells were changes in splicing patterns, we treated primary cardiomyocytes treated with the PKC/PKD inhibitor Gö6976 (10 μM) and the CaMK inhibitor with inhibitors blocking the function of these kinases, Gö6976 KN93 (1 μM) individually or in combination. (B) In addition to NaCl or KCl, targeting PKC/PKD and KN93 targeting CaMK, in conjunction cells were treated with 200 pmol of siRNA targeting PKD1 or CaMKIIδ in- with the KCl treatment. Interestingly, as shown in Fig. 3A, the dividually or in combination. A scrambled nontargeting siRNA was used as = KCl-induced splicing pattern change of Nf1 exon 23a was sig- a control. n 3. Error bars represent standard deviations. nificantly reduced only when the CaMK and PKC/PKD pathways were inhibited simultaneously, but not when the pathways were and Ank2 exon 21 was also abolished when the cells were treated inhibited individually. These results suggest that the CaMK and with siRNAs targeting both CaMKIIδ and PKD1, whereas the PKC/PKD signaling pathways have redundant functions in reg- splicing pattern of Capzb exon 8 was not affected (Fig. S4C). BIOCHEMISTRY ulating calcium-induced splicing changes of Nf1 exon 23a. These results support the hypothesis that the CaMKIIδ and PKD1 We next carried out siRNA knockdown experiments to further pathways play an important role in regulating multiple alternative investigate the role of these two signaling pathways in splicing splicing events in response to changes in calcium levels. regulation. Both the CaMK and PKC/PKD protein kinase families δ contain multiple members. We chose to knock down CaMKII and Histone Hyperacetylation in Depolarized Cardiomyocytes. Up-regu- PKD1, because their roles in regulating gene expression in car- lation of the CaMKIIδ and PKD1 pathways has been shown to diomyocytes have been well-established (21, 23, 25). We found that increase cytoplasmic localization of class II histone deacetylases δ siRNA-mediated depletion of both CaMKII and PKD1 com- HDACs 4, 5, 7, 9) under hypertrophic stress in neonatal pletely abolished the effect of KCl on Nf1 exon 23a alternative rat cardiomyocytes (20, 22–24). To determine whether the splicing, whereas individual knockdown had no effect (Fig. 3B), depolarization-induced calcium-level increase leads to export corroborating the inhibitor study. The siRNA knockdown efficiency δ of class II HDACs from the nucleus to the cytoplasm, we is shown in Fig. S4 A and B. siRNA knockdown of CaMKII and performed an immunofluorescence staining assay to examine the PKD1, either individually or in combination, had no effect on the cellular localization of HDACs in NaCl- or KCl-treated cells and alternative splicing of Nf1 exon 23a in cardiomyocytes treated with in KCl-treated cells followed by recovery. Using an antibody that theNaClcontrol(Fig.3B). These results indicate the important role δ recognizes HDACs 4, 5, and 7, we found that KCl treatment of CaMKII and PKD1 in calcium-regulated alternative splicing. significantly depleted these class II HDACs from the nucleus, We further tested whether depletion of these kinases affected which was reversed after recovery, whereas the level of these alternative splicing of the other calcium-responsive exons shown HDAC proteins remained constant (Fig. S5A). The cellular in Fig. 2. We found that KCl-induced skipping of Ktn1 exon 36 localization change is likely specific to class II HDACs, because we did not observe any change with HDAC2, a class I HDAC (Fig. S5B). Furthermore, Western blot analysis using nuclear and cyto- Ktn1 e36 Ank2 e21 Enah e5 Mef 2A e9 Capzb e8 plasmic protein fractions indicated that the levels of the class II Na K R Na K R Na K R Na K R Na K R HDACs are reduced in the nucleus and increased in the cytoplasm (Fig. S5C). To examine the effect of HDAC translocation on histone acetylation, we performed Western blot analysis using acid- Avg % Inc 48 15 47 11 1 11 30 10 32 9111 15 14 15 extracted histones from primary cardiomyocytes and antibodies against acetylated histone H3 and H4. We observed a 2.5-fold 100 100 100 100 100 increase of histone acetylation after depolarization for both histones H3 and H4 (Fig. 4A). The total protein levels of histone 50 50 50 50 50 H3 and H4 remained unchanged (Fig. 4A). Histone acetylation was

% Inclusion Inclusion % 0 0 0 0 0 completely restored to basal levels 24 h after KCl removal (Fig. 4A). Similar results were obtained using ES cell-derived cardiomyocytes, Fig. 2. Calcium-mediated dynamic changes in alternative splicing of other whereas no change was observed with undifferentiated ES cells alternative exons. Semiquantitative RT-PCR analysis using total RNA isolated (Fig. S6A). Histone hyperacetylation induced by KCl was blocked from primary cardiomyocytes treated with 100 mM NaCl (Na), KCl (K), or KCl followed by recovery (R). Primers annealing to exons surrounding the al- when inhibitors of PKC/PKD and CaMK were used simultaneously ternative exons were used. Products representing inclusion (open circles) or but not individually (Fig. S6B). These results demonstrate that de- skipping (solid circles) of an alternative exon are indicated. n = 3. Error bars polarization of cardiomyocytes leads to an increase in global acet- represent standard deviations. ylation of histone H3 and H4.

