CAAT/enhancer-binding protein epsilon (C/EBPε) is known to be a critical transcription factor mediating terminal differentiation of granulocytes and a target to boost killing of bacteria by the innate immune system. However, our data found that ∼9 month old C/EBPε knockout (KO) mice developed a novel phenotype similar to the myeloproliferative disorder. In bone marrow (BM) of C/EBPε KO mice, colonies of granulocyte-macrophage progenitor (CFU-GM) were increased (143±21 vs. 59±13, P<0.01) while erythroid progenitors (BFU-E) were decreased (8±2 vs.14±4, P<0.01) as compared to age- and sex- matched wild type controls. Flow data showed that the percentage of BM immature myeloid (Mac1+Gr1int) were much higher (48%±3% vs. 9%±5%, P<0.01) while Ter119+ erythrocytes were less (13%±4% vs. 47%±1%, P<0.01) than control. In addition, the ratio of spleen/ body weight in C/EBPε KO mice was augmented (0.81%±0.16% vs. 0.25%±0.03%, P<0.01) and spleen H&E staining showed extramedullary hemopoiesis. Compared to spleen samples in control mice, CFU-GM (97±16 vs. 9±2, P<0.01), BFU-E (20±5 vs. 4±1, P<0.01) and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitors (CFU-GEMM, 11±3 vs. 2±1, P<0.01) in C/EBPε KO mice were significantly increased. Consistent with spleen CFU assay, the percentage of Mac1+Gr1int immature myeloid cells (18%±8% vs. 3%±1%, P<0.05) and Ter119+ erythrocytes (38%±1% vs. 12%±2%, P<0.01) were raised in KO than in control mice by flow cytometry analysis. Taken together, C/EBPε deletion in mice blocks myeloid differentiation and impairs erythoid development in BM as well as leads to extramedullary hemopoiesis in spleen.
To investigate regulatory networks controlled by C/EBPε, we performed genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) in wild type and C/EBPε KO mice BM using N- and C-terminal C/EBPε antibodies. We have identified ∼15000 significant C/EBPε binding sites and as with the majority of transcription factors, C/EBPε targets predominantly distal intergenic and intronic regulatory regions. However, 15% of significant binding sites are localized within gene promoters and some of them are known to play a critical role in hematopoietic lineage differentiation such as PU.1, RUNX1 and SCL. Interestingly, C/EBPε also binds known distal regulatory regions as the PU.1 enhancer, and our data has highlighted novel potential intergenic and intronic enhancers for RUNX1. The ChIP-PCR results of these transcription factors are consistent with ChIP-seq.
In summary, our study suggests that C/EBPε can have a profound effect on myeloid development by regulating key genes and its deregulation can produce a myeloproliferative abnormality.
Citation Format: Tong Yin, Touati Benoukraf, Phillip H. Koeffler. Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2013-5448
Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.