Exploring the role of Zeb1 in the haematopoietic system

Zeb1 is a zinc finger transcription factor that plays a critical role in embryonic development, tissue regeneration, and stem cell biology. While Zeb1 has been suggested to be involved in regulating the self-renewal and differentiation of hematopoietic stem cells (HSCs), the cellular and molecular mechanisms by which Zeb1 may regulate HSC function are not fully understood. We initially identified potential ZEB1 target genes in HSCs by RNA-sequencing of adult HSCs after Mx1-Cre mediated deletion of Zeb1. We found Zeb1 regulates cell adhesion and cytoskeletal organization related genes as well as lipid metabolism related genes. Our studies identified cell adhesion molecule EpCAM as a critical ZEB1 target in HSCs. We analysed the impact of Zeb1 mediated regulation of EpCAM expression on the transcriptional programming and molecular fate of HSCs and found that EpCAM expression in Zeb1 knock-out (KO) HSCs increased cell survival and decreased transcriptional markers for mitochondrial metabolism, RNA synthesis, and differentiation. To investigate the role of Zeb1 in the long-term maintenance of HSCs, we conditionally deleted Zeb1 using the Vav-iCre system, which deletes Zeb1 gene expression in HSCs during embryonic development at E11.5. Zeb1 KO mice were born with reduced Mendelian inheritance, demonstrating Zeb1 KO in HSCs during embryonic development causes perinatal lethality. We found Zeb1 KO mice that survived into young adulthood (8-12 weeks) displayed expanded HSC numbers but with reduced repopulation activity in transplantation experiments. Young adult Zeb1 KO HSCs displayed widespread myeloid and lymphoid differentiation defects including B-cell maturation and erythroid differentiation. Disrupted erythroid differentiation in Zeb1 KO HSCs led to the development of anaemia. In addition to the well-described critical role for Zeb1 in T-cell development, we found that loss of Zeb1 in HSCs impaired positive selection of thymocytes and the cell survival of T-cell subpopulations, resulting in a pronounced expansion in effector and memory T-cell frequency and a reduction in naive T-cells. Our RNA-seq analysis of young adult Zeb1 KO HSCs revealed altered transcriptional programming related to erythropoiesis, immunodeficiency, HSC maintenance pathways and inflammation. Heightened expression of v inflammatory cytokines IL-6, TNF-alpha and IFN-gamma in the plasma of young adult Zeb1 KO mice was observed, identifying Zeb1 as a critical mediator of inflammation. Ageing of adult Zeb1 KO mice lead to fatality with almost complete penetrance, and evidence of profound anaemia and preliminary evidence suggesting the development of pre-leukaemia (MDS/MPN-like) was observed. In addition, a single aged Zeb1 KO mouse appeared to develop a CD4+ T-cell driven lymphoproliferative syndrome. This suggests that Zeb1 depletion in HSCs can drive both myeloid and lymphoid malignancies/pre-malignancies. Overall, these data support the notion that Zeb1 acts as a tumour suppressor in select haematologic malignancies. In summary, this study reveals that Zeb1 is critical regulator of long-term HSC integrity, and that lack of Zeb1 in HSCs leads to the development of haematologic malignancies. These findings shed light on the complex molecular mechanisms underlying Zeb1 regulation of HSC function, which may have important implications for the development of novel therapies for hematopoietic disorders where ZEB1 expression is downregulated