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EBF1 and PAX5 exert dose dependent epigenetic repression of T-lineage potential [ChIPmentation]

ABSTRACT: B-lymphocyte development is dependent on the coordinated action of transcription factors controlling the expression of stage and lineage specific genes. To better understand gene regulation and changes in the epigenetic landscape in early B-cell development, we conducted combined SC-RNA/ATAC seq analysis of bone marrow progenitor populations, allowing us to build a developmental trajectory based on changes in epigenetic accessibility. This analysis identified a rapid shift in DNA accessibility, where T-lineage primed progenitors gained the epigenetic landscape of B-lymphocytes. The epigenetic switch correlated well with initiation of EBF1 and PAX5 transcription as well as their functional activity. As ectopic expression of EBF1 in Pax5-/- pro-B cells was not sufficient to mediate either the full activation of the B-lineage program of silencing of T-lineage associated genes, we conclude that PAX5 and EBF1 are independently essential for the establishment of the epigenetic landscape in normal B-cell development. This idea was supported by the finding that pro-B cells from mice with combined heterozygote deletion of the Ebf1 and Pax5 genes, carried a non-differentiated epigenetic landscape and expressed T-lineage associated genes. The importance of epigenetic silencing for the preservation of B-cell fate was supported by the finding that inhibition of the histone methylases EZH1 and EZH2 in pro-B cells, allowed for the activation of T-lineage genes and generation of T-lineage cells in response to Notch signaling. Hence, B-lymphoid commitment is associated with a transcription factor dose dependent epigenetic switch, silencing an inherent T-lineage potential in early lymphoid progenitors.

ORGANISM(S): Mus musculus

PROVIDER: GSE295901 | GEO | 2025/11/25

REPOSITORIES: GEO

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Project description:In order to investigate how transcription factor dose impacts B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. While combined reduction of Pax5 and Ebf1 dose had minimal impact on the development of the earliest CD19+ progenitors, these cells displayed an increased T-cell potential in vivo and in vitro. Alteration in lineage fate depended on a Notch1 mediated conversion process while no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5+/-Ebf1+/- pro-B cells was reflected in the transcriptional response because even though cells of both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, only the Pax5+/-Ebf1+/- pro-B cells down-regulated genes central for the preservation of stable B-cell identity. This report stresses the importance of transcription factor dose in lymphocyte development and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling after the generation of activated intracellular Notch1. This provides an insight to how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation. EBF-1 ChIP-seq: Cultivated CD43+IgM- cells (ProB) cells from Wt, EBF-1 +/-, PAX-5 +/- and EBF-1 +/- PAX-5 +/- (TH) were assessed for EBF-1 binding by ChIP-seq. Replicate Ebf1 ChIP-seq runs on each genotype (Wt, TH, Ebf1+/- and Pax5+/-) and corresponding inputs were pooled into one dataset and analyzed as one combined sample per genotype. RNA-seq no treatment: Briefly, ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4), WT (n=4), Ebf1+/- (n=2) and Pax5+/- (n=2) were sorted and RNA extracted with Qiagen RNeasy Micro Kit. RNA was sent to UCLA Microarray Core for library preparation and were subsequently for 50 cycles of HiSeq 2000 SBS sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)). RNA-seq 1 day on OP9DL1 and OP9: In short, in vitro expanded ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4) and WT (n=4) were exposed either on OP9 or OP9-DL1 stromal cells for 24 hours and RNA extracted with Qiagen RNeasy Micro Kit. Due to low reads, two Wt and Ebf1+/-Pax5+/- were sequenced twice. Libraries were constructed using Nugen's Ovation Ultralow Library systems and were subsequently for 50 cycles of NextSeq500 sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)).

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EBF1 and PAX5 exert dose dependent epigenetic repression of T-lineage potential [ChIPmentation]

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