The role of DNA methylation modifiers in shaping the hematopoietic differentiation topology

造血分化是一个随机过程，它以确定性的方式提供多种不同的细胞类型。 固定频率这些对立面通过分化的拓扑表示得到调和： 如果每个干细胞的命运决定是随机的，那么景观的形状将决定干细胞的命运。 分化群体的频率。然而，微分拓扑是如何编码的？ DNA甲基化（DNAme）可能是形成分化拓扑结构的关键因素。DNA是 最稳定遗传的表观遗传标记，因此是编码拓扑信息的强有力候选者。 此外，人类的克隆造血，一种构成显著拓扑破坏的状态，通常 涉及DNAme修饰物中的体细胞突变，包括Dnmt 3a、Tet 2和Idh 2。 为了研究DNA甲基化如何重塑分化拓扑结构，我们进行了单细胞RNAseq， > 50，000个来自Tet 2、Idh 2、Dnmt 3a突变和野生型小鼠的骨髓祖细胞。Tet 2敲除显示 红系定向祖细胞减少，单核细胞定向祖细胞增加。值得注意的是， 红细胞对单核细胞的命运决定偏差是由早期未定型HSC的引发破坏引起的。 此外，Dnmt 3a缺失导致甲基化的相反作用（低甲基化），也导致 相反的拓扑倾斜。这就提出了一个问题，即什么是随机之间的机械联系， 全基因组DNAme变化和确定性拓扑结构偏斜（例如单核细胞与红细胞）。 我们假设，全基因组DNA的获得或丢失可能会影响命运的决定，通过固有的偏见， TF基序CpG富集。事实上，我们对谱系定义转录因子（TF）结合基序的分析 发现红细胞基序显示出CpG含量的显著富集，与单核细胞基序相比。 为了进一步探讨这一假设，我们将使用以下方法来研究TF基序的DNAme对HSC启动的影响： 两种新的互补方法。首先，ATAC-seq与亚硫酸氢盐测序偶联， 研究开放染色质的位点，这些位点对引发至关重要，同时研究它们的甲基化状态。第二，直接链接 DNA me和HSC的转录状态，我们将应用联合单细胞亚硫酸氢盐测序和RNA测序， 在单细胞水平上评价TF结合基序DNAme和引发之间的相互作用。 为了进一步确定DNAme基序对TF结合的影响，我们将评估红细胞和单核细胞TF 使用ChIP-bisulfite-seq.从功能上检查扩散结合的影响 基序DNAme改变，我们将应用具有靶向结合基序本身的引导RNA的表观遗传编辑。 最后，为了研究人类HSC分化中的这一现象，我们将应用我们新的单细胞多克隆抗体， 组学平台共同捕获单细胞甲基化组、转录体和基因型。因此，我们将比较 在具有克隆造血的同一个体内，突变型与野生型HSC的分化拓扑， 并确定DNAme在重塑人类HSC分化中的作用。

Hematopoietic differentiation is a stochastic process that delivers multiple distinct cell types at deterministically fixed frequencies. These opposites are reconciled through a topological representation of differentiation: even if the fate decision of each individual stem cell is stochastic, the shape of the landscape will determine the frequencies of the differentiated populations. However, how is the differentiation topology encoded? DNA methylation (DNAme) may be a key contributor to shaping the differentiation topology. DNAme is the most stably inherited epigenetic mark and therefore a strong candidate for encoding topological information. Moreover, clonal hematopoiesis in humans, a state that constitutes a significant topological disruption, often involves somatic mutations in modifiers of DNAme, including Dnmt3a, Tet2 and Idh2. To study how DNA methylation reshapes the differentiation topology we preformed single-cell RNAseq of >50,000 bone marrow progenitors from Tet2, Idh2, Dnmt3a mutated and wildtype mice. Tet2 knockout showed a decrease in erythroid-committed progenitors, and increase in monocyte-committed progenitors. Notably, this erythroid vs. monocyte fate-decision skew is caused by disruption in priming of early, uncommitted HSCs. Moreover, Dnmt3a deletion, which causes the opposite effect on methylation (hypomethylation), also results in opposite topological skews. This raises the question of what is the mechanistic link between stochastic genome-wide DNAme changes and deterministic topology skews (e.g. monocyte vs. erythroid). We hypothesize that genome-wide DNAme gain or loss may affect fate-decision through inherent biases in the TF motif CpG enrichment. Indeed, our analysis across lineage-defining transcription factor (TF) binding motifs uncovered that erythroid motifs show a marked enrichment in CpG content, compared with monocytic motifs. To further explore this hypothesis, we will examine the impact of DNAme of TF motifs on HSC priming, using two novel complementary approaches. First, ATAC-seq coupled with bisulfite sequencing will simultaneously study the sites of open chromatin critical to priming together with their methylation state. Second, to directly link DNAme and the transcriptional state of HSCs, we will apply joint single-cell bisulfite sequencing and RNAseq, to evaluate, at the single cell level, the interplay between TF binding motif DNAme and priming. To further define the impact of motif DNAme on TF binding, we will evaluate erythroid and monocytic TF binding in relation to DNAme using ChIP-bisulfite-seq. To functionally examine the impact of diffuse binding motif DNAme changes, we will apply epigenetic editing with guide RNAs targeting the binding motif itself. Finally, to examine this phenomenon in human HSC differentiation, we will apply our novel single-cell muti- omics platforms to jointly capture single-cell methylome, transcriptone and genotype. Thus, we will compare within the same individual with clonal hematopoiesis, the differentiation topology of mutant vs. wildtype HSCs, and define the role of DNAme in reshaping HSC differentiation in humans.