In Vivo Deletion of the Cebpa +37 Kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis

The C/EBPα transcription factor is a key mediator of normal myeloid differentiation, and its reduced expression or activity is central to myeloid transformation, as recently reviewed (Friedman, Int. J. Hematol. 2015). The murine Cebpa gene contains a conserved, 450 bp, +37 kb regulatory element that displays the enhancer specific H3K4me1 and the activating H3K27Ac histone modifications at increasing levels as LT-HSC progress to ST-HSC, CMP, and GMP, increases promoter activity 6-fold when introduced into 32Dcl3 myeloid cells linked to a luciferase reporter, directs murine hCD4 transgene expression preferentially to GMP, myeloid CFUs, and functional LT-HSC, and is bound and activated by RUNX1, PU.1, SCL, GATA-2, C/EBPα, and c-Myb (Guo et al, Blood 2012; Guo et al, J. Leuk. Biol., 2014; Cooper et al, PLoS One 2015).  In addition, CRISPR/Cas9 mediated enhancer mutation markedly reduces Cebpa transcription in 32Dcl3 cells (Cooper et al, 2015), and in AML samples harboring t(8;21) the RUNX1-ETO oncoprotein interacts with the human CEBPA locus specifically at the homologous +42 kb enhancer. We have now characterized mice harboring floxed Cebpa +37 kb enhancer alleles. Utilizing a B6 BAC encompassing the Cebpa locus, we assembled a targeting vector containing a 5.5 kb 5’ homology arm, the + 37 kb enhancer flanked by loxP sites, a PGK-Neo cassette flanked by frt sites, and a 0.9 kb 3’ homology arm. After introduction into a B6 mESC line, several G418-resistant subclones were isolated, and homologous, heterozygous targeting was confirmed by 3’ PCR and 5’ and 3’ Southern blotting. Two ESC lines were utilized to develop germ line chimeras, and upon breeding with B6 albino mice, one yielded several offspring with germ line transmission of the targeted allele, which were then bred to homozygosity.  In an initial set of experiments, marrow from wild-type (WT) and Enh(f/f) mice exposed to 5-FU was transduced with pBabePuro or pBabePuro-Cre followed by puromycin selection and in vitro analysis. Cebpa mRNA was reduced 10-fold in the lineage-negative subset upon Enh(f/f) marrow transduction with Cre. CFU-G were reduced 8-fold, whereas CFU-M and CFU-GM were unaffected, and enhancer deletion enabled serial replating of myeloid CFUs for >16 generations, though these remained dependent on IL-3. Morphologically, enhancer deletion led to depletion of mature granulocytes and accumulation of CD11b^-Gr-1^- blasts after four days of culture in IL-3, IL-6, and SCF. We next generated Enh(f/f);Mx1-Cre mice and subjected these and Enh(f/f) littermates to pIpC injections, followed by analysis four weeks after the last injection.  Cebpa mRNA was reduced 12-fold in GMP, 13-fold in CMP, and >20-fold in the LSK marrow subset.  Peripheral blood and marrow neutrophils were reduced 3- to 8-fold, whereas monocytes, RBCs, platelets, and B or T lymphocytes were unaffected. GMP as a percentage of Lin^-Sca-1^-c-kit⁺ progenitors were reduced 3-fold, CMP were increased 1.5-fold, and MEP and CLP were unaffected. CFU-G were reduced 3-fold, CFU-M were increased 2-fold, and BFU-E were increased 4-fold. LSK as a percentage of Lin^- cells were increased 3-fold, and MPP and ST-HSC as a percentage of LSK were unaffected, whereas LSK/SLAM were reduced 10-fold, and enumeration of functional LT-HSC by competitive transplantation is in progress. We also generated Enh(f/f);Vav-Cre mice; these demonstrated similarly impaired granulopoiesis, but were obtained at only 12% of the Mendelian ratio, suggestive of embryonic lethality as early death was not observed.  In addition, we used FLPo-Cre to delete the Neo cassette; homozygous deletion did not alter myeloid CFU numbers or FACS-defined progenitors. Finally, we utilized CMV-Cre to develop germ line enhancer-deleted mice. As with Vav-Cre, homozygous offspring were obtained far below the predicted numbers.  Nevertheless, these expressed Cebpa mRNA at levels equivalent to WT controls in liver, lung, adipose, kidney, small intestine, skeletal muscle, and cardiac tissues, and these organs displayed normal cellular architecture upon hematoxylin-eosin staining.  In summary, conditional deletion of the +37 kb Cebpa enhancer in adult mice demonstrates that it acts as a critical, hematopoietic specific enhancer of Cebpa gene expression and that marked Cebpa mRNA down-modulation upon enhancer deletion impairs granulopoiesis and enables serial, cytokine-dependent myeloid CFU replating, a pre-leukemic phenotype.