Runx1 deletion or dominant inhibition reduces Cebpa transcription via conserved promoter and distal enhancer sites to favor monopoiesis over granulopoiesis

Abstract

Deletion of Runx1 in adult mice produces a myeloproliferative phenotype. We now find that Runx1 gene deletion increases marrow monocyte while reducing granulocyte progenitors and that exogenous RUNX1 rescues granulopoiesis. Deletion of Runx1 reduces Cebpa mRNA in lineage-negative marrow cells and in granulocyte-monocyte progenitors or common myeloid progenitors. Pu.1 mRNA is also decreased, but to a lesser extent. We also transduced marrow with dominant-inhibitory RUNX1a. As with Runx1 gene deletion, RUNX1a expands lineage ؊ Sca-1 ؉ c-kit ؉ and myeloid cells, increased monocyte CFUs relative to granulocyte CFUs, and reduced Cebpa mRNA. Runx1 binds a conserved site in the Cebpa promoter and binds 4 sites in a conserved 450-bp region located at ؉37 kb; mutation of the enhancer sites reduces activity 6-fold in 32Dcl3 myeloid cells. Endogenous Runx1 binds the promoter and putative ؉37 kb enhancer as assessed by ChIP, and RUNX1-ER rapidly induces Cebpa mRNA in these cells, even in cycloheximide, consistent with direct gene regulation. The ؉37 kb region contains strong H3K4me1 histone modification and p300-binding, as often seen with enhancers. Finally, exogenous C/EBP␣ increases granulocyte relative to monocyte progenitors in Runx1-deleted marrow cells. Diminished CEBPA transcription and consequent impairment of myeloid differentiation may contribute to leukemic transformation in acute myeloid leukemia cases associated with decreased RUNX1 activity. (Blood. 2012; 119(19):4408-4418) IntroductionThe Runx1 transcription factor contributes to formation of pluripotent adult HSCs from hemogenic endothelium during embryogenesis. [1][2][3] Deletion of floxed Runx1 alleles in adult mice preserves pluripotent HSCs and erythroid cells but leads to thrombocytopenia and a marked reduction in B-and T-lymphoid cells and their precursors. [4][5][6] In addition, these mice develop an expansion of myeloid progenitors, with increased numbers of lineage Ϫ Sca-1 ϩ c-kit ϩ (LSK) cells, common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), myeloid CFUs, and mature myeloid cells. 4,5 This myeloproliferative phenotype is transplantable and so intrinsic to the hematopoietic system. 5 The myeloid expansion associated with absence of Runx1 may be relevant to myeloid leukemogenesis. Reduced RUNX1 activity occurs commonly in acute myeloid leukemia (AML), often because of expression of the RUNX1-ETO or core binding factor ␤ (CBF␤)-SMMHC oncoproteins or as a result of RUNX1 genomic mutation. [7][8][9] It has been suggested that these and other "type II" alterations, including CEBPA mutation, contribute to myeloid transformation by inhibiting myeloid differentiation, with coincident "type I" alterations, for example, activation of N-Ras or receptor tyrosine kinases, inhibiting apoptosis while stimulating proliferation. 10 In this study, we further assess the effect of Runx1 gene deletion on myeloid differentiation in adult mice. Comparison of Runx1-deleted myeloid CFUs with those of control mice uncovered a marked increase in m...

Logical modeling of lymphoid and myeloid cell specification and transdifferentiation

“…The B-cell lineage is further controlled by Mef2c, the interleukine 7 receptor (IL7r), Ets1, Foxo1, Ebf1, and Pax5 (15,16,17). The specification of the myeloid GMPs depends on C/EBPα (6), which is regulated by Runx1 (runt-related transcription factor 1) (18). Macrophage specification further relies on the macrophage colony-stimulating factor (M-CSF) receptor (CSF1r), on the up-regulation of PU.1, and on Cebpb and the Id proteins (including Id2) (19,20).…”

Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis

“…Cycloheximide was provided at 50 g/ml. 32Dcl3 cells (29) cultured in Iscove's modified Dulbecco's medium (IMDM) with 1 ng/ml murine interleukin 3 (IL-3, Cell Signaling) and penicillin/ streptomycin were subjected to retroviral transduction as described (4). Subclones were obtained by limiting dilution.…”

“…Plasmids-(Runx1) 4 TKLUC, CMV-␤Gal, pCEFL-Src(E381G), pBabePuro-Runx1-ER(T), CMV-CBF␤, CMV-FLAG-HDAC1, CMV-FLAG-HDAC3, CMV-HA-Cdc20, CMV-HA-Cdh1, pMIG, and pMIGC were previously described (4,16,23,26,27,30,31). CMV-HA-ubiquitin was obtained commercially (Addgene).…”

“…Runx1/AML1 directs the emergence of long term adult hematopoietic stem cells from hemogenic endothelium during development and subsequently contributes to lymphoid, myeloid, and megakaryocyte lineage development (1)(2)(3)(4). Runx1 contributes to myeloid lineage specification in part via induction of Pu.1 via its Ϫ14-kb enhancer and of Cebpa via its ϩ37-kb enhancer (4 -7).…”

“…Runx1 contributes to myeloid lineage specification in part via induction of Pu.1 via its Ϫ14-kb enhancer and of Cebpa via its ϩ37-kb enhancer (4 -7). Runx1 gene deletion in adult mice reduces both Pu.1 and Cebpa RNA levels in marrow myeloid progenitors, with consequent reduced granulopoiesis and increased monopoiesis (4). In addition to directing differentiation, Runx1 favors G 1 to S cell cycle progression, in part via induction of cdk4 and cyclin D3, and inhibits apoptosis, in part due to suppression of p53 induction and stimulation of Bcl-2 expression (8 -11).…”