DNMT3A and TET2 Compete and Cooperate to Repress Differentiation Lineage-Specific Factors in Hematopoietic Stem Cells

Mutations in the epigenetic modifiers DNMT3A and TET2 non-randomly co-occur in lymphoma and leukemia despite their epistasis in the methylation-hydroxymethylation pathway. Using double knock-out (DKO) mice in which malignancy development is accelerated, we show that the DKO methylome reflects regions of independent, competitive and cooperative activity. Expression of lineage-specific transcription factors, including the erythroid regulator Klf1 is upregulated in DKO HSCs. DNMT3A and TET2 both repress Klf1 suggesting a model of cooperative inhibition by the epigenetic modifiers. These data demonstrate a dual role for TET2 in promoting and inhibiting HSC differentiation, loss of which, along with DNMT3A, obstructs differentiation leading to transformation. Overall design: I)Whole genome bisulfite sequencing of Tet2 conditional knockout hematopoietic stem cells, Tet2 and DNMT3a double knockout hematopoietic stem cells using Illumina HiSeq 2000.II)Whole genome CMS-enriched bisulfite sequencing of Tet2 conditional knockout hematopoietic stem cells, Tet2 and DNMT3a double knockout hematopoietic stem cells using Illumina HiSeq 2000.III)Whole genome mRNA profiles of Tet2 conditional knockout hematopoietic stem cells, Tet2 and DNMT3a double knockout hematopoietic stem cells using Illumina HiSeq 2000. IV)Genome wide histone modification H3K27me3 profiling of Tet2 conditional knockout hematopoietic stem cells, Tet2 and DNMT3a double knockout hematopoietic stem cells using Illumina HiSeq 2000

表观遗传修饰因子DNMT3A与TET2的突变在淋巴瘤和白血病中呈非随机共现，尽管二者在甲基化-羟甲基化通路中存在上位性（epistasis）。本研究利用恶性肿瘤发生加速的双基因敲除（double knock-out, DKO）小鼠模型，证实DKO的甲基化组可反映出独立、拮抗与协同发挥作用的基因组区域。在DKO造血干细胞（hematopoietic stem cells, HSCs）中，包括红细胞系调控因子Klf1在内的谱系特异性转录因子表达出现上调。DNMT3A与TET2均可抑制Klf1的表达，这提示了表观遗传修饰因子协同抑制的作用模型。上述数据表明，TET2在造血干细胞分化过程中兼具促进与抑制的双重功能；其与DNMT3A的缺失会阻碍细胞分化，进而引发细胞转化。

总体实验设计：
I) 采用Illumina HiSeq 2000测序平台，对Tet2条件性敲除造血干细胞以及Tet2与DNMT3a双基因敲除造血干细胞开展全基因组亚硫酸氢盐测序；
II) 采用Illumina HiSeq 2000测序平台，对上述两种细胞开展全基因组CMS富集亚硫酸氢盐测序；
III) 采用Illumina HiSeq 2000测序平台，对上述两种细胞开展全基因组mRNA表达谱分析；
IV) 采用Illumina HiSeq 2000测序平台，对上述两种细胞开展全基因组组蛋白修饰H3K27me3谱分析。