GSE124822
DNA methylation disruption reshapes the hematopoietic differentiation landscape
Somatic mutations in genes implicated in DNA methylation (DNAme; e.g., TET2, DNMT3A and IDH2), are frequently observed in hematological malignancies, as well as in clonal hematopoiesis (CH). Yet, how these mutations disrupt the hematopoietic differentiation topology remains largely unknown.  By applying complementary single-cell sequencing approaches to murine bone marrow hematopoietic stem and progenitor cells, we observe that mutations in DNAme modifiers result in significantly altered differentiation topology. In particular, we find shifts in the frequencies of erythroid vs. myelo-monocytic progenitors, which can be traced back to fate-priming skews in the earliest uncommitted hematopoietic stem cells (HSCs). DNAme analysis both in open chromatin regions and at single-cell resolution, demonstrate that methylation changes resulting from DNAme modifier disruption are distributed stochastically across the genome. To reconcile the distributed nature of DNAme changes with the deterministic erythroid vs. myelo-monocytic topological skews, our data implicate differential sensitivity of key transcription factors to global methylation changes due to biases in CpG enrichment in their binding motifs. Finally, by coupling single-cell whole transcriptome analysis with targeted genotyping, we observe a similar skew in early transcriptional priming impact human CH bone marrow progenitors in an individual with DNMT3A-755S mutation. Thus, our results provide evidence that DNAme encodes topological information for hematopoietic differentiation. Overall design: We performed multiple single cell assays, including single cell RNA sequencing, single cell ATAC-seq and single cell RNA plus reduced representation bisufite for Mx1-Cre (WT), Mx1-Cre Tet2fl/fl, Mx1-Cre Dnmt3afl/fl and Mx1-Cre Idh2WT/R140Q bone marrow hematopoietic progenitors (Lineage negative or Lineage negative, c-Kit positive as indicated). To induce recombination of the conditional alleles, 16-20 week-old male Mx1-Cre, Mx1-Cre Tet2fl/fl, Mx1-Cre Dnmt3afl/fl and Mx1-Cre Idh2R140Q/+ mice were treated with three doses of polyinosinic-polycytadylic acid (pIpC; 12 mg/kg/day; GE Healthcare, Chicago, IL) every other day via intra-peritoneal injection. Primary mouse bone marrow (BM) cells were isolated into cold phosphate-buffered saline (PBS), without Ca2+ and Mg2+, and supplemented with 2% bovine serum albumin (BSA) to generate single cell suspensions. Red blood cells (RBCs) were removed using ammonium chloride-potassium bicarbonate (ACK) lysis buffer, resuspended in PBS/2% BSA, and filtered through a 40µm cell strainer. Cells were then stained with streptavidin-conjugated secondary antibody, and live (DAPI-negative) lineage-negative cells were purified by flow cytometry on a BD Aria (BD Bioscience, San Jose, CA). For single cell RNA sequencing assays, a total of 10 WT, 10 Tet2 KO, 4 Dnmt3a KO and 3 Idh2-R140Q mice were used. GRANT: DP2-CA239065 awarded to Dan Landau GRANT: R01HL145283-01 awarded to Dan Landau