Sharma et al. PNAS Early Edition | 3of9 Downloaded by guest on September 25, 2021 AB that show a change in alternative splicing pattern. To test this Na K R hypothesis, we focused on two HATs, CBP and p300, which are Na K R Na K R H3K9ac known to play important roles in heart functions (36–38). We H4 H3 H3K27ac first examined their association with the three genes discussed H4ac H3ac H3K14ac 3 3 above, Nf1, Ktn1, and Capzb. Our ChIP analysis using antibodies H3K36me3 against CBP or p300 showed that, in mouse primary car- 2 2 H3K4me3 diomyocytes treated with KCl, association of CBP and p300 1 1 H3 H4K16ac along the Nf1 and Ktn1 genes increased moderately by up to 1.5- 0 0 H4 fold (Fig. 5A and Fig. S7A), whereas their association with the Capzb gene did not change (Fig. 5B). Statistical analysis in-

CDNaCl KCl dicated that the moderate increase of CBP and p300 along Nf1 Nf1 Capzb and Ktn1 is statistically significant (Fig. 5A and Fig. S7A). 6 3 * * * * * * * * To determine whether the activities of CBP and p300 are re- sponsible for the calcium-induced histone hyperacetylation on 2 H3 3 Nf1 and Ktn1, we carried out an siRNA knockdown assay to 1 reduce the protein level of CBP or p300 (Fig. 5C, Right). The H3ac/ H3ac/H3 RT-PCR splicing analyses indicated that the KCl-induced splic- 0 0 ing changes for Nf1 exon 23a and Ktn1 exon 36 were at least

Fig. 4. Histone hyperacetylation is induced by elevated intracellular calcium A CBP Nf1 NaCl KCl p300 Nf1 2 levels. (A) Primary cardiomyocytes were treated as described in Fig. 2. Total * * * * * * * 2 * * * * * * * histones were acid-extracted from the treated cells. Western blot analysis 1.5 1.5 was carried out using antibodies specific for total or pan-acetylated histone 1 1 H3 or H4. The graphs show the fold differences between KCl- and NaCl- treated cells. n = 3. Error bars represent standard deviations. (B) Western 0.5 0.5 CBP association blot analysis using histones prepared as described in A and specific anti- 0 p300 association 0 bodies as indicated. Arrows indicate hyperacetylation at specific lysines. (C and D) ChIP analysis using primary cardiomyocytes treated with NaCl (light gray bars) or KCl (black bars). IP was performed using antibodies against pan-acetylated H3 or total H3 and real-time PCR assay using primers tar- geting different regions of Nf1 (C)orCapzb (D) as indicated. The ratio of B CBP Capzb 2 p300 Capzb acetylated to total histone H3 was plotted in the graphs with the value for 2 NaCl KCl 1.5 * each region in NaCl-treated cells set at 1. n = 3. The P values of the Student t 1.5 * 1 test for each region were calculated, and the asterisk represents a statisti- 1 −8 cally significant P value (<8.9 × 10 ). 0.5 0.5 CBP association 0 p300 association 0 To study acetylation of specific lysines on histone H3, we ex- amined H3 acetylated at lysine 9, 14, and 27 and H4 acetylated at lysine 16, and found similar change patterns for all of the examined lysines (Fig. 4B). We also examined the histone methylation status C --+- -+sip300 -+-- +-siCBP siControl siCBP of H3K4 and H3K36 and found no changes (Fig. 4B). These results Na Na Na K K K suggest that depolarization of cardiomyocytes induces histone Na K Na K hyperacetylation at multiple lysines. CBP γTubulin The seemingly global hyperacetylation result predicts that 68 67 70 15 45 48 Avg % Inc. hyperacetylation occurs on broad instead of localized chromo- 100 siControl sip300 somal regions. Thus, we hypothesize that for any given gene, 80 Na K Na K hyperacetylation occurs along the gene body instead of on a lim- 60 p300 ited region such as the promoter region. To test this hypothesis, 40 γTubulin

we performed a chromatin immunoprecipitation (ChIP) assay Inclusion % 20 comparing Nf1 and Ktn1, the two genes that contain calcium- 0 responsive alternative exons, with Capzb, the gene that contains a nonresponsive alternative exon (Fig. 2). Our results showed Fig. 5. Role of the HATs p300 and CBP in a calcium-induced alternative splicing pattern change in primary cardiomyocytes. (A and B)ChIPanalysisto a positive correlation between histone hyperacetylation through- examine the association of the HATs CBP and p300 with the Nf1 (A)orCapzb out the gene body and calcium responsiveness, with approximately (B) genes in NaCl- or KCl-treated cells. Cells were treated with 100 mM NaCl or fourfold increase of histone