BRD4 Contributes to the Regulation of Alternative Splicing

BRD4 有助于选择性剪接的调控

• 批准号: 10926375
• 负责人: Dinah S. Singer
• 金额: $ 17.35万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926375
• 关键词: 3' Splice Site; ATP phosphohydrolase; Acute Myelocytic Leukemia; Affect; Alternative Splicing; Binding; Bromodomain; CD34 gene; CSPG6 gene; Cell Cycle; Cell Cycle Regulation; Cells; Chromatin; Chromatin Structure; Co-Immunoprecipitations; Complex; DNA; DNA Binding; Data Set; Development; Disease; Ensure; Epigenetic Process; Event; Exons; Frequencies; Gene Expression Regulation; Genes; Genetic Transcription; Genome; HCT116 Cells; Head; Heterodimerization; Heterogeneous-Nuclear Ribonucleoproteins; Human; Inflammation; Lead; Link; Malignant Neoplasms; Mediating; Metabolism; Modeling; Molecular; Mus; Mutation; Normal Cell; Patients; Pattern; Play; Point Mutation; Process; Promoter Regions; Protein Family; Proteins; RNA Polymerase II; RNA Splicing; Regulation; Resistance; Ribonucleoproteins; Role; Sampling; Series; Shapes; Sister Chromatid; Spliceosomes; System; Thymus Gland; Trans-Activators; Transcript; acute T-cell lymphoblastic leukemia cell; acute myeloid leukemia cell; cancer cell; cofactor; cohesin; embryonic stem cell; exon skipping; homologous recombination; in vivo; novel; posttranscriptional; prevent; promoter; recruit; transcription factor

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.

我们已经确定BRD4有助于在体内和癌细胞中正常细胞中剪接的模式。通过与基因组上的剪接机械的直接相互作用，BRD4通过调节外显子使用而优先起作用。这些发现导致BRD4功能的模型可以整合染色质结构，转录和剪接的过程，以确保对基因表达的适当调节。在胸腺中记录了BRD4在调节体内替代剪接中的作用，在胸腺中，BRD4的缺失导致替代剪接模式发生明显变化。此外，我们发现从一个发育阶段到另一个发育阶段的过渡 - dn到ISP到DP到sp-伴随着剪接模式的变化，表明剪接在百里香中受到发育的调节。在每个阶段中以大致相等的水平表达的BRD4都会有助于这种调节，因为删除BRD4会导致分化每个阶段的剪接模式的变化，优先影响跳过的外显子事件，但不会影响剪接因子的表达。与其在癌症中的作用一致，BRD4还有助于T-All细胞中替代剪接的模式，在很大程度上影响了与细胞周期调节有关的基因。有趣的是，BRD4的降解会导致剪接模式的变化要大得多，而BRD4通过其溴化群的抑制与染色质结合。这表明BRD4介导的替代剪接调节并不完全取决于其通过其溴结构域与染色质结合。对该角度的进一步支持来自于发现BRD4与剪接因子的共定位并不受JQ1的干扰，JQ1阻止了与染色质结合的结合。此外，尽管BRD4的降解靶向启动子和基因体，但JQ1通过JQ1与染色质的相互作用阻止了BRD4溴化群介导的相互作用，这在很大程度上仅限于基因的启动子区域，并进一步支持BRD4与长制和剪接机器的相互作用的结论不依赖于其成果。我们发现BRD4直接与FUS和HNRNPM相互作用的发现表明它可以作为剪接机械的组成部分参与。 BRD4促进剪接调节的确切机制仍有待确定。基于BRD4和粘着素之间的功能相似之处，我们检查了粘蛋白还可以单独或与BRD4结合的替代剪接的可能性。我们发现粘蛋白调节纤维素和体内的剪接。粘着蛋白的耗竭会导致剪接模式改变。在粘蛋白亚基SMC1中引入小鼠胚胎干细胞中的单点突变，都会改变其与剪接因子的关联和剪接模式。从机械上讲，粘蛋白与剪接机械的核心成分相互作用，包括U1-70，调节因子FUS和HNRNPM。重要的是，在原发性AML患者样品中，与正常的CD34+细胞或没有内粘着蛋白或剪接因子突变的正常CD34+细胞或AML细胞相比，粘蛋白突变与剪接模式的改变有关。与粘着蛋白在AML中的替代剪接中的直接作用一致，与AML相关的粘着蛋白突变不再与剪接因子相互作用。令人惊讶的是，在两个不同的主要AML样品数据集中与粘蛋白突变相关的剪接模式的改变导致代谢扰动。粘蛋白介导的剪接模式与仅由BRD4调节的剪接不同。重要的是，粘蛋白和BRD4共同有助于单独的剪接模式，与任何一个因素不同。与它们的剪接，粘蛋白和BRD4的共同调节直接相互作用，并存在于HCT116细胞中的复合物中。这些研究确定了粘蛋白在调节替代剪接方面的新作用，无论是单独或与BRD4结合在一起，并对人AML癌的表征具有影响。

项目成果

Cohesin regulates alternative splicing.

• DOI: 10.1126/sciadv.ade3876
• 发表时间: 2023-03
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Singh, Amit K.;Chen, Qingrong;Nguyen, Cu;Meerzaman, Daoud;Singer, Dinah S.
• 通讯作者: Singer, Dinah S.