Multi-omics dissection of the ZEB axis during hematopoietic differentiation
Regulatory regions in the genome are recognized by specific transcription factors to control the expression of target genes. Identifying transcription factor activity across the genome helps elucidate mechanisms of gene regulation in both physiological and pathological contexts. Although many transcription factors have been implicated in blood diseases, the full spectrum of transcription (de)regulation in hematopoiesis remains incompletely understood. Here, we aimed to identify the full spectrum of transcription factors involved in normal hematopoiesis, to subsequently assess their relevance in hematopoietic malignancies.
Using the Blueprint project data, which provides an extensive epigenetic dataset mapping the epigenomes of human hematopoietic cell types at various differentiation stages, we performed motif enrichment analysis in conjunction with transcription factor expression data to identify activity during hematopoietic differentiation. Focusing on the ZEB transcription factors, we conducted DNA pulldown assays, protein binding affinity experiments, and immunoprecipitation to characterize their binding context and associated protein partners, as well as ChIP-seq and RNA-seq experiments combined with perturbations to identify their gene program.
Motif enrichment analysis revealed lineage-specific activity of the ZEB motif, triggering further investigation into the molecular activities of ZEB1 and ZEB2, the transcription factors binding this motif. Biochemical assays showed differential binding of ZEB1 and ZEB2 to the ZEB motif in lymphoid and myeloid contexts, respectively. Integration of ChIP-seq and RNA-seq data enabled profiling of ZEB2 transcriptional output in hematopoietic cells, established its genome-wide binding and revealed colocalization with the repressive NuRD complex. Targeted protein degradation of ZEB1 and ZEB2 in an erythrocyte/megakaryocyte leukemia model allowed functional validation of predicted ZEB2 targets and the deduction of the biological consequences of ZEB2 signalling in hematopoietic cells.
Our findings showcase the role and importance of ZEB1 and ZEB2 in hematopoietic differentiation and their potential involvement in leukemic processes. This study expands the understanding of ZEB-mediated regulation in blood cell development and provides a foundation for exploring ZEB family members as therapeutic targets in hematologic malignancies.