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Dnmt3a-Mutant Hematopoietic Stem cells are resistant to inflammatory stress

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE168807 | GEO | 2022/02/22

REPOSITORIES: GEO

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Project description:Since DNMT3A is a de novo DNA methyltransferase, transcriptional differences in WT versus Dnmt3a-/- HSCs upon infection suggest a possible role for epigenetic regulation in the HSC transcriptional response to inflammation. Therefore, we wanted to investigate if differences in stress and presence of Dnmt3a affect the methylome overall, and specific IFNg responses genes. 100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/ Dnmt3-/-_x0001_ mice (n = 7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after ‘‘methylated’’ adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylationlightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/ sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads). Global methylation of Dnmt3a-/- cells was more divergent upon infection compared to WT. There were vastly more hyper- and hypomethylated regions in infected versus naive Dnmt3a-/- HSCs compared to WT. Among the altered regions, both WT and Dnmt3a-/- HSCs showed slightly more hypomethylated than hypermethylated regions, although changes in both directions were present. In the WT background, regions of hyper- and hypomethylation were mostly restricted to CpG islands. By contrast, highly significant differences in hypomethylation were found in the Dnmt3a-/- HSCs in CpG islands, shores, enhancers, and promoters DNMT3A is highly active in HSCs during infection and that the loss of DNMT3A function results in significant global changes in methylation, including both hyper- and hypomethylation, in genome regions that likely affect gene transcription. we examined the methylation of Batf2, Jun, and Fos. All 3 of these prodifferentiation genes showed increased methylation in the promoter region in the Dnmt3a-/- background as compared to the WT background. Hypermethylation of these promoter regions is consistent with their transcriptional repression.

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Dnmt3a-Mutant Hematopoietic Stem cells are resistant to inflammatory stress

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