基于组学数据的整合分析探究胚胎干细胞分化中KZNF家族的分工调控模式及其关键成员分子

KRAB-domain-containing zinc finger protein (KZNF) family originated lately during evolution, then expaned and divergenced quickly, and there are strong specificity across species. KZNF family is the largest family of transcription factors in human (There are more than 400 KZNF genes in human genome). It is known that KZNFs play important roles in the repression of retroelements in embryonic stem cells (ESCs). However, the regular regulation patterns of KZNFs in the definitive differentiation of ESCs are largely unkonwn. In this study, with the improved integrated method for the analysis of KZNF regulation network, we will comprehensively analyze the important evolutionary events of KZNFs and the clade-specific KZNFs will be further classified. The omic data of gene expression and regulation during mouse and human ESC differentiation will be collected, and the expression and regulation pattern of KZNFs will be investigated. The target genes of KZNFs and the related signal pathways will be mined and we can infer the common and lineage-specific regulation networks and modules, from which we can understand the divergence of the regulation pattern of ESC differentiation between mouse and human. What's more, the key KZNF genes contributing to the regulation of ESC differentiation will be selected for the deep experimental validation. Their regulating function and regulation mechanisms will be confirmed by various experiments. Meanwhile, the database and search platform specified for KZNF family will be developed. This study will substantially enrich the information of the regulating functions and evolutionary mechanisms of KZNFs and is of great significance for the understanding of the mechanisms of ESC differentiation, which are of great theoretical significance and potential application value.

KRAB型锌指蛋白（KZNF）家族起源晚、进化快、物种特异性强，是人类最大的转录调控因子家族（含400多KZNF基因）。KZNF在胚胎干细胞（ESC）中抑制反转录转座子，对于ESC的稳定至关重要；前期研究提示KZNF在ESC向各谱系分化中呈现分工调控趋势，但具体分工模式和关键分子尚未明晰。本研究将针对KZNF家族特征，基于多组学数据，建立KZNF基因表达调控网络分析方法，挖掘KZNF基因表达和受调控模式、KZNF的靶基因及其参与的信号通路；构建KZNF在ESC分化中“共同”和“谱系特异”的表达调控网络，探索人和小鼠ESC分化的分子水平差异；挖掘起重要调控作用的KZNF分子，并针对部分KZNF进行功能和机制验证；同时，构建KZNF家族进化与表达调控信息数据库和检索平台，为研究者提供重要信息。本项目利于全面认识KZNF这一大家族的功能和进化特征及ESC分化机制，具有重要理论意义和潜在应用价值。

KRAB型锌指蛋白家族（KZNF）是哺乳动物中最大的转录因子家族，具有起源晚、进化快、物种特异性强的特征。一些KZNF在胚胎干细胞（ESC）中抑制反转录转座子，并且一些KZNF对于ESC的干性和分化至关重要。然而，由于仍有许多KZNF的功能是未知的，使得在ESC及其分化过程中KZNF的具体分工模式和关键分子尚未完全明晰。本项目基于多组学数据，重点针对KZNF家族的特征、其在ESC中的调控模式和关键分子进行了系统性分析和挖掘。首先通过分析KZNF家族的进化、结构和表达特征，发现了保守且特殊的“矛盾”，即：年轻蛋白倾向于特异性表达，KRAB结构域和KZNF进化中出现较晚，但却是倾向于完全结构化和高度结构化；年轻基因和编码结构化蛋白的基因常为特异性且低丰度表达，KZNF基因大部分是较年轻的基因，且KZNF倾向于高度结构化，但却倾向于普遍且中丰度表达。其次，通过整合ChIP-seq/ChIP-exo数据、ATAC-seq数据和表达信息，获取了ESC向内、中胚层分化过程中265个KZNF的潜在靶基因，构建了KZNF-靶基因调控网络，并搭建了KZNF家族基因进化和表达调控信息检索和分析平台（KEEP）。基于表达信息和构建的KZNF-靶基因调控网络，筛选得到了在ESC向中胚层过程中重要的分子ZNF611，并通过实验验证了在ESC中ZNF611能够结合并正调控STK38的表达，进一步的调控机制及细胞表型相关实验仍在进行中。综上结果，本项目为全面认识KZNF家族的进化、结构、表达和功能特征及其快速进化原因提供了基础，并为深入探究ESC分化中KZNF的功能提供了重要的线索。项目资助发表论著10篇（包括5篇SCI论文、5篇中文核心期刊论文）；获软件著作权3项；培养博士生3名（两名已毕业，一名在读），硕士生3名（均已毕业）。