H3 acetylation along the Nf1 and Ktn1 100 mM KCl for 24 h. ChIP analysis was carried out using anti-CBP or anti-p300 genes and no change along the Capzb gene after depolarization antibodies and real-time PCR using primers targeting the indicated regions of (Fig. 4 C and D and Fig. S6C). Note that throughout the manu- Nf1 or Capzb. The coimmunoprecipitated gene fragments were normalized to script, each ChIP analysis was carried out in three independent the input value. Fold differences of the HAT association with Nf1 or Capzb in experiments (i.e., n = 3) and each real-time PCR assay was carried NaCl- or KCl-treated cells are shown in the graphs. n = 3. The P values of the Student t test for each region were calculated, and the asterisk represents out in duplicate (two PCR assays with each experiment). For ChIP − a statistically significant P value (<8.9 × 10 5). (C)Cardiomyocytesweretreated analysis, the IP values were normalized to input. with siRNAs targeting CBP or p300. A nonspecific siRNA was used as a control. All cells were then treated with 100 mM KCl for 24 h or 100 mM NaCl, followed The Role of Histone Acetyltransferases in Calcium-Induced Alternative by semiquantitative RT-PCR to examine the Nf1 exon 23a splicing pattern. Splicing Changes. We reasoned that the calcium-induced trans- Expression of CBP and p300 protein was detected by Western blot analysis location of class II HDACs would provide more access for his- using anti-CBP or anti-p300 antibodies. γ-Tubulin was used as a loading con- tone acetyltransferases (HATs) to acetylate histones on genes trol. n = 3. Error bars represent standard deviations.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1408964111 Sharma et al. Downloaded by guest on September 25, 2021 partially reversed when the CBP or p300 level was reduced (Fig. A PNAS PLUS 5C, Left, and Fig. S7B). Knockdown of both CBP and p300 in Nf1 Capzb NaCl KCl NaCl KCl KCl-treated cells led to a moderately stronger reversal of the 4 4 splicing phenotype of Nf1 exon 23a (Fig. S7D). As expected, no 3 change was observed in the splicing of Capzb exon 8 (Fig. S7C). 2 2 The NaCl-treated cells did not show any changes in splicing of 1 these exons when CBP or p300 was knocked down (Fig. 5C and 0 0

Fig. S7 B and C). Furthermore, interestingly, the increased as- RNAPII occupancy RNAPII occupancy sociation of CBP and p300 on Nf1 and Ktn1 was abolished when kinase inhibitors were used (Fig. S8). These results suggest that increased activity of HATs, such as CBP and p300, along gene bodies is responsible for histone hyperacetylation, which leads to B C --++CPT increased skipping of alternative exons when cells are stimulated NaCl KCl by calcium. 8 * Na K Na K 6 The Role of Histone Deacetylases in the Specificity of Calcium-Induced * Alternative Splicing Changes. An important question in calcium- 4 72 9 68 64 Avg % Inc induced alternative splicing change is how gene specificity is 100 2 achieved. In other words, why is the splicing pattern for Capzb 80 exon 8 not changed after KCl treatment? One hypothesis is that 0 60 class II HDACs are differentially recruited to specific genes and, Elongation rate (Kb/min) Nf1 Ktn1 Capzb 40

thus, only those genes that are associated with class II HDACs in Inclusion % 20 untreated or NaCl-treated cells will show splicing pattern change 0 after the KCl treatment, which induces the cytoplasmic trans- location of these HDACs. Our ChIP analyses using antibodies Fig. 6. Calcium-induced RNAPII elongation rate increase. (A) RNAPII occu- specific for the class II HDACs HDAC4 or HDAC5 or the class I pancy examined by ChIP analysis using H5 antibody. The coimmunoprecipi- HDAC HDAC2 indicate that the baseline levels of the HDAC tated gene fragments were normalized to the input value. The graphs indicate the relative level of RNAPII accumulation at the indicated regions association are similar with the three genes (Fig. S9). Interestingly, BIOCHEMISTRY along the Nf1 or Capzb genes in primary cardiomyocytes treated with however, when cardiomyocytes were treated with KCl, the associ- 100 mM NaCl (light gray) or KCl (black). n = 3. (B) The RNAPII transcriptional ation of class II HDACs was reduced along Nf1 and Ktn1,butnot elongation rate along three genes in ES cell-derived cardiomyocytes treated Capzb, and the reduction was blocked by use of the two kinase with 100 mM NaCl or KCl. The elongation rate was measured as described in inhibitors, whereas the association of HDAC2 does not change on Materials and Methods. n = 3. The asterisk represents a statistically signifi- − any of the three genes (Fig. S10). These results suggest that the cant P value (<2.5 × 10 4). (C) Semiquantitative RT-PCR to examine the Nf1 differential dissociation of class II HDACs provides the specificity exon 23a splicing pattern in primary cardiomyocytes that were treated with of the KCl-induced splicing change. 