BRWD3蛋白复合物参与基因转录调控的机理

The BRWD3 gene is localized to Xq21, encoding a protein consisting of a bromodomain and several WD repeats. It plays a role in transcription as a chromatin-modifying factor. Abnormal expression of BRWD3 is associated with B-cell chronic lymphocytic leukemia, breast cancer and mental retardation. However, its causal pathogenesis is unknown. To understand the pathogenesis, it is fundamental to uncover the normal regulatory mechanism about BRWD3. According to its structure characters, BRWD3 may function through WD40 assembling proteins into a regulatory complex, which then specifically bind target sites with the bromodomain. Although BRWD3 functions a global regulation at the chromatin level, the composition of its regulatory complex and the related downstream genes remain unclear. In this project, the BRWD3 interacting proteins will be screened from human embryonic brain cDNA library with yeast two-hybrid. The composition of BRWD3 regulatory protein complex will be identified through GST pull-down, cell two hybrid and co-immunoprecipation. The binding target genes will be identified with chromatin co-immunoprecipation combining with DNA sequencing. The affinity among BRWD3 complex and the target sites will be confirmed with Real-Time Polymerase Chain Reaction. Based on the mutations of BRWD3 obtained in our previous research, the influence of abnormal BRWD3 will be analyzed on transcription regulation of target genes. Through comparing the wild type and the mutants, the composition of the regulatory protein complex and the downstream genes will be confirmed. This pilot study will provide not only theoretical support for regulatory mechanism of BRWD3 protein complex, also clues for understanding the pathogenic mechanism of mental retardation and breast cancer. In addition, this research is meaningful for developing small molecular inhibitors of bromodomain as well as for screening target sites and other intervention manner.

Bromodomain and WD repeat-containing protein 3 (BRWD3)异常与精神发育迟滞和肿瘤等疾病发生密切相关。根据其结构与功能特点，BRWD3可能以染色质重塑复合物形式结合到靶位点发挥转录调控作用，正常转录调控和异常致病机理不明。本项目拟采用酵母双杂交的方法，从人胚胎脑cDNA文库中筛选与BRWD3相互作用的蛋白，采用经典蛋白质相互作用研究手段确认调控复合物组成；采用染色质免疫共沉淀和测序方法解析调控复合物结合靶基因；利用我们在精神发育迟滞患者中筛选到的突变位点，进一步证实BRWD3蛋白复合物及与下游基因之间的相互作用。研究结果将有助于阐明BRWD3在染色质水平大范围调控转录的过程和机理，初步揭示BRWD3突变导致精神发育迟滞发生的可能原因，也对目前受关注的bromodomain靶向小分子抑制剂的研发、筛选靶点和干预措施等研究有实际参考价值。

包含bromodomain结构域的蛋白发挥转录调控或辅助激活作用，常常参与胚胎发育、能量代谢、脂肪形成进而炎症反应，异常状态常与癌症、糖尿病、肥胖、神经发育畸形、炎症等多种疾病相关。BRWD3与个体正常发育以及癌症、智力低下等相关，机理不明。本项目在中国智力低下高发区—秦巴山区，探究BRWD3突变情况，分析BRWD3突变对人认知功能影响及其分子机制。结果：检测到4种类型的突变，其中2种是丧失WD40和bromodomain这2类结构域的截短突变；这2类结构域均可以与其它蛋白结合，形成蛋白复合物，为探究BRWD3截短突变导致智力低下发生的机制，采用酵母双杂交，在人脑cDNA文库中筛选与BRWD3相互作用蛋白，筛选出的12个蛋白中，NUFUIP1据报道与智力低下相关；经生物膜干涉技术和CO-IP，证实BRWD3与NUFIP1具有特异性相互作用；采用染色质免疫共沉淀结合测序方法，筛选出BRWD3结合的基因；采用QPCR方法，分析BRWD3转录水平变化对功能注释智力低下基因转录水平影响，SETD5、SMARCB1、CTNNB1、ELP2、KAT6A、MEF2C、RLIM以及SOBP在BRWD3转录水平被敲低以后，转录水平显著降低；因BRWD3和NUFIP1相互作用，分析上述基因是否同时受NUFIP1调控，采用RNAi法敲低NUFIP1，分析候选基因是否受这2个转录因子转录水平的调控。结果显示SETD5、SMARCB1 、RLIM 、SOBP 、ELP2、MEF2C等基因转录水平显著受到BRWD3和NUFIP1水平影响；采用过表达NUFIP1，ChIP后半定量PCR法分析，结果显示MEF2C受BRWD3和NUFIP1共同调节；采用xCELLigence® DP实时细胞分析仪分析BRWD3对细胞活动影响，初步揭示BRWD3转录水平变化会影响细胞的侵袭转移能力。本次研究首次从转录调控角度，诠释BRWD3突变导致智力低下发生的机理：BRWD3结构域缺失的截短突变，影响与NUFIP1等蛋白的相互作用，影响海马依赖性学习与记忆能力相关基因MEF2C转录；另外，单纯改变BRWD3转录水平，可改变细胞运动能力，可能会改变人言语认知和类比推理能力，导致智力低下表型出现。