The cebpa +37-kb enhancer directs transgene expression to myeloid progenitors and to long-term hematopoietic stem cells

C/EBPα is expressed preferentially in myeloid compared with lymphoid or erythroid cells and directs myeloid lineage specification. C/EBPα is also expressed at lower levels in HSCs and in several nonhematopoietic tissues. The Cebpa gene has a conserved, 450-bp segment at +37 kb that harbors enhancer-specific epigenetic marks and is activate in a myeloid cell line. Herein, we characterize transgenic C57BL/6 mice, in which the Cebpa enhancer and 845-bp promoter regulate a hCD4 reporter. FACS analysis, in vitro colony assays, and in vivo competitive and secondary transplantation revealed that myeloid but not MEPs or lymphoid progenitors and also functional LT-HSCs are found almost exclusively in the Cebpa-hCD4⁺ compared with hCD4^- marrow population. hCD4⁺ CMP yielded predominantly myeloid, whereas hCD4 CMP^- generated mainly Meg/E colonies. Providing insight into control of CMP maturation, Cebpa and Pu.1 RNAs were preferentially expressed in hCD4⁺ CMP, Scl, Gata2, Gata1, Klf1, Ets1, and Fli1 predominated in hCD4^- CMP, and Runx1, Myb, HoxA9, and Erg levels were similar in both. Cebpa-hCD4 transgene expression was lacking in multiple nonhematopoietic tissues. In summary, the +37-kb Cebpa enhancer and promoter are sufficient for marrow myeloid progenitor and LT-HSC-specific expression.