3.5 μM CPT in addition to the NaCl or KCl treatment. n = 4. Error bars rep- resent standard deviations. Elevated Intracellular Calcium Levels Correlate with Increased Transcriptional Elongation Rate in Cardiomyocytes. These results, combined with our observation that altered calcium levels did presence of KCl using a procedure initially described by Singh not affect either the total protein level or cellular localization and Padgett and modified by our laboratory (41, 42). In this pattern of the splicing regulators known to affect Nf1 exon 23a experiment, ES cell-derived cardiomyocytes were treated with β alternative splicing (Fig. S11), prompted us to test the role of the 5,6-dichloro-1- -D-ribofuranosylbenzimidazole (DRB) to inhibit kinetic coupling model in the calcium-induced skipping of al- transcription. After removal of DRB, cells were collected at 0, ternative exons. The kinetic coupling model posits that the 15, 30, 60, 75, and 90 min, and pre-mRNA accumulation of RNAPII elongation rate affects the splicing outcome of an al- specific regions of a given gene was analyzed by real-time RT- ternative exon by changing the time allowed for an alternative PCR using several sets of primer pairs spanning the entire gene exon to be recognized by the splicing machinery assembled on of interest (Figs. S12–S14). The transcriptional elongation rate a transcribing, nascent pre-mRNA (18, 39, 40). The higher was calculated using the pre-mRNA accumulation data, as de- elongation rate leads to increased skipping of alternative exons, scribed (41). Interestingly, the transcriptional elongation rate for which are typically associated with suboptimal splicing signals, Nf1 and Ktn1 nearly doubled in KCl-treated cells compared with due to a shorter window of time for the exons to be defined. NaCl-treated cells, from 3.2 kb/min to 6.8 kb/min for Nf1 (Fig. We hypothesized that calcium-induced hyperacetylation led 6B and Fig. S12) and from 2.4 kb/min to 4.14 kb/min for Ktn1 to a more relaxed chromatin configuration, allowing for faster (Fig. 6B and Fig. S13). Most importantly, the transcriptional RNAPII elongation and, thus, increased exon skipping. elongation rate for Capzb did not show a statistically significant To test this hypothesis, we examined the RNAPII occupancy change, from 1.93 kb/min to 2.08 kb/min (Fig. 6B and Fig. S14). The on the three genes discussed above by ChIP analysis using the H5 increased elongation rate is likely throughout the affected genes, antibody specific to Ser2 phospho-C-terminal domain (CTD) of because the calculated rate for Nf1 is the same using data of pre- the large subunit of RNAPII, a hallmark of elongating RNAPII. We observed a significant reduction of RNAPII occupancy on mRNA accumulation in more than one region of the gene. the Nf1 and Ktn1 genes when cells were depolarized (Fig. 6A and These data establish a strong correlation between hyper- Fig. S7E), but no changes on Capzb (Fig. 6A). These observa- acetylation, faster transcriptional elongation, and increased tions indicate an inverse correlation between RNAPII occupancy skipping of alternative exons. To further strengthen the conclu- and exon skipping in depolarized primary cardiomyocytes, and sion on transcriptional elongation, we treated depolarized pri- are consistent with our hypothesis that RNAPII moves faster on mary cardiomyocytes with camptothecin (CPT), a drug used to Nf1 and Ktn1 in depolarized cells. slow down transcriptional elongation (41, 43). Use of CPT in To provide definitive evidence to support our model, we combination with KCl completely restored the splicing pattern of measured the transcriptional elongation rate of RNAPII on the Nf1 exon 23a (Fig. 6C) and Ktn1 exon 36 (Fig. S15A), but had no three genes in ES cell-derived cardiomyocytes in the absence or effect on Capzb exon 8 (Fig. S15B).

