染色质构象调控lncRNA在造血干细胞红系分化中的作用机制研究

Currently, directional differentiation of stem cells is the hot spot in the field of regenerative medicine and stem cell research, and erythroid differentiation of hematopoietic stem cells is one of the important topics. Previous studies indicated that erythroid differentiation is regulated by multiple factors, however, the role of lncRNA in erytroid differentiation still remains unclear. Our previous generated transcriptome data revealed that dozens of lncRNAs specially expressed at each stage during erythroid differentiation, which implied that these lncRNAs may play a role in differentiation regulating. Further analyses on ENCODE data implied that chromatin conformation may involved in regulation of these lncRNAs. In this project, we intend to employ cell lines and primary hematopoietic stem cells (CD34+CD38-) extracted from human cord blood, which can differentiate and develop into erythrocytes, as the study model to study the regulatory functions of the candidate lnRNAs. Firstly, we plan to evaluate the function of candidate lncRNAs by comparing the erythroid differentiation progress between controls and lncRNA ectopic expressed cells. Then, we reveal the correlation between chromatin interactions and lncRNAs expression during differentiation by using 4C technique. Finally, we investigate the regulation mechanism of lncRNAs through RNA pull-down and RIP. We aim to construct a dynamic regulation network consist of chromatin interaction, lncRNAs, proteins and erythroid differentiation by comprehensively analyzing results from all mentioned studies. This study will not only enrich the regulation mechanisms of erythroid differentiation, but also may provide a new perspective for other studies in directional differentiation of stem cells.

干细胞定向分化是当前干细胞与再生医学领域的研究热点，而造血干细胞的红系分化是其中重要的课题之一。以往的成果表明红系分化受到多重因素调控，而lncRNA在其中发挥的作用仍待研究。项目组已有的转录组学数据分析结果提示多个lncRNA潜在调控红系分化，相关ENCODE数据的挖掘分析提示染色质构象参与上述lncRNA的表达调控。本研究将利用红系细胞株诱导分化模型和人脐带血来源的造血干细胞定向红系分化模型，通过干扰候选lncRNA表达，观察其对红系分化的影响；利用4C技术探讨染色质构象对候选lncRNA的调控作用；采用RNA pull-down、RIP等技术研究候选lncRNA的调控机制。综合染色质构象、lncRNA和红系分化三方面研究结果，构建“染色质-lncRNA-结合蛋白”对红系分化的动态调控网络。该网络不仅将丰富红细胞分化的调控机制，也可望为干细胞定向分化相关领域的研究提供新思路。

红系分化是一个动态的、复杂的、精确调控的过程。以往的研究揭示了细胞因子、转录因子、microRNAs等在调控红系分化方面的作用及其机制，但是lncRNAs和染色质可及性相互作用在红系分化过程中的调控机制还缺乏全面的认识。..本研究以脐带血来源的造血干细胞红系分化为研究模型，通过深入挖掘红系分化过程中不同阶段的多组学数据，分析了染色质可及性和基因表达的动态特征，对出现变化的染色质可及性和基因的功能进行了鉴定。本研究鉴定了红系分化晚期阶段（Erythroblast）LncRNA、转录因子和关键调控基因的相互作用网络，并对新鉴定的候选 lncRNA的生物学功能进行验证。本研究也对巨核红系祖细胞阶段的LncRNA进行了功能验证。本研究重点围绕LncRNA通过染色质动态变化调控红系造血的分子机制进行。..项目组通过对染色质可及性和长非编码RNA（LncRNA）在红系分化过程中的调控规律进行了深入解析，发现染色质可及性和LncRNA在不同红系分化阶段具有阶段特异性的特征，EB阶段变化最为显著。染色质可及性和LncRNA的相互作用网络参与调控了红系分化进程。我们特别鉴定了红系分化晚期的分子网络，并鉴定了该网络中几个关键的调控红系分化晚期的候选LncRNAs，如LINC01128、LINC01133、CYTOR及PCED1B-AS1，以及可能的作用途径。通过对红系分化特定阶段的LncRNA进行功能验证，发现这些LncRNA可通过染色质的动态变化调控红系分化进程。本项目首次发现了LncRNA与染色质可及性的相互作用调控红系造血的分子机制。..我们的发现为人类红细胞生成中lncRNA和染色质的动态相互作用提供了新的见解，这可能导致发现预防或治疗红细胞生成或造血疾病的生物标志物。