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

Hematopoietic stem and progenitor cells can differentiate into all types of blood cells. Regulatory mechanisms underlying pluripotency in progenitors, such as the ability of lymphoid progenitor cells to differentiate into T-lineage, remain unclear. We have previously reported that LIM domain only 2 (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 remains obscure. Here, we identified approximately 600 Lmo2-interacting molecules in a lymphoid progenitor cell line by two-step affinity purification with LC-MS/MS analysis. Zinc finger and BTB domain containing 1 (Zbtb1) and CBFA2/RUNX1 partner transcriptional corepressor 3 (Cbfa2t3) were found to be the functionally important binding partners of Lmo2. We determined CRISPR/Cas9-mediated acute disruption of Zbtb1 or Cbfa2t3 in the lymphoid progenitor or bone marrow-derived primary hematopoietic progenitor cells causes significant defects in the initiation of T-cell development when Notch signaling is activated. Our transcriptome analysis of Zbtb1- or Cbfa2t3-deficient lymphoid progenitors revealed that Tcf7 was a common target for both factors. Additionally, ChIP-seq analysis showed that Lmo2, Zbtb1, and Cbfa2t3 cobind to the Tcf7 upstream enhancer region, which is occupied by the Notch intracellular domain/RBPJ transcriptional complex after Notch stimulation, in lymphoid progenitors. Moreover, transduction with Tcf7 restored the defect in the T-lineage potential of Zbtb1-deficient lymphoid progenitors. Thus, in lymphoid progenitors, the Lmo2/Zbtb1/Cbfa2t3 complex directly binds to the Tcf7 locus and maintains responsiveness to the Notch-mediated inductive signaling to facilitate T-lineage differentiation.

Effects of the Zbtb1 Gene on Chromatin Spatial Structure and Lymphatic Development: Combined Analysis of Hi-C, ATAC-Seq and RNA-Seq.

Zbtb1 (zinc finger and BTB domain containing 1) is a member of mammalian zbtb gene family. A series of bioinformatics analysis was carried out for the EL4 cell and the Zbtb1-deficient EL4 cell by Hi-C, ATAC-seq and RNA-seq techniques. Finally, Hi-C results showed that the intensity of chromatin interaction in the deletion group decreased with distance, the degree of chromosome interaction decreased significantly, the AB division region changed significantly, and the compactness of TAD structure decreased; The results of ATAC-seq showed that the open area and degree of chromatin in the deletion group decreased; 7778 differentially expressed mRNAs were found by RNA-seq. Our experimental results for the first time expounded the significance of Zbtb1 gene for T cell development, lymphocyte production and apoptosis from the aspects of chromosome spatial structure and chromatin opening degree, and provided relevant theoretical basis and data support for the in-depth study of related Zbtb1 genes in the future.

Analysis of lncRNAs and mRNA Expression in the ZBTB1 Knockout Monoclonal EL4 Cell Line and Combined Analysis With miRNAs and circRNAs.

In previous experiments, we identified the effect of deletion of the Zbtb1 gene on circRNAs and microRNAs. In this study, we examined the expression profiles of lncRNAs and mRNAs using the RNA-seq method for Zbtb1-deficient EL4 cells and performed a clustering analysis of differentially expressed lncRNAs and mRNAs. GO term histograms and KEGG scatter plots were drawn. For the experimental results, a joint analysis was performed, which predicted the regulatory relationships among lncRNAs, mRNAs, microRNAs and circRNAs. For the regulatory relationship between lncRNAs and target genes, the chromatin structure and the degree of openness were verified for the possible target gene locations regulated by lncRNA using experimental methods such as Hi-C and ATAC-seq. Ultimately, the possible differential regulation of the Brcal and Dennd5d genes by lncRNAs and the differential changes in transcription factor binding sites in the promoter region were identified. For neRNA-regulated target genes with significantly differentially expressed mRNAs, a combined screen was performed, and the final obtained candidate target genes were subjected to GO and KEGG term enrichment analyses. Our results illustrate that the Zbtb1 gene can not only function as a regulatory factor but also regulate EL4 cells from multiple perspectives based on ceRNA theory.

MicroRNA and circRNA Expression Analysis in a Zbtb1 Gene Knockout Monoclonal EL4 Cell Line.

Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, and a member of the mammalian Zbtb gene family. Previous studies have shown that Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by the control and gene deletion groups were determined by RNA-seq. In general, 24 differentially expressed microRNA and 16 differentially expressed circRNA were found between normal group and gene deletion group. Through further analysis of differentially expressed genes, GO term histogram and KEGG scatter plot were drawn, and three pairs of miRNA and circRNA regulatory relationships were found. This study describes the differentially expressed microRNA and circRNA in normal and Zbtb1-deficient EL4 cell lines, thus providing potential targets for drug development and clinical treatment of T-cell lymphoma.

A novel tumor suppressor ZBTB1 regulates tamoxifen resistance and aerobic glycolysis through suppressing HER2 expression in breast cancer.