Sharma et al. PNAS Early Edition | 5of9 Downloaded by guest on September 25, 2021 Taken together, the results presented here provide strong calcium-mediated epigenetic changes can regulate gene ex- support for the RBP-independent model that, in response to pression at the level of alternative splicing in cardiomyocytes. increased calcium in cardiomyocytes, the RNAPII elongation rate is increased, leading to more skipping of alternative exons. RBP-Independent vs. RBP-Dependent Calcium-Responsive Mechanisms. Furthermore, our studies reveal the mechanistic link between In cardiomyocytes, we have discovered an alternative splicing increased calcium and RNAPII elongation rate change. As mechanism that appears to be splicing regulator-independent. diagrammedinFig.7A, the elevated intracellular level of calcium We show that increased calcium levels lead to changes in chro- increases the activity of CaMKIIδ or PKD1, which induces the matin modification, which in turn lead to exon skipping. We find nucleus-to-cytoplasm translocation of class II HDACs. As no evidence that this mechanism depends on calcium-induced a consequence, the association of HATs with the genes con- changes of splicing regulator levels, posttranslational mod- taining calcium-responsive alternative exons is increased, re- ifications, or cellular localization (Fig. S11). We posit that this sulting in hyperacetylation of H3 and H4 along the gene body of seemingly RBP-independent mechanism functions beyond, yet in the context of, classical RBP–RNA interactions. these genes. This allows for faster RNAPII elongation rate on However, it is unlikely that the mechanism we describe here is these genes (Fig. 7B). Note that this conclusion is based on the only mechanism induced by calcium. Instead, it is likely to studies of two genes that contain calcium-responsive exons (Nf1 work in conjunction with, and/or in parallel to, other RBP-driven and Ktn1). We believe that it can be applied to other genes that splicing regulatory mechanisms. In this context, we can envision contain calcium-responsive exons, such as those shown in Fig. 2. several scenarios. First, when CaMKII and PKD are activated, Indeed, at least Ank2 appears to be regulated similarly, as use of they may phosphorylate RBPs, which would lead to changes in kinase inhibitors also reversed the KCl-induced splicing change their activity and splicing patterns of their target pre-mRNAs. (Fig. S4C). It will be interesting to study whether histone hyper- An excellent example of this is the depolarization-induced acetylation and increased transcriptional elongation rate occur skipping of the stress axis-regulated exon (STREX) of the Slo1 with Ank2 and other calcium-responsive exon-containing genes. gene. Membrane depolarization of cerebellar neurons induces phosphorylation of hnRNP L at Ser513 by CaMKIV, which Discussion enhances the interaction of hnRNP L with the CaMKIV- Our analysis of calcium-mediated splicing regulation in car- responsive RNA element (CaRRE1) upstream of STREX. As diomyocytes has revealed a previously unidentified regulatory a result, U2AF binding is reduced and STREX is skipped (44). mechanism that integrates histone modifications, transcription, Second, histone hyperacetylation can cause up-regulation of and splicing. We also demonstrate that in addition to transcription, specific splicing regulators at the transcription level, which will lead to either inclusion or skipping of their target alternative exons (Fig. 7B). Third, if epigenetically induced skipping of an exon occurs in a splicing regulator, alternative splicing changes of target pre-mRNAs can then occur as a secondary event (Fig. 7B). A potential example of this is the Rbfox1-mediated splicing pattern change in depolarization of P19 cell-derived neurons (17). In this case, depolarization induces skipping of exon 19 of Rbfox1, which increases nuclear localization of Rbfox1. As a re- sult, several alternative exons change their splicing patterns (17). It is not known how Rbfox1 exon 19 is skipped in depolarized cells. Because this exon is not associated with any CaRRE motifs, it is tantalizing to speculate that the epigenetic mecha- nism described here is responsible for the initial skipping event in an alternative splicing cascade. In our study, use of a calcium chelator in conjunction with an NMDA receptor antagonist in KCl-treated cardiomyocytes re- lieved Nf1 exon skipping without causing an apparent change of transcript level (Fig. S3 A and B). This result appears to imply that additional, possibly RBP-mediated mechanisms exist in calcium-induced splicing control.

