转录因子GATA1调控lncRNA参与红系分化的作用及机制研究

Currently, directional differentiation of stem cells is the hot spot in the field of regenerative medicine and stem cell research, and erythroid differentiation is one of the important topics. Previous studies indicated that transcription factor GATA1 is one of the most essential regulators of erythroid differentiation. However, most GATA1 mechanism researches focused on digging the correlation with coding genes, and ignored noncoding RNA, especially lncRNA. Our previous generated transcriptome data revealed that dozens of lncRNAs including MIAT and RP11-960L18.1 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 revealed that GATA1 binds at the gene locus of MIAT and RP11-960L18.1. In this project, we intend to employ inducible cell lines as the study model to investigate how GATA1 binding at MIAT and RP11-960L18.1 gene locus during erythroid differentiation by performing data analyses, dual-luciferase assay and ChIP. We are going to evaluate function of MIAT and RP11-960L18.1 during erythroid differentiation through interfering their expression. RNA-seq of control and lncRNA manipulated cell line will be performed to gain global effects caused by lncRNA and further provide information for mechanism studies. We plan to preliminary investigate further mechanism of MIAT and RP11-960L18.1 by analyzing data and performing chromatin conformation capture and RNA pull-down. We aim to construct a dynamic network of GATA1 regulating lncRNA during erythroid differentiation. 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，红系分化是其中重要的方向之一。转录因子GATA1是调控红系分化的重要分子，既往针对GATA1作用机制的研究主要集中在编码基因的调控功能。通过对项目组之前的转录组数据和相关ENCODE数据的整合分析，发现GATA1还可能通过非编码的lncRNA MIAT、RP11-960L18.1等参与调控红系分化。本研究将利用红系分化细胞模型，通过ChIP实验鉴定候选lncRNA上的GATA1结合变化；通过干扰表达实验，研究lncRNA的红系分化调控功能；利用转录组测序分析，构建lncRNA调控红系分化的分子网络，并为下一步分子机制研究提供数据基础；结合生信数据与功能实验，初步探讨lncRNA的分子作用机制。以期构建GATA1通过lncRNA调控红系分化的分子网络。希望通过上述工作丰富红系分化调控机制的理论体系，也为干细胞定向分化相关领域研究提供新思路。

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，红系分化是其中重要的方向之一。转录因子GATA1是调控红系分化的重要分子，既往针对GATA1作用机制的研究主要集中在其对编码基因的调控功能。然而，随着越来越多的研究表明非编码RNA 在红系分化中也发挥重要调控功能，GATA1 通过lncRNA 调控红系分化的分子机制尚不清楚。项目组通过整合分析转录组数据和ENCODE中的ChIP-seq数据，系统筛选了全基因组lncRNAs，并鉴定了系列LncRNAs，其中最为显著的包括PCED1B-AS1。通过ChIP-qPCR和干扰GATA1表达，我们实验验证了GATA1对PCED1B-AS1的调控，以及PCED1B-AS1对红系分化调控功能。我们还结合公共数据对PCED1B-AS1的作用机制进行了探讨。本项目中，我们筛选到具有潜在调控红系分化的lncRNA PCED1B-AS1，验证GATA1对lncRNA PCED1B-AS1的调控作用，以及PCED1B-AS1促进红系分化。我们还确定了GATA1并不完全通过编码基因调控红系分化，GATA1也可通过非编码RNA在红系分化中发挥重要调控功能。通过本项目的GATA1 动态调控lncRNA 研究，补充了GATA1 调控红细胞分化发育的理论体系。同时，GATA1 调控lncRNA 参与红系分化的研究，将帮助完善红细胞分化发育的理论体系，为进一步揭示异常分化所引发的血液疾病的机理提供参考。