Zeb2 DNA-binding sites in neuroprogenitor cells reveal autoregulation and affirm neurodevelopmental defects, including in Mowat-Wilson Syndrome

Abstract

Perturbation and mechanistic studies have shown that the DNA-binding transcription factor Zeb2 controls cell fate decision, differentiation and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs) Zeb2 strong upregulation is necessary for their exit from primed pluripotency and entering neural and general differentiation. We engineered mouse ESCs to produce epitope-tagged Zeb2 from one of its two endogenous alleles. Using crosslinking ChIP-sequencing, we mapped for the first time 2,432 DNA-binding sites of Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major site maps promoter-proximal to Zeb2 itself, and its homozygous removal demonstrates that Zeb2 autoregulation is necessary to elicit proper Zeb2 effects in ESC toNPC differentiation. We then cross-referenced all Zeb2 DNA-binding sites with transcriptome data from Zeb2 perturbations in ESCs, ventral forebrain in