Calcium-Induced Histone Modification Changes That Regulate Alternative Splicing. Hyperacetylation of histones leads to more re- laxed chromatin configurations, which support more rapid RNAPII transcription (45, 46). Although it is well-known that histone hyperacetylation at the promoter region leads to increased rates of transcription initiation (47), the impact of hyperacetylation on transcription along the gene body has not been extensively studied. Here we measured transcriptional elongation rate on three genes, two containing calcium-responsive alternative exons and one con- Fig. 7. Model illustrating how increased intracellular calcium levels lead to taining a calcium-nonresponsive alternative exon. We uncovered chromatin changes, which result in changes in alternative splicing patterns. a very strong correlation between histone hyperacetylation, tran- (A) Increased calcium levels activate CaMK and PKD, which phosphorylate scriptional elongation rate, and exon skipping. Significantly, use of class II HDACs, causing their translocation from the nucleus to the cytoplasm. – CPT to slow down transcriptional elongation abolished the effect (B) The disrupted HAT HDAC balance leads to histone hyperacetylation, of increased calcium levels on alternative splicing (Fig. 6). which relaxes chromatin, resulting in an increase in RNAPII transcriptional elongation rate. The dashed arrow indicates that if exon skipping occurs in A number of recent studies have linked the rate of RNAPII an RBP, it may change the ability of the RBP to regulate downstream al- transcriptional elongation and the regulation of alternative ternative splicing events. Black ovals indicate acetyl marks on histones. txn, splicing of the pre-mRNA being transcribed, although tran- transcription. scriptional elongation rate was not measured directly in most of

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1408964111 Sharma et al. Downloaded by guest on September 25, 2021 these studies (42, 48–55). Several distinct mechanisms that reg- In the cardiovascular system, activation of the calcium- PNAS PLUS ulate transcriptional elongation rate to impact alternative splic- CaMKII-HDAC and calcium-PKD-HDAC pathways has been ing have been characterized (39, 56). The RNAPII elongation shown to cause cardiac hypertrophy using genetic mouse models rate can be regulated by association of heterochromatic proteins (21, 25, 58). Deletion of one of the class II HDAC kinases is suffi- induced by siRNA or small RNA (sRNA) (48, 50, 53), in- cient to rescue pathological cardiac remodeling (21). In this teraction of chromatin remodeling complexes with histone study, CaMKIIδ null mice were viable and displayed no obvious marks or methylated DNA (49, 51, 52), or structural changes in cardiac structural or functional abnormalities. These mutant RNAPII (54, 55). A recent study from our laboratory demon- mice showed reduced phosphorylation of a class II HDAC strated that alternative splicing of Nf1 exon 23a can also be (HDAC4), suggesting a diminished kinase activity. When chal- regulated at the chromatin level in a Hu protein-dependent lenged with pressure overload hypertrophic stress, these CaMKIIδ manner in neurons (42), which is a distinct mechanism from the null mice were protected against hypertrophy and fibrosis compared one presented here. In this case, binding of Hu proteins to its with their wild-type littermates (21). Furthermore, in vivo studies target sequences on pre-mRNA blocks HDAC activity, leading to using a p300 transgenic mouse model demonstrated a significant a local histone hyperacetylation surrounding exon 23a, increasing role of p300 in the pathology of cardiac hypertrophy (59). the RNAPII elongation rate of the subsequent rounds of tran- At the molecular level, all of the previous studies have focused scription (42). We showed that neither the expression level nor on up-regulation of gene expression at the transcription initia- cellular localization of HuR, the Hu protein family member tion level such as for Myocyte enhancer factor 2 (Mef2), Nuclear expressedincardiomyocytes,ischangedinKCl-treatedcells(Fig. factor of activated T cells (Nfat), GATA transcription factors, S11). In addition, in neurons, Nf1 exon 23a is predominantly Cardiac homeobox transcription factors (Csx/Nkx2–5), Atrial skipped, and depolarization would not further increase skipping. natriuretic factor (Anf), and so forth (60–63). Our studies reveal These observations highlight the distinct regulatory mechanisms a previously unidentified layer of regulation, supporting a model on the same alternative exons in different cell types or in in which activation of calcium-CaMKII/PKD-HDAC pathways responding to different external conditions. also changes gene expression at the alternative splicing level. However, we predict that the mechanism we uncovered in Interestingly, the developmental remodeling of the heart is cardiomyocytes exists in other cell types such as neurons, al- facilitated by both transcriptional changes (64–66)andchangesin though the specific players of the pathways may be different. To alternative splicing patterns (35, 67). These changes facilitate ad- date, only one other example, exon 18 of NCAM, was reported aptation of the developing heart to adult functions. It has been

