Dnmt3a-Mutant Hematopoietic Stem cells are resistant to inflammatory stress

Dnmt3a-mutant mouse HSCs, but not control HSCs are able to maintain clonality during type II inteferson signaling. To investigate the molecular mechanism of this observation, we investigated IFNg-incuded changes in DNA methylome of mouse HSCs in a mouse model. In the model, we transplanted 10% CD45.2 donor BM cells, 10% CD45.1/2-GFP competitors and 80% CD45.1 BM cells into CD45.1 recipients. In the treatment group, we transplated the CD45.1 BM cells with the genotype of IFNgrKO; rtTA-M2; Tet-O-IFNg to induce a chronic IFNg exposure. A physiological relevant level of IFNg was induced by 1250ppm doxycycline chow every other week during 5-24 weeks post-transplant. In the control group, we transplanted CD45.1 cells with the genotype of IFNgrKO; rtTA-M2. All the mice in the control group were also on the same schedule of doxycycline chow. Recipients were sacrificed 26-weeks post-transplant and 3x10^5 donor derived BM cells from each group were sorted for gDNA extraction. Consistent with literature, our results showed a gradient loss of DNA methylation from Dnmt3a-HET to Dnmt3a-KO compared to Vav-cre controls. Chronic IFNg exposure, however, did not change the global DNA methylation levels. All the IFNg-induced changes were locus-specific. We conclud that IFNg-induced changes in DNA methylome cannot explain the protection of Dnmt3a-mutant HSCs against type II inteferon signaling. Overall design: Whole genome-wide bisulphite sequencing. Mice of different genotypes (Vav-cre: Control; Vav-cre;Dnmt3af/f: 3aKO) were retro-orbitally injected with two doses of 10ug of recombinant mouse IFNg with 24h apart. Mice were sacrificed for bone marrow cell harvest 2h post the second injection. Gender-, age-, genotype- matched mice recieved volume-equivalent PBS as control groups. Bone marrow cells were stained with lineage markers, as well as antibodies against cKIT and EPCR. Lin-cKIT+EPCR+ cells were sorted and used for scRNA-seq library construction. Data were processed with CellCounter and analyzed with Seurat 3.0. Hematopoietic stem cells (HSCs) were sorted from Vav-CRE:Control, Vav-CRE:Dnmt3a-KO, Vav-CRE:Tet2-KO mice (treated with IFNg or PBS) for RNA-seq to determine gene expression