H2AZ Orchestrates Neural Progenitor Cell Proliferation and Differentiation via Setd2-mediated H3K36me3 Modification [RNA-seq]

Purpose:To gain a deeper insight into how H2AZ regulates embryonic neurogenesis, RNA-sequencing (RNA-seq) was performed to analyze the genome-wide changes by H2AZ deletion at E12. Methods: Total RNA was extracted from E12 telencephalic tissue of H2AZcKO and H2AZfl/fl mice. Then total RNA was quality controlled and quantified using an Agilent 2100 Bioanalyzer. After converting to cDNA and building library, high-throughput sequencing was performed using the Illumina HiSeq 2500 platform in Annoroad Genomics. Results: Approximately approximately one thousand transcripts showed differential expression between the H2AZfl/fl and H2AZcKO brain, with a fold change >= 1.5 and p value <0.05. Gene ontology (GO) analysis showed that the down-regulated genes were enriched in the terms related to transcription and cortex development, such as regulation of transcription, cerebellar cortex formation, forebrain development, and cerebellum development. Up-regulated genes showed a significant enrichment of terms involved in negative regulation of cell differentiation, neuron migration, and cognition. These results reflected the importance of H2AZ in cortical development. Conclusions: We conclude that RNA-seq based transcriptome characterization would provide a framework for understanding how a disruption in the H2AZ gene may contribute to cortical development. Total RNA was extracted from E12 telencephalic tissue of H2AZcKO and H2AZfl/fl mice and generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.研究目的：为深入探究H2AZ如何调控胚胎神经发生，本研究通过RNA测序（RNA-sequencing，RNA-seq）分析了E12时期H2AZ基因敲除后全基因组的表达变化。研究方法：从H2AZcKO及H2AZfl/fl小鼠的E12端脑组织中提取总RNA，随后采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）对总RNA进行质量控制与定量。将总RNA反转录为cDNA并构建测序文库后，于诺禾致源基因（Annoroad Genomics）使用Illumina HiSeq 2500平台完成高通量测序。研究结果：在H2AZfl/fl与H2AZcKO小鼠脑组织中，约有1000个转录本的表达差异倍数≥1.5且p值<0.05。基因本体（Gene Ontology，GO）富集分析显示，下调基因显著富集于转录调控与皮层发育相关的功能条目，包括转录调控、小脑皮层形成、前脑发育及小脑发育。上调基因则显著富集于细胞分化负调控、神经元迁移及认知功能相关条目。上述结果印证了H2AZ在皮层发育过程中的重要作用。研究结论：本研究通过基于RNA测序的转录组表征，为解析H2AZ基因缺失如何影响皮层发育提供了研究框架。本研究所用的总RNA提取自H2AZcKO与H2AZfl/fl小鼠的E12端脑组织，并采用Illumina HiSeq 2500平台完成了三次生物学重复的深度测序。