The RUNX1-Regulated Cebpa +37 Kb Enhancer Directs Human CD4 Transgene Expression To Long-Term Hematopoietic Stem Cells and Preferentially To Myeloid Compared With Lymphoid Or Erythroid Progenitors

We found that adult mice lacking RUNX1 have increased monopoiesis relative to granulopoiesis correlated with reduce Cebpa RNA expression in myeloid progenitors and that RUNX1 binds and activates Cebpa reporters via two sites in the Cebpa promoter and four conserved sites in a 450 bp +37 kb enhancer (Guo et al, Blood, 2012). This enhancer interacts with p300 and contains the H3K4me1 enhancer mark in myeloid cells. Previously reported ChIP-Seq data had demonstrated interaction of RUNX1 with the +37 kb enhancer, along with GATA2, SCL, Gfi-1b, MEIS1, Fli-1, ERG, PU.1, Lyl1, and LMO2. We have now generated transgenic C57BL/6 mice in which the Cebpa +37 kb enhancer positioned upstream of its 845 bp promoter (-720 to +125) directs expression of cytoplasmically truncated human CD4 (hCD4). Two founders with germ line transmission have been identified – both have detectable hCD4 RNA in marrow, at approximately 10-fold increased levels in one line compared to the other, with hCD4 expression detectable by FACS only in the higher expressing line. Analysis of 4-7 mice reveals that hCD4 expression is detected only in a small subset of mature hematopoietic cells, present in 6% of Mac-1⁺Gr-1⁺ neutrophils, 31% of Mac-1⁺Gr-1^- monocytes, 4% of CD3⁺ T cells, 3% of CD19⁺ or 7% of B220⁺ B-cells, and 5% of Ter119⁺ erythroid cells. 6.5+/-1.9% of total marrow mononuclear cells and 13+/-4% of Lin^- marrow cells express hCD4. At the progenitor level, hCD4 was evident in 56+/-7% of CMP, 70+/-5% of GMP, 36+/-9% of CLP, and only 1.5+/-1.0% of MEP. There was>100-fold enrichment of myeloid CFU but a 2-fold decrease in B-lymphoid colonies comparing hCD4+ versus hCD4- fractions of total marrow. Staining LSK cells for CD34 and FcγR demonstrated that 91+/-10% of multi-potent progenitors (MPP), 61+/-8% of ST-HSC, and 17+/-8% of LT-HSC express hCD4. Similarly, staining for the SLAM markers demonstrated that 19+/-9% of LSK-CD48^-CD150⁺ LT-HSC express hCD4. Mean fluorescence index of hCD4 staining was about 2-fold higher in LSK, MPP, ST-HSC, CMP, and GMP compared with CLP and LT-HSC and about 3-fold higher than mature blood myeloid cells. 2E6 hCD4+ or hCD4- CD45.2 marrow cells were transplanted into lethally irradiated CD45.1 syngeneic recipients. 5/5 mice receiving hCD4- cells died at 2 weeks, reflecting lack of even progenitor engraftment, whereas 5/5 mice receiving hCD4+ cells were alive at 20 weeks, reflecting expression of the transgene in functional LT-HSC, with secondary transplanted mice viable at 4 week to date. The marrow cells of surviving mice were almost exclusively CD45.2, with hCD4+ expression in Lin^- or Lin^-c-kit⁺ marrow cells similar to that of donor mice. Although our second founder does not allow hCD4 detection by FACS, analysis of hCD4 RNA in marrow stem and progenitor subsets for this line is in progress, as is competitive transplantation of hCD4- versus hCD4+ marrow cells from the higher-expressing line. Murine E13.5 fetal liver hematopoietic stem and progenitor cells express hCD4 in a pattern similar to adult marrow. In zebrafish, the murine Cebpa +37 kb enhancer directs ZsGreen expression specifically to the caudal hematopoietic territory (CHT), analogous to the murine fetal liver, at 72 hour post-fertilization. Murine Cebpa +37 kb enhancer:ZsGreen transgene expression, lacking the Cebpa promoter, overlaps in the CHT with that of murine Runx1 +23 kb enhancer:mCherry in compound transgenic fish. The Cebpa and Runx1 enhancers direct similar tdTomato expression from a Sleeping Beauty vector in the K562 cell line, and these vectors are being compared for expression in hESC derived hematopoietic cells. In summary, the Cebpa gene contains an enhancer active in both LT-HSC and myeloid progenitors. Further characterization of its regulation may provide insights into the development of these hematopoietic stem and progenitor subsets and into how Cebpa expression is impaired in myelodysplastic syndromes or in acute myeloid leukemia. In addition, the Cebpa enhancer may prove useful in efforts to optimize LT-HSC formation from hESC.