Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells

Hematopoietic stem and progenitor cells are able to differentiate into all blood cell types. Regulatory mechanisms underlying pluripotency in progenitors, such as the ability of lymphoid progenitor cells to differentiate into T-lineage, are not fully understood. We have previously reported that Lmo2, a bridging factor in large transcriptional complexes, is essential to retain the ability of lymphoid progenitors to differentiate into T-lineage. However, biochemical characterization of Lmo2 protein complexes in physiological hematopoietic progenitors has remained obscure. In this study, we identified around 600 of Lmo2 interacting molecules in a lymphoid progenitor cell line by two-step affinity purification with LC-MS/MS analysis. Among them, we found that Zbtb1 and Cbfa2t3 are functionally important binding partners of Lmo2. CRISPR/Cas9-mediated acute disruption of Zbtb1 or Cbfa2t3 in a lymphoid progenitor line or BM-derived primary hematopoietic progenitors caused significant defects in the initiation of T cell development when Notch signal is activated. Transcriptome analysis of Zbtb1- or Cbfa2t3-deficient lymphoid progenitors reveled that Tcf7, one the earliest Notch-target genes, is a common target of both factors. ChIP-seq analysis clearly showed that Lmo2, Zbtb1 and Cbfa2t3 co-bind to the Tcf7 upstream enhancer region, where is occupied by Notch intracellular domain/RBPJ transcriptional complex after Notch stimulation, in lymphoid progenitors. Moreover, transduction of Tcf7 restored the defect in the T-lineage potential of Zbtb1-deficient lymphoid progenitor cells. Thus, in lymphoid progenitors, Lmo2/Zbtb1/Cbfa2t3 complex directly binds to the Tcf7 locus and maintains responsiveness to the Notch-mediated inductive signaling for the T-lineage program. ChIP-seq of LP cells for Zbtb1 and Cbfa2t3 RNA-seq of LP cells with CRISPR/Cas9-mediated disruption of LMO2, Zbtb1, Cbfa2t2 or Cbfa2t3.

造血干细胞和祖细胞（hematopoietic stem and progenitor cells）能够分化为所有血细胞类型。祖细胞多能性背后的调控机制，例如淋巴祖细胞向T细胞谱系分化的能力，尚未完全阐明。我们此前曾报道，大型转录复合物中的桥接因子Lmo2（Lmo2）是维持淋巴祖细胞向T细胞谱系分化能力的必需因子。然而，在生理状态的造血祖细胞中，对Lmo2蛋白复合物的生化表征仍不明晰。本研究通过两步亲和纯化结合液相色谱-串联质谱（LC-MS/MS）分析，在淋巴祖细胞系中鉴定出约600个Lmo2互作分子。其中，我们发现Zbtb1与Cbfa2t3是Lmo2功能上至关重要的结合伴侣。在淋巴祖细胞系或骨髓（BM）来源的原代造血祖细胞中，经CRISPR/Cas9介导的Zbtb1或Cbfa2t3急性敲除，会在Notch信号激活时显著损伤T细胞发育的起始过程。对Zbtb1缺陷或Cbfa2t3缺陷的淋巴祖细胞的转录组分析显示，作为最早的Notch靶基因之一的Tcf7，是这两个因子的共同靶标。染色质免疫共沉淀测序（ChIP-seq）分析清晰显示，在淋巴祖细胞中，Lmo2、Zbtb1与Cbfa2t3共同结合于Tcf7的上游增强子区域；该区域在Notch刺激后会被Notch胞内结构域/RBPJ转录复合物占据。此外，过表达Tcf7可修复Zbtb1缺陷型淋巴祖细胞的T细胞谱系分化潜能缺陷。综上，在淋巴祖细胞中，Lmo2/Zbtb1/Cbfa2t3复合物可直接结合Tcf7基因座，并维持对Notch介导的诱导信号的响应性，从而调控T细胞谱系程序。本研究的实验数据集包含两类：一是针对淋巴祖细胞（LP cells）的Zbtb1与Cbfa2t3染色质免疫共沉淀测序数据；二是经CRISPR/Cas9介导敲除LMO2、Zbtb1、Cbfa2t2或Cbfa2t3的淋巴祖细胞的RNA测序（RNA-seq）数据。