RNA募集染色质重塑复合物NuRD的分子机制

NuRD (Nucleosome Remodeling and Deacetylation) complex plays a key role in various cellular processes, including transcription regulation, DNA damage responses, embryo development and carcinogenesis. However, the recruiting mechanism is still not clear. Currently it is known that NuRD is recruited to target genes by interacting with transcription factors (TF) or other proteins. But recent ChIP-seq data shows majority of CHD4 target genes do not contain certain TF binding sites. In our previous papers, we have shown a stress-induced long noncoding RNA-PAPAS, which is transcribed from the antisense direction of ribosomal RNA genes (rDNA), interacts with CHD4 and recruits NuRD complex to rDNA promoter, and triggers epigenetic silencing of rDNA by histone deacetylation and shifting the promoter-bound nucleosome into a position that is refractory to transcription initiation. In this study, we will map the minimal interacting region of PAPAS with CHD4 by RNA pulldown with truncated PAPAS and try to find the specific sequence motif or secondary structure. Meanwhile, we would also perform CLIP-seq (crosslinking-immunprecipitation and high-throughput sequencing) with CHD4 to identify the interacting RNAs, then analyse general characteristics of these RNAs. Therefore, we can predict which RNA can interact with CHD4. Furthermore, we will identify post-translational modifications of CHD4 upon heat shock to investigate how the RNA binding ability is regulated. The ultimate aim of this proposal is to discover novel rectuiting model of chromatin remodeling complex by specific chromatin-bound RNAs.

染色质重塑复合物NuRD在基因转录调控、DNA损伤修复、胚胎发育和癌症发生等过程中有重要作用，但是其具体的募集机制尚不清楚。目前认为NuRD通过结合转录因子而定位于靶基因，从而发挥其功能。但是ChIP-seq的结果显示，转录因子并不能完全解释染色质重塑复合物NuRD识别序列的特异性。前期研究发现CHD4可以直接结合细胞应激诱导的LncRNA，并募集NuRD复合物至核糖体RNA基因的启动子区，调节染色质结构。本项目拟定以CHD4为出发点，通过RNA删切与突变等，找到CHD4识别RNA的特异性。同时利用CLIP-seq探究所有结合的RNA的序列或结构特点，从而预测与之结合的RNA. 进一步利用热休克为模型，揭示CHD4翻译后修饰如何调控与RNA的结合，从而影响NuRD复合物的招募。本课题将验证RNA募集NuRD复合物的广泛性，进一步从新的视角揭示染色质重塑复合物的募集特异性。

本项目以CHD4为出发点，通过RNA删切与突变等，找到CHD4识别RNA的特异性。同时利用CLIP-seq探究所有结合的RNA的序列或结构特点，从而预测与之结合的RNA。进一步利用热休克为模型，揭示CHD4翻译后修饰如何调控与RNA的结合，从而影响NuRD复合物的招募。我们研究表明PAPAS直接与DNA相互作用，形成DNA-RNA三重结构，将PAPAS 连接到增强子区域内的嘌呤伸展，从而将相关的CHD4/NuRD（核小体重塑和去乙酰化）引导至rDNA启动子。蛋白质-RNA相互作用实验结合RNA二级结构作图表明，CHD4的N端部分与PAPAS中的非结构化富含A区相互作用。该序列的缺失或突变消除了与CHD4的相互作用。PAPAS的压力依赖性上调伴随着CHD4在三个丝氨酸残基的去磷酸化，这增强了CHD4/NuRD与RNA的相互作用并加强了对rDNA转录的抑制。结果同样表明lncRNA 引导染色质重塑复合物至特异性基因组位点的功能，并揭示了CHD4/NuRD介导的转录调控的磷酸化依赖性机制。