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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE199696 | GEO | 2022/09/10

REPOSITORIES: GEO

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Project description:Epigenetic modifications must underlie lineage-specific differentiation since terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Hematopoiesis provides a well-defined model of progressive differentiation in which to study the role of epigenetic modifications in cell fate decisions. Multi-potent progenitors (MPPs) can differentiate into all blood cell lineages, while downstream progenitors commit to either myeloerythroid or lymphoid lineages. While DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs {Broske, 2009 #6}, a comprehensive DNA methylation map of hematopoietic progenitors, or of any cell lineage, does not exist. Here we have generated a mouse DNA methylation map, examining 4.6 million CpG sites throughout the genome including all CpG islands and shores, examining MPPs, all lymphoid progenitors (ALPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Interestingly, differentiation towards the myeloid lineage corresponds with a net decrease in DNA methylation, while lymphoid commitment involves a net increase in DNA methylation, but both show substantial dynamic changes consistent with epigenetic plasticity during development. By comparing lineage-specific DNA methylation to gene expression array data, we find many examples of genes and pathways not previously known to be involved in lymphoid/myeloid differentiation, such as Gcnt2, Arl4c, Gadd45α, and Jdp2. Several transcription factors, including Meis1 and Prdm16 were methylated and silenced during differentiation, suggesting a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome appears to be important in hematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation, often correlating with gene expression changes, and define a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions. mRNA expression of 8 hematopoietic progenitor populations [MPPFL-(5), MPPFL+(3), CMP(3), GMP(3), CLP(3), DN1(3), DN2(3), DN3(3)] were compared

Project description:Growth factor independent 1 (Gfi1) is a transcriptional repressor originally identified as a common integration site in Moloney-murine-leukemia-virus-induced T-cell leukemia. Gfi1-/- mice display increased apoptosis of developing thymocytes and T lymphopenia; however, there are contradictory reports of the absolute number of Gfi1-/- early T lineage progenitors. We used floxed alleles of Gfi1 crossed to various T-cell-specific Cre transgenes to map the requirements for Gfi1 during lymphoid priming and development. We show that Gfi1 is necessary for the proper formation and function of both lymphoid-primed multipotent progenitors and early T lineage progenitors. These defects correlate with a global inability of Gfi1-/- progenitors to enforce the activation of lymphoid genes including IL7R, Rag1, Flt3 and Notch1. Forced expression of intracellular Notch1 fails to rescue the Gfi1-/- defective lymphoid gene signature or Gfi1-/- T cell development. Instead, activation of Notch1 in Gfi1-/- cells results in a potent synthetic lethal phenotype that is most dramatic in immature thymocytes, but absent in mature peripheral T cells where developmental transcriptional programs are silent. Moreover, we find that the requirement for Gfi1-transcriptional integration of Notch-driven lymphoid transcriptional programs is cell autonomous. Our data indicate that Gfi1 is required at multiple independent stages of lymphoid development. In hematopoietic progenitors Gfi1 is necessary to integrate Notch1 signaling, mediate lymphoid priming, the formation of early T lineage progenitors and subsequent T lineage commitment. Lineage negative cells were purified by magnetic beads from RosaCreERT2 Gfi1 ex4-5 floxed mice and an activated Notch1 signal was introduced using a GFP+ retroviral vector. GFP+ progenitors were FACS-sorted and cultured in semi-solid media for one week to allow sufficient time to to instruct lymphoid differentiation, then replated in 1uM 4-OHT or EtOH control. After an additional 7 days, CFU were disrupted and RNA was isolated for global gene expression using microarrays.

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

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