HISTONE DEACETYLASES 1 AND 2 TARGET GENE REGULATORY NETWORKS OF NEPHRON PROGENITORS TO CONTROL NEPHROGENESIS [RNA-seq]

Our studies demonstrated the critical role of Histone deacetylases (HDACs) in the regulation of nephrogenesis. To better understand the key pathways regulated by HDAC1/2 in early nephrogenesis, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) of Hdac1/2 on isolated nephron progenitor cells (NPCs) from mouse E16.5 kidneys. Our analysis revealed that 11802 (40.4%) of Hdac1 peaks overlap with Hdac2 peaks, further demonstrates the redundant role of Hdac1 and Hdac2 during nephrogenesis. Common Hdac1/2 peaks are densely concentrated close to the transcriptional start site (TSS). GREAT Gene Ontology analysis of overlapping Hdac1/2 peaks reveals that Hdac1/2 are associated with metanephric nephron morphogenesis, chromatin assembly or disassembly, as well as other DNA checkpoints. Pathway analysis shows that negative regulation of Wnt signaling pathway is one of Hdac1/2’s most significant function in NPCs. Known motif analysis indicated that Hdac1 is enriched in motifs for Six2, Hox family, and Tcf family members, which are essential for self-renewal and differentiation of nephron progenitors. Interestingly, we found the enrichment of HDAC1/2 at the enhancer and promoter regions of actively transcribed genes, especially those concerned with NPC self-renewal. Hdac1/2 simultaneously activate or repress the expression of different genes to maintain the cellular state of nephron progenitors. We used the Integrative Genomics Viewer to visualize these target genes associated with each function and found that Hdac1/2 co-bound to the enhancers or/and promoters of genes associated with nephron morphogenesis, differentiation, and cell cycle control. Taken together, our ChIP-Seq analysis demonstrates that Hdac1/2 directly regulate the molecular cascades essential for nephrogenesis. Transcriptomic profiling of Hdac1 and Hdac2 conditional mutant cap mesenchyme cells at E16.5.本研究证实了组蛋白去乙酰化酶（Histone deacetylases, HDACs）在肾发生调控中的关键作用。为深入解析HDAC1/2在早期肾发生过程中调控的核心通路，我们对小鼠胚胎16.5天（E16.5）肾脏分离得到的肾单位祖细胞（nephron progenitor cells, NPCs）开展了HDAC1/2的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-Seq）。分析结果显示，11802个HDAC1结合峰（占比40.4%）与HDAC2结合峰存在重叠，进一步证明了HDAC1与HDAC2在肾发生过程中的功能冗余性。HDAC1/2的共同结合峰密集富集于转录起始位点（transcriptional start site, TSS）附近。对重叠的HDAC1/2结合峰进行GREAT基因本体论（Gene Ontology）分析发现，HDAC1/2与后肾肾单位形态发生、染色质组装与去组装以及各类DNA检验点相关。通路富集分析显示，Wnt信号通路的负调控是HDAC1/2在NPCs中最为关键的功能之一。已知基序富集分析显示，HDAC1富集于Six2、Hox家族及Tcf家族成员的结合基序，而这些因子对肾单位祖细胞的自我更新与分化至关重要。值得注意的是，我们发现HDAC1/2富集于活跃转录基因的增强子与启动子区域，尤其是那些与NPC自我更新相关的基因。HDAC1/2可同时激活或抑制不同基因的表达，以维持肾单位祖细胞的细胞稳态。我们借助整合基因组查看器（Integrative Genomics Viewer）对各功能相关的靶基因进行了可视化分析，发现HDAC1/2共同结合于肾单位形态发生、分化及细胞周期调控相关基因的增强子或启动子区域。综上，本研究的ChIP-Seq分析证实，HDAC1/2可直接调控肾发生所必需的分子级联反应。此外，我们还完成了对E16.5天HDAC1与HDAC2条件性突变帽状间充质细胞的转录组谱分析。