组蛋白甲基化转移酶NSD2在多发性骨髓瘤中的转录调控研究

DNA in eukaryotic cells wraps on proteins called histones to form chromatin. Histone modifications which include methylation, acetylation, ubiquitylation, etc., affect different aspects of DNA functions, such as transcription. Among all histone modifications and molecules involved in the processes, methylation and its modifying enzymes, histone methyltransferases, are the most complicated and important. Many histone methyltranferases play important roles in tumorigensis through their regulation of transcription. .Histone methyltransferase, NSD2, is up-regulated in about 15% of multiple myeloma due to t (4, 14) translocation. It is also up-regulated in many hematopoietic and lymphoid tumors and solid tumors, with high levels of NSD2 expression positively correlating with high grades of tumors and poor prognosis. Although strong evidence supports that NSD2 plays a key role in tumorigenesis, the mechanism of NSD2 mediated transcriptional regulation is poorly understood. .Our recent work showed that NSD2 catalyzes H3K36 di-methylation (H3K36me2) and its specificity depends on the nature of the substrate, i.e. DNA stimulates NSD2 activity towards H3K36me2, while in the absence of DNA, NSD2 activity is low and nonspecific. This study clarified the discrepancy that has misled the field for a long time and clearly linked NSD2 with H3K36me2. .Our understanding of the role of H3K36 methylation in transcription has been limited to H3K36me3 and its catalyzing enzyme SET2, which associates with RNA polymerase II (RNAPII) and catalyzes H3K36me3 along the body of active genes. However, evidence suggests that NSD2 regulates transcription in a different way: it has been found binding at promoter regions of multiple genes to repress transcription, it has also been reported to enhance androgen receptor dependent gene activation. Moreover, NSD2 associated with different proteins in mouse embryonic stem cells and embryonic heart. Our preliminary data suggest that it binds with yet a different set of proteins from those two situations in HT1080 human fribrosarcoma cells. . We hypothesize that NSD2 is targeted to different gene regions by their associating proteins and regulates transcription in different ways. In this project, we propose to perform ChIP-seq for NSD2, H3K36me2, and RNAPII and expression Chip in multiple myeloma cells to identify their targets, explore their positioning on the genes, and study the different binding patterns of the three and associate them with transcriptional activity of the target genes. We will also purify NSD2 interacting proteins to study how NSD2 complexes are targeted to genes and affect transcription in different phases (initiation and/or elongation) of transcription. .Our research will not only broaden understanding of the function of H3K36 methylation in transcription but also provide valuable information regarding NSD2 target genes and NSD2 mediated transcriptional change in the tumorigenesis of Multiple Myeloma.

组蛋白甲基化转移酶通过对染色质的共价修饰在基因转录和人类疾病中起重要作用。组蛋白甲基化转移酶NSD2在含有t(4,14) 染色质异位的多发性骨髓瘤细胞中高表达，促进瘤细胞的生长与小鼠体内肿瘤形成。而且,它在多种淋巴造血组织肿瘤与实体瘤中高表达，且表达水平与肿瘤的恶性度与预后相关。虽然大量研究支持NSD2在肿瘤形成中的重要作用，NSD2在肿瘤中的转录调控机制还不清楚。申请人新近工作表明NSD2催化H3K36二甲基化，其作用方式与经典的H3K36甲基化酶十分不同。本项目拟在多发性骨髓瘤细胞系中用ChIP-seq与基因表达芯片相结合的方法研究NSD2，H3K36me2，RNA聚合酶II的基因定位，探讨NSD2在基因转录中的作用环节和转录活性，寻找NSD2调控的关键靶基因；用蛋白纯化法获得NSD2相互作用蛋白，探讨NSD2复合物的基因定位与转录调控机制，为H3K36甲基化与转录调控提供新思路。

组蛋白甲基化转移酶通过对染色质的共价修饰在基因转录和人类疾病中起重要作用。组蛋白甲基化转移酶NSD2催化H3k36me2并且与疾病关系密切，在儿童中，NSD2的单倍体不足导致以神经发育不全为特征的儿童发育疾病Wolf–Hirschhorn 综合征，在成年人中，NSD2在含有t(4,14)染色质异位的多发性骨髓瘤细胞中高表达，被认为是肿瘤发生的驱动基因。虽然大量研究支持NSD2在肿瘤形成中的重要作用，NSD2在肿瘤中的转录调控机制还不清楚。在本项目中，我们使用了带有t(4,14)染色体异位的多发性骨髓瘤细胞系，建立了稳定的shRNA-NSD2 细胞系。运用ChIP－seq实验，我们发现NSD2与其催化的H3k36me2修饰在基因的启动子与基因体区都有结合。NSD2的目的基因中包括大量肿瘤相关通路基因。结合基因表达谱，我们发现NSD2主要与目的基因的转录活化相关。通过亲和纯化与质谱技术，我们发现了一些NSD2结合蛋白，其中与BRD4的结合对NSD2在部分目的基因的启动子区域的募集起重要作用。除此之外，我们还发现NSD2在弥漫大B细胞淋巴瘤中高表达，而且NSD2的表达水平与弥漫大B细胞淋巴瘤的分期成正相关与病人的预后成负相关，NSD2在弥漫大B细胞淋巴瘤发生中可能有相似的致癌机制。我们还建立了小鼠胚胎干细胞体外神经分化体系，拟进一步研究与比较NSD2在个体发育的生理状态下的作用机制。本研究扩展了对组蛋白甲基化转移酶NSD2的致癌机制的了解，为针对NSD2的小分子抑制剂的抑癌作用研究奠定了基础。