Transcriptional repressor zinc finger and BTB domain containing 1 (ZBTB1) is required for DNA repair. Because DNA repair defects often underlie genome instability and tumorigenesis, we determined to study the role of ZBTB1 in cancer. In this study, we found that ZBTB1 is down-regulated in breast cancer and this down-regulation is associated with poor outcome of breast cancer patients. ZBTB1 suppresses breast cancer cell proliferation and tumor growth. The majority of breast cancers are estrogen receptor (ER) positive and selective estrogen receptor modulators such as tamoxifen have been widely used in the treatment of these patients. Unfortunately, many patients develop resistance to endocrine therapy. Tamoxifen-resistant cancer cells often exhibit higher HER2 expression and an increase of glycolysis. Our data revealed that ZBTB1 plays a critical role in tamoxifen resistance in vitro and in vivo To see if ZBTB1 regulates HER2 expression, we tested the recruitments of ZBTB1 on HER2 regulatory sequences. We observed that over-expressed ZBTB1 occupies the estrogen receptor α (ERα)-binding site of the HER2 intron in tamoxifen-resistant cells, suppressing tamoxifen-induced transcription. In an effort to identify potential microRNAs (miRNAs) regulating ZBTB1, we found that miR-23b-3p directly targets ZBTB1. MiR-23b-3p regulates HER2 expression and tamoxifen resistance via targeting ZBTB1. Finally, we found that miR-23b-3p/ZBTB1 regulates aerobic glycolysis in tamoxifen-resistant cells. Together, our data demonstrate that ZBTB1 is a tumor suppressor in breast cancer cells and that targeting the miR-23b-3p/ZBTB1 may serve as a potential therapeutic approach for the treatment of tamoxifen resistant breast cancer.

Regulation of the Development and Function of B Cells by ZBTB Transcription Factors.

The large ZBTB family comprises a diverse group of transcriptional factors. Several ZBTB proteins have emerged as critical factors that regulate the lineage commitment, differentiation, and function of lymphoid cells as well as many other developmental events. For instance, dysfunctions of ZBTB20 or ZBTB24 have been linked to multisystem failures in humans. Within the B-cell lineage, BCL6, ZBTB7A, ZBTB17, and ZBTB1 regulate the development/differentiation of B cells in both bone marrow and peripheral lymphoid organs, while ZBTB20 and ZBTB32 seem to mainly impact the maintenance of terminal plasma cells. Given the importance of B cells in the prevention and treatment of infectious or autoimmune disorders, we herein summarize the roles of seven ZBTB family members (BCL6, ZBTB7A, ZBTB17, ZBTB20, ZBTB32, ZBTB1, and ZBTB24) in the development, differentiation, and function of B cells as well as the underlying molecular mechanisms.

Zbtb1 controls NKp46+ ROR-gamma-T+ innate lymphoid cell (ILC3) development.

Innate lymphoid cells (ILCs) play a central role conferring protection at the mucosal frontier. In this study, we have identified a requirement of the transcription factor Zbtb1 for the development of RORγt+ ILCs (ILC3s). Zbtb1-deficient mice lacked NKp46+ ILC3 cells in the lamina propria of the small and large intestine. This requirement of Zbtb1 was cell intrinsic, as NKp46+ ILC3s were not generated from Zbtb1-deficient progenitors in bone marrow chimeras and Zbtb1-deficient RORγt+ CCR6-NKp46- ILC3s didn't generate NKp46+ ILC3s in co-cultures with OP9-DL1 stroma. In correlation with this impairment, Zbtb1-deficient ILC3 cells failed to upregulate T-bet expression, and to acquire IFN-γ production characteristic of NKp46+ cells. Finally, absence of NKp46+ILC3 cells combined with the absence of T-cells in Zbtb1-deficient mice, led to a transient susceptibility to C. rodentium infections. Altogether, these results establish that Zbtb1 is essential for the development of NKp46+ ILC3 cells.

Zbtb1 prevents default myeloid differentiation of lymphoid-primed multipotent progenitors.

Zbtb1 is a transcription factor that prevents DNA damage and p53-mediated apoptosis in replicating immune progenitors, affecting lymphoid as well as myeloid development when hematopoietic progenitors are in competition in mixed bone marrow chimeras. However, Zbtb1-deficient mice do not have an apparent myeloid deficiency. We report here that Zbtb1-deficient lymphoid-primed multipotent progenitors (LMPPs) are biased to develop towards the myeloid fate in detriment of lymphoid development, contributing to the apparent unaffected myeloid development. Zbtb1 expression was maintained during lymphoid development of LMPP cells but downregulated during myeloid development. Deficiency of Zbtb1 in LMPP cells was sufficient to direct a myeloid fate in lymphoid-inducing conditions and in the absence of myeloid cytokines as shown by upregulation of a myeloid gene signature and the generation of myeloid cells in vitro. Finally, biased myeloid differentiation of Zbtb1-deficient LMPP cells was not due to increased p53-dependent apoptosis as it was not reverted by transgenic Bcl2 expression or p53 deficiency. Altogether, our results show that Zbtb1 expression prevents activation of a default myeloid program in LMPP cells, ensuring the generation of lymphoid cells.