Integrated analysis of transcriptional and chromatin alteration in Jak2V617F and Asxl1-/-Jak2V617F BM c-kit+ cells

We performed bulk RNA-seq, transposase-accessible chromatin using sequencing (ATAC-Seq) and chromatin immunoprecipitation (ChIP)-seq on Asxl1-/-Jak2VF and Jak2VF BM c-kit+ cells to elucidate the transcriptional and associated epigenetic alterations after Asxl1 deletion. GSEA showed that the upregulated genes in Asxl1-/-Jak2VF were significantly associated with bona fide PcG target genes, as identified by the overlap between H3K27me3 and H2AK119ub1 ChIP-seq­ experiments . Integrated analysis of RNA-seq and ATAC-seq data showed that there existed a significant increase of chromatin accessibility associated with upregulated genes, and these sites with gained accessibility were enriched with increased levels of H3K4me1 and H3K27ac, histone marks of active enhancers in Asxl1-/-Jak2VF BM c-kit+ cells. Moreover, PcG target gene Egr1 was upregulated in BM c-kit+ cells compared with other three genotypes and considered as key gene for disease phenotypes. Examination of chromatin accessibility, histone modifications and mRNA expression in BM c-kit+ cells.我们对Asxl1基因敲除（Asxl1-/-）且携带Jak2VF突变的骨髓（bone marrow, BM）c-kit阳性细胞，以及仅携带Jak2VF突变的骨髓c-kit阳性细胞，开展了批量RNA测序（bulk RNA-seq）、转座酶可及性染色质测序（ATAC-Seq）与染色质免疫共沉淀测序（ChIP-seq），旨在阐明Asxl1缺失后发生的转录组及相关表观遗传改变。基因集富集分析（Gene Set Enrichment Analysis, GSEA）结果显示，Asxl1-/-Jak2VF细胞中的上调基因与真正的多梳蛋白家族（Polycomb Group, PcG）靶基因显著相关；这类靶基因通过H3K27me3与H2AK119ub1的ChIP-seq实验重叠结果得以鉴定。对RNA-seq与ATAC-seq数据的整合分析表明，与上调基因相关的染色质可及性显著升高；这些染色质可及性升高的位点，在Asxl1-/-Jak2VF骨髓c-kit+细胞中富集了活性增强子的组蛋白修饰标记——H3K4me1与H3K27ac水平的显著上调。此外，相较于其余三种基因型的细胞，多梳蛋白家族靶基因Egr1在骨髓c-kit+细胞中表达上调，且被认定为疾病表型的关键基因。本研究针对骨髓c-kit+细胞的染色质可及性、组蛋白修饰及mRNA表达水平展开了检测。