BRD4 Contributes to the Regulation of Alternative Splicing

Division Of Basic Sciences - Nci

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Abstract

We have established that BRD4 contributes to patterns of splicing both in normal cells in vivo and in cancer cells. Through its direct interaction with the splicing machinery on the genome, BRD4 acts preferentially by modulating exon usage. These findings lead to the model that BRD4 functions to integrate the processes of chromatin structure, transcription and splicing to ensure proper regulation of gene expression. The role of BRD4 in regulating alternative splicing in vivo was documented in the thymus where deletion of BRD4 resulted in marked changes in the patterns of alternative splicing. Furthermore, we have found that the transitions from one developmental stage to another - DN to ISP to DP to SP - are accompanied by changes in the patterns of splicing, indicating that splicing is developmentally regulated in the thymus. BRD4, which is expressed at approximately equal levels in each of these stages, contributes to this regulation since deletion of BRD4 results in changes in the splicing patterns at each stage of differentiation, preferentially affecting skipped exon events, but not affecting the expression of splicing factors. Consistent with its role in cancer, BRD4 also contributes to patterns of alternative splicing in T-ALL cells, largely affecting the genes involved in cell cycle regulation. Interestingly, degradation of BRD4 results in much larger changes in splicing patterns than the inhibition of BRD4 binding to chromatin through its bromodomains. This suggests that BRD4 mediated regulation of alternative splicing does not depend entirely on its binding to chromatin through its bromodomains. Further support for this perspective comes from the finding that BRD4's co-localization with splicing factors is not perturbed by JQ1, which prevents binding to chromatin. In addition, whereas degradation of BRD4 targets both promoters and gene bodies, blocking of BRD4 bromodomain-mediated interactions with chromatin by JQ1 is largely confined to the promoter regions of genes, lending further support to the conclusion that BRD4's interaction with the elongation and splicing machineries does not depend on its bromodomains. Our finding that BRD4 directly interacts with both FUS and HnRNPM, suggests that it can participate as an integral component of the splicing machinery. The precise mechanism(s) by which BRD4 contributes to the regulation of splicing remain to be determined. Based on the functional parallels between BRD4 and cohesin, we examined the possibility that cohesin also regulates alternative splicing, either alone or in combination with BRD4. We find that cohesin regulates splicing in cellulo and in vivo. Depletion of cohesin leads to altered patterns of splicing. Introduction of a single point mutation in the cohesin subunit, SMC1, in mouse embryonic stem cells, alters both its association with splicing factors and the pattern of splicing. Mechanistically, cohesin interacts with core components of the splicing machinery including U1-70, the regulatory factors FUS and HNRNPM. Importantly, in primary AML patient samples, cohesin mutations are associated with altered splicing patterns compared with either normal CD34+ cells or AML cells without cohesin or splicing factor mutations. Consistent with a direct role of cohesin in alternative splicing in AML, AML-associated cohesin mutations no longer interact with splicing factors. Strikingly, the alterations in splicing patterns associated with cohesin mutations in two distinct datasets of primary AML samples result in perturbations in metabolism. The patterns of splicing mediated by cohesin are distinct from those regulated by BRD4 alone. Importantly, cohesin and BRD4 together contribute to a pattern of splicing that is distinct from either factor alone. Consistent with their co-regulation of splicing, cohesin and BRD4 directly interact and exist in a complex in HCT116 cells. These studies establish a novel role for cohesin in regulating alternative splicing, either alone or in conjunction with BRD4 and have implications in the characterization of human AML cancer.