to undergo chromatin-level regulation in response to increased shown that in cardiac hypertrophy and heart failure, the gene ex- BIOCHEMISTRY calcium levels, albeit in a different cell type, depolarized neurons pression program often reverts from the adult to fetal patterns (68– (11). Comparing the NCAM study with our results, we notice 70). In our study, we noticed a similar trend for the alternative both similarities and differences. Even though transcriptional exons in Ktn1 (exon 36), Ank2 (exon 21), Enah (exon 5), and Mef2A elongation was not measured directly, the data for pre-mRNA (exon 9) when the calcium level was increased (Fig. 2). It is thus accumulation of regions downstream and upstream of NCAM tempting to speculate that such changes contribute to the physiology exon 18 imply that the elongation rate was increased after de- of the stressed heart, although additional studies need to be con- polarization, leading to more skipping of this exon (11). How- ducted to examine the differential functions of specific alternatively ever, although the increased transcriptional elongation rate for spliced isoforms. Nf1 and Ktn1 appears to be global, it was suggested that the elongation rate increase for the NCAM gene is local, because the Materials and Methods localized increase of H3K9 acetylation and H3K36 trimethyla- Cell Culture, Drug Treatment, and siRNA Transfection. Mouse ventricular car- tion after depolarization correlated with the increased elonga- diomyocytes were prepared from hearts of 1- to 3-d-old newborn CD1 mice tion rate and skipping of exon 18 (11). Interestingly, we did not (Charles River Laboratories) and cultured as previously described (71) with observe any differences in H3K36Me3 or H3K4Me3 after de- minor modifications. Following the procedure, we obtained cells with 90–95% purity, indicated by immunofluorescence staining using polarization of cardiomyocytes (Fig. 4B). anti-cardiac troponin T (cTNT) antibody (Developmental Studies Hybridoma Another important question is how specificity is achieved. Our Bank). The Institutional Animal Care and Use Committee at Case Western results show that genes containing calcium-responsive exons are Reserve University approved these experiments and confirmed that all associated with more HATs, including CBP and p300, whereas experiments conformed to the relevant regulatory standards. a gene containing a calcium-nonresponsive exon does not show Mouse embryonic stem cells (R1) were obtained from the Case Transgenic enhanced association with these HATs in response to increased and Targeting Facility and were cultured as described (29). After two pas- calcium levels (Fig. 5 and Fig. S7). What induces enhanced HAT sages, the cells were differentiated into cardiomyocytes using the hanging association is an open question that remains to be investigated. drop method as previously described (72). The cells started beating ap- Results of our preliminary experiment suggest that KCl induces proximately on day 7 of differentiation and were used by day 10. differential dissociation of class II HDACs, causing reduced class Drug treatment was carried out for 24 h in all cases. In double treatment experiments, KCl/NaCl and drugs were added at the same time. Gö6976 (Sigma) II HDAC association only from those genes containing calcium- was used at a concentration of 10 μM, whereas KN93 (Sigma) was used at 1 μM. responsive exons. It is possible that chromatin remodelers are Nifedipine (Fisher), caffeine (Fisher), AP5 (R&D Systems), and BAPTA-AM (Life mediated by calcium, which removes class II HDACs only from Technologies) were all added at the concentrations indicated in the figures. a subset of genes. It is also possible that the rate of nucleosome Camptothecin (Fisher) was used at a concentration of 3.5 μM. replacement during transcriptional elongation is differentially reg- Primary cardiomyocytes were transfected with scrambled control siRNA or ulated on those genes that contain calcium-responsive exons (57). siRNA targeting a specific gene (200 pmol for 5 × 106 cells) using Lipofect- amine 2000 (Life Technologies) as described by the manufacturer. The Gene Expression Regulation by the Calcium-CaMK/PKD-HDAC Pathways. sequences of siRNAs are shown in Table S1. Our results indicate that the CaMKII and PKD signaling pathways play redundant roles in calcium-mediated splicing regulation, as Semiquantitative RT-PCR, Western Blot, and Immunofluorescence Analyses. Total RNA extraction and RT-PCR analysis were performed as described either one of the two is sufficient to promote the downstream effect: – translocation of class II HDACs from the nucleus to the cytoplasm, previously (73). Low-cycle number PCR (16 20) was performed to determine – the relative abundance of individual isoforms of RNA transcripts. Primer which perturbs HAT HDAC balance. The increased association of information is included in Table S2. Quantification of exon inclusion was de- HATs including CBP and p300 with Nf1 and Ktn1 causes histone termined using a Typhoon Trio Variable Mode Imager (GE Healthcare). The hyperacetylation of these genes, enhancing the rate of transcrip- results shown are representative of results from at least three independent tional elongation and exon skipping (Fig. 5). experiments. Total protein extraction, Western blotting (73), immunofluores-

Sharma et al. PNAS Early Edition | 7of9 Downloaded by guest on September 25, 2021 cence (74), and acid extraction of total histones (75) were performed as pre- nologies). The reverse-transcription and real-time PCR analyses were viously described. Antibody information is included in Table S3. described previously (42). Primers targeting exon–intron or intron–exon junctions were used. Accumulation of pre-mRNA containing different ChIP and Real-Time PCR. The ChIP experiments were performed with a ChIP regions of a gene at each time point was plotted comparing NaCl-treated Assay Kit (Millipore; 17-295) using 6 × 107 cells that were fixed with 1% with KCl-treated cells. The rate of transcriptional elongation was calculated formaldehyde for 30 min at 37 °C as described (42). Immunoprecipitation by dividing a specific length of transcript by the time taken for its tran- was carried out overnight at 4 °C with 15 μg of H5 antibody (Covance) or scription. For example, the Nf1 transcriptional elongation rate in KCl-treated anti–acetyl-histone H3 (Millipore), -CBP (Santa Cruz Biotechnology), or -p300 cells was calculated using exon 40, because it is expressed after 30 min of (Santa Cruz Biotechnology) antibodies. Nonspecific rabbit IgG (Sigma) was transcription, but at the same time point exon 45, which is only 3 kb away, is used as a control. Cross-linking of bound DNA fragments was then re- not expressed. The primers targeting exon 40 are 205 kb away from the versed using 5 M NaCl, and DNA was dissolved in 100 μL Tris (10 mM)/EDTA promoter, which gives the transcriptional elongation rate of 6.8 kb/min. (1 mM) and used as a template for real-time PCR. Real-time PCR was car- Primers targeting exon 17 at the 30-min time point were used to measure ried out as previously described (42). Primers used in the PCR analyses are the elongation rate for Nf1 in NaCl-treated cells. To calculate the tran- described in Table S2. Each primer set has one primer annealing to an scriptional elongation rate for Ktn1, exon 30 at the 15-min time point and intronic region and the other annealing to an exonic region. For data exon 13 at the 15-min time point were used for KCl-treated and NaCl- analysis, each sample was quantified in duplicate and at least three in- treated cells, respectively. Last, to calculate the transcriptional elongation dependent ChIP analyses were performed. The ChIP signals were normal- rate for Capzb, primers targeting Capzb exon 7 at the 45-min time point and ized to input. P values were calculated by conducting a Student t test exon 5 at the 45-min time point were used for KCl-treated and NaCl-treated comparing values obtained from NaCl and KCl treatments for each in- cells, respectively. dicated region of a specific gene. ACKNOWLEDGMENTS. The authors thank Dr. James Stévenin (Institut de Transcriptional Elongation Analysis. Inhibition and reinitiation of tran- Génétique et de Biologie Moléculaire et Cellulaire) for the anti-SC35 anti- scription and bromouridine (BrU) labeling were performed as described body. We also thank Drs. Helen Salz, Saba Valadkhan, and Jo Ann Wise (41, 42). For this experiment, ES cell-derived cardiomyocytes were trea- for critical reading of the manuscript and members of the H.L. laboratory for helpful discussions. We thank Dr. Saptarsi Haldar for stimulating ted with 50 μM DRB (Sigma) for 8 h before 2 mM BrU (Sigma) was added discussions. This work was supported by the National Institutes of Health into the medium and the cells were cultured for another hour. For [NS049103 (to H.L.), and S10RR021228 and S10RR024536 National Center samples that were treated with KCl, it was added at the same time as for Research Resources Shared Instrumentation Grants for the Leica BrU. Following the BrU incubation, cells were collected at 0, 15, 30, 45, DM6000 wide-field microscope and the GE Healthcare Typhoon Trio Var- 60, 75, and 90 min and total RNA was isolated using TRIzol (Life Tech- iable Mode Imager].

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