Comparision of Differential methylation between Control and mutant Dnmt3a

Dnmt3a is the most recurrently mutated gene in clonal hematopoiesis (CH), and it is a critical regulator of hematopoietic stem cells (HSCs). Conditional deletion of Dnmt3a in mouse HSC results in enhanced self-renewal but impaired differentiation. Dnmt3a encodes for a de novo DNA methyltransferase enzyme but both mouse and human cells with loss of Dnmt3a show minimal change in DNA methylation levels which do not correlate with gene expression differences. To understand if there are methylation differences between control and mutant (R878H) Dnmt3a, we are performing WGBS. Overall design: To investigate the differential DNA methylation in hematopoietic cells expressing Dnmt3a mutant with reduced DNA methyltransferase activity, we established Cre-mediated knock in mouse model. Competitive HSC transplantation was performed. Cells were isolated 18-weeks post-transplant for analysis of DNA methylation patterns by whole genome bisulfite sequencing.

Dnmt3a是克隆性造血 (clonal hematopoiesis, CH) 中最频发发生突变的基因，同时是造血干细胞 (hematopoietic stem cells, HSCs) 的关键调控因子。在小鼠造血干细胞中条件性敲除Dnmt3a，会导致其自我更新能力增强但分化功能受损。Dnmt3a编码一种从头DNA甲基转移酶，但无论是小鼠还是人类细胞，在Dnmt3a缺失后其DNA甲基化水平仅出现极细微变化，且该变化与基因表达差异无相关性。为探究对照组与Dnmt3a突变体（R878H）之间是否存在甲基化差异，我们正在开展全基因组亚硫酸氢盐测序 (whole genome bisulfite sequencing, WGBS) 实验。整体实验设计：为研究DNA甲基转移酶活性降低的Dnmt3a突变体在造血细胞中引发的差异DNA甲基化情况，我们构建了Cre介导的敲入小鼠模型，并开展了竞争性造血干细胞移植实验。于移植后18周分离细胞，通过全基因组亚硫酸氢盐测序分析其DNA甲基化模式。