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Aberrant DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [CUT&RUN]

ABSTRACT: Background: Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by a heterozygous germline mutation inRUNX1. The patients with FPDMM show not only thrombocytopenia with platelet dysfunction, but also a high level of developing hematological malignancies, strongly suggesting that FPDMM is in a precancerous state. However, the DNA methylation status of FPDMM has not yet been elucidated due to no animal models for FPDMM and the difficulty in obtaining the patient-derived samples. Results: We found that differentiation efficiencies into HPCs and megakaryocytes was reduced in the FPDMM-mimicking cells, which were established by genome editing for human iPS cells as a FPDMM-model, compared with those of wild-type cells. The FPDMM-mimicking HPCs were subjected to DNA methylation analysis, and the HPCs showed the distinct DNA methylation patterns compared to wild-type HPCs. Furthermore, we identified FLI1 as a putative causative transcription factor of the differential DNA hypermethylation involved in both promoting the binding site-directed DNA demethylation and regulating megakaryopoiesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE270829 | GEO | 2024/07/01

REPOSITORIES: GEO

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Project description:The transcription factor cMyb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that cMyb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which cmyb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. mRNA and miRNA expression for each sample were profiled by Affymetrix GeneAtlas U219 strip array and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. RNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control (NegCTR) synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments.

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Aberrant DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [CUT&RUN]

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