Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of alternative splicing. p300 promotes genome-wide exon inclusion in both a transcription-dependent and -independent manner. Using CD44 as a paradigm, we found that p300 regulates alternative splicing by modulating the binding of splicing factors to pre-mRNA. Using a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates alternative splicing by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. p300-mediated CD44 alternative splicing reduces cell motility and promotes epithelial features. Our findings reveal a chromatin-related mechanism of alternative splicing regulation and demonstrate its impact on cellular function.

Enhancer demethylation in leukemia has been shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in cancer progression. Since transcription and splicing are coupled, we wondered whether VEGFA enhancer activity can also regulate the gene’s alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6a and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients present with high expression of +157 eRNA and inclusion of VEGFA exons 6a and 7. In addition, our results show that the positive regulator of RNAPII transcription elongation, CCNT2, binds VEGFA’s promoter and enhancer, and its silencing promotes exclusion of exons 6a and 7 as it slows down RNAPII elongation rate. Thus our results suggest that VEGFA’s +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates for the first time a connection between an endogenous enhancer and alternative splicing regulation of its target gene.

Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 demethylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 265 following DNA damage, and that the phosphorylation is important for proper cell-cycle regulation. We demonstrated that SAT1 alternative splicing, regulated by KDM3A, plays a role in cell-cycle regulation. Furthermore we found that KDM3A's demethylase activity is not needed for SAT1 alternative splicing regulation. In addition, we identified KDM3A's protein partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor binding. Our work reveals chromatin sensing of the environment in the regulation of alternative splicing.

7Running head: KDM3A regulates alternative splicing of cell-cycle genes 8 Keywords: pre-mRNA splicing, alternative splicing, chromatin 9 10 2 ABSTRACT 11 Changes in the cellular environment result in chromatin structure alteration, which in turn 12 regulates gene expression. To learn about the effect of the cellular environment on the 13 transcriptome, we studied the H3K9 de-methylase KDM3A. Using RNA-seq, we found that 14 KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle 15 and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 16 265 following DNA damage, and that the phosphorylation is important for a proper cell cycle 17 regulation. We demonstrated that SAT1 alternative splicing, regulated by KDM3A, plays a 18 role in cell cycle regulation. Furthermore we found that KDM3A's demethylase activity is not 19 needed for SAT1 alternative splicing regulation. In addition, we identified KDM3A's protein 20 partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing 21 and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results 22suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor 23 binding. Our work reveals chromatin sensing of the environment in the regulation of 24 alternative splicing. 25 26 27 30 gene, isoforms that can have different functions, all contributing to the cell's diverse tasks 1 . 31Traditionally the regulation of alternative splicing was attributed to regulatory elements in 32 the pre-mRNA. Recently we and others have shown the link between chromatin structure and 33 epigenetic modifications in splicing regulation 2-10 . The data linking chromatin and alternative 34 splicing is not enough to explain the physiological causes and consequences of chromatin-35 mediated changes in alternative splicing. Furthermore, a major unanswered question in the 36 field is how, and to what extent, environmental changes shape the cell transcriptome via 37 modulation of alternative splicing.38 Cellular environment has been shown to modulate splicing in several ways. The main one is 39 the induction of post-translational modifications of splicing factors. Phosphorylation of SR 40 splicing factors affects their protein-protein interactions as well as regulating protein activity. 41A specific example is activation of the Fas receptor leading to dephosphorylation of SR 42 proteins that promote pro-apoptotic isoforms of two key regulators, Bcl-X and caspase-9 11 . 43Another example is cell-cycle-dependent splicing patterns, which are also modulated by SR 44 protein phosphorylation by specific kinases and phosphatases such as NIPP1 12 . Another mode 45 of action for alternative splicing regulation following cellular environmental changes could 46 be by modulating chromatin, as changes in the environment are sensed by cells and 47 transduced into changes in gene expression, leading to specific cellular responses. 48The histone demethylase KDM3A has been shown to ...

Enhancer demethylation in leukemia and lymphoma was shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in vascularization and cancer progression. Since transcription and splicing are coupled in space and time, we hypothesized that the VEGFA enhancer can also regulate the gene’s alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6b and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients display high enhancer activity and inclusion of VEGFA exons 6b and 7. To search for a key protein connecting transcription with alternative splicing, we analyzed 161 chromatin immunoprecipitation (ChIP)-seq experiments for DNA binding proteins and found that PML and CCNT2 bind VEGFA’s promoter and enhancer. CCNT2 is a positive regulator of RNA polymerase II (RNAPII) transcription elongation and indeed its silencing promotes exclusion of exons 6b and 7. Slowing down RNAPII elongation promotes exclusion of exons 6b and 7. Thus our results suggest that VEGFA’s +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates the importance of the interplay between transcription and alternative splicing.