Abstract

Distinct, lineage-biased subsets of multipotent progenitor cells (MPP) dynamically respond to the demands of the hematopoietic system to replenish mature hematopoietic cells as needed. It currently remains unclear as to whether distinct epigenetic mechanisms regulate lineage-specific expansion and differentiation from MPPs. Focusing on lymphoid-primed multipotent progenitor cells (LMPP/MPP4), we performed a lentiviral shRNA screen of 15 epigenetic factors, selected based on differential expression between myeloid-restricted and lymphoid-restricted progenitors. Following a 48 hour infection with lentiviral shRNA constructs or a non-targeting control, the lineage potential of lymphoid-primed multipotent progenitors was interrogated by myeloid and lymphoid colony forming unit (CFU) assays. From this screen, knockdown of the lysine methyltransferase Kmt5a most dramatically altered lineage output from lymphoid-primed multipotent progenitors through an expansion of myeloid lineage colonies without altering lymphoid colony production. To confirm target specificity, two independent shRNA hairpins targeting distinct locations of the Kmt5a transcript demonstrated that knockdown of Kmt5a (97.1% and 99.5% versus non-targeting control shRNA) increased macrophage colony production by 1.94 and 1.95 fold, respectively (P < 0.01 and P < 0.05, n = 3). Preliminary single cell culture experiments support that the enhanced myeloid lineage output from lymphoid-primed multipotent progenitors occurs at the single-cell level through increased cloning efficiency of myeloid-biased cells. Our results suggest that Kmt5a functions to restrict myeloid lineage output from lymphoid-primed multipotent progenitors. Mechanistically, KMT5A is responsible for monomethylation of histone H4K20 and the methylation of non-histone proteins (ex. p53K376). Our ongoing work aims to distinguish between these histone and non-histone targets to determine the precise mechanisms restricting myeloid lineage output from lymphoid-primed multipotent progenitors. This work has direct implications for a better understanding of the molecular drivers of transient myeloid lineage reprogramming of lymphoid-primed multipotent progenitors during hematopoietic regeneration, age associated myeloid lineage skewing of hematopoiesis, and myeloid malignancies.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.