Abstract
RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MK) to characterized Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1 bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1 occupied genomic regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. Megakaryocytic specificity of Runx1/P300 bound enhancers was validated by transfection mutagenesis and Runx1/P300 co-bound regions of two key megakaryocytic genes Nfe2 and Selp were tested by in vivo transgenesis. The data provides the first example of genome wide Runx1/p300 occupancy in maturating primary FL-MK, unravel the Runx1-regulated program controlling MK maturation in vivo and identify a subset of its bona fide regulated genes. It advances our understanding of the molecular events that upon RUNX1mutations in human lead to the predisposition to familial platelet disorders and FPD-AML.
Highlights
The RUNX1 transcription factor (TF) is a key gene expression regulator in embryonic and adult hematopoiesis [1]
FL-derived primary MK obtained from Runx1F/F/platelet factor 4 (Pf4)-Cre mice (FL-MKRunx12/2) provided unique means for studying Runx1 function explicitly during terminal stages of megakaryocytic maturation
The megakaryopoiesis process leading to production of platelets involves profound cell morphological changes and is transcriptionally regulated at multiple stages
Summary
The RUNX1 transcription factor (TF) is a key gene expression regulator in embryonic and adult hematopoiesis [1]. Over-expression of RUNX1 in myeloid cell lines induces megakaryocytic differentiation [6,7], while knockdown impairs megakaryopoiesis [8]. Haploinsufficiency of RUNX1, due to heterozygous loss-of-function mutations, is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML) [9,10]. While the importance of RUNX1 in megakaryopoiesis is well-established [5,12,13] information about its role in driving the regulatory program of MK maturation and platelet formation in the in vivo milieu is lacking, as is the information about RUNX1-direct target genes during the advanced stages of megakaryocytic differentiation
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