Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
Department of Physiology, China Medical University, Shenyang, China.
PLoS One. 2020 Dec 17;15(12):e0244161. doi: 10.1371/journal.pone.0244161. eCollection 2020.
C/EBPα is required for formation of granulocyte-monocyte progenitors (GMP) and also participates in B lymphopoiesis. The common lymphoid progenitor (CLP) and preproB populations but not proB cells express Cebpa, and pan-hematopoietic deletion of the +37 kb Cebpa enhancer using Mx1-Cre leads not only to reduced GMP but also to 2-fold reduced marrow preproB and >15-fold reduced proB and preB cells. We now show that IL7Rα-Cre-mediated deletion of the +37 kb Cebpa enhancer, which occurs in 89% of Ly6D+ and 65% of upstream Ly6D- CLP, leads to a 2-fold reduction of both preproB and proB cells, and a 3-fold reduction in preB cells, with no impact on GMP numbers. These data support a direct role for C/EBPα during B lineage development, with reduced enhancer deletion in Ly6D- CLP mediated by IL7Rα-Cre diminishing the effect on B lymphopoiesis compared to that seen with Mx1-Cre. Amongst mRNAs encoding key transcriptional regulators that initiate B lymphoid specification (PU.1, E2A, IKAROS, EBF1, FOXO1, and BACH2), only Ebf1 levels are altered in CLP upon Mx1-Cre-mediated Cebpa enhancer deletion, with Ebf1 reduced ~40-fold in Flt3+Sca-1intc-kitintIL7Rα+ CLP. In addition, Cebpa and Ebf1 RNAs were 4- and 14-fold higher in hCD4+ versus hCD4- CLP from Cebpa-hCD4 transgenic mice. Histone modification ChIP-Seq data for CLP indicate the presence of active, intronic Ebf1 enhancers located 270 and 280 kb upstream of the transcription start sites. We identified a cis element in this region that strongly binds C/EBPα using the electrophoretic mobility shift assay. Mutation of this C/EBPα-binding site in an Ebf1 enhancer-TK-luciferase reporter leads to a 4-fold reduction in C/EBPα-mediated trans-activation. These findings support a model of B lymphopoiesis in which induction of Ebf1 by C/EBPα in a subset of CLP contributes to initiation of B lymphopoiesis.
C/EBPα 对于粒细胞-单核细胞祖细胞（GMP）的形成是必需的，并且也参与 B 淋巴细胞生成。共同淋巴祖细胞（CLP）和前 B 细胞群，但不是前 B 细胞，表达 Cebpa，使用 Mx1-Cre 对 +37 kb Cebpa 增强子进行全血造血缺失不仅导致 GMP 减少，而且导致骨髓前 B 细胞减少 2 倍，前 B 和前 B 细胞减少 >15 倍。我们现在表明，IL7Rα-Cre 介导的 +37 kb Cebpa 增强子缺失，其发生在 89%的 Ly6D+和 65%的上游 Ly6D- CLP 中，导致前 B 和前 B 细胞减少 2 倍，前 B 细胞减少 3 倍，而对 GMP 数量没有影响。这些数据支持 C/EBPα 在 B 细胞谱系发育过程中的直接作用，IL7Rα-Cre 介导的 Ly6D- CLP 中减少的增强子缺失与 Mx1-Cre 相比，对 B 淋巴细胞生成的影响较小。在起始 B 淋巴细胞特异性的关键转录调节因子的 mRNAs 中（PU.1、E2A、IKAROS、EBF1、FOXO1 和 BACH2），只有在 Mx1-Cre 介导的 Cebpa 增强子缺失后，CLP 中的 Ebf1 水平发生改变，Flt3+Sca-1intc-kitintIL7Rα+ CLP 中的 Ebf1 减少约 40 倍。此外，Cebpa 和 Ebf1 RNA 在 Cebpa-hCD4 转基因小鼠的 hCD4+与 hCD4- CLP 之间分别高 4 倍和 14 倍。CLP 的组蛋白修饰 ChIP-Seq 数据表明，在转录起始位点上游 270 和 280 kb 处存在活性内含子 Ebf1 增强子。我们在该区域中鉴定出一个顺式元件，该元件通过电泳迁移率变动分析强烈结合 C/EBPα。Ebf1 增强子-TK-荧光素酶报告基因中该 C/EBPα 结合位点的突变导致 C/EBPα 介导的转激活减少 4 倍。这些发现支持 B 淋巴细胞生成的模型，其中 C/EBPα 在 CLP 的一个亚群中诱导 Ebf1 有助于 B 淋巴细胞生成的起始。
