title: 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.
abstract: The murine Cebpa gene contains a +37 kb, evolutionarily conserved 440 bp enhancer that directs high-level expression to myeloid progenitors in transgenic mice. The enhancer is bound and activated by Runx1, Scl, GATA2, C/EBPα, c-Myb, Pu.1, and additional Ets factors in myeloid cells. CRISPR/Cas9-mediated replacement of the wild-type enhancer with a variant mutant in its seven Ets sites leads to 20-fold reduction of Cebpa mRNA in the 32Dcl3 myeloid cell line. To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer. CMV-Cre mediated germline deletion resulted in diminution of the expected number of viable Enh(f/f);CMV-Cre offspring, with 28-fold reduction in marrow Cebpa mRNA but normal levels in liver, lung, adipose, intestine, muscle, and kidney. Cre-transduction of lineage-negative marrow cells in vitro reduced Cebpa mRNA 12-fold, with impairment of granulocytic maturation, morphologic blast accumulation, and IL-3 dependent myeloid colony replating for >12 generations. Exposure of Enh(f/f);Mx1-Cre mice to pIpC led to 14-fold reduction of Cebpa mRNA in GMP or CMP, 30-fold reduction in LSK, and <2-fold reduction in the LSK/SLAM subset. FACS analysis of marrow from these mice revealed 10-fold reduced neutrophils, 3-fold decreased GMP, and 3-fold increased LSK cells. Progenitor cell cycle progression was mildly impaired. Granulocyte and B lymphoid colony forming units were reduced while monocytic and erythroid colonies were increased, with reduced Pu.1 and Gfi1 and increased Egr1 and Klf4 in GMP. Finally, competitive transplantation indicated preservation of functional long-term hematopoietic stem cells upon enhancer deletion and confirmed marrow-intrinsic impairment of granulopoiesis and B cell generation with LSK and monocyte lineage expansion. These findings demonstrate a critical role for the +37 kb Cebpa enhancer for hematopoietic-specific Cebpa expression, with enhancer deletion leading to impaired myelopoiesis and potentially preleukemic progenitor expansion.
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Guo
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Stacy
authors.initials: S
authors.lastname: Cooper
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Friedman
mesh_terms: D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000172:deficiency / Q000235:genetics*; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005260:Female; D005786:Gene Expression Regulation; D006098:Granulocytes / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D038041:Lymphopoiesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D023461:Myeloid Progenitor Cells / Q000166:cytology* / Q000378:metabolism; D038042:Myelopoiesis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C530718:CEBPA protein, mouse; C486109:Egr1 protein, mouse; C079873:Gfi1 protein, mouse; C000715468:Klf4 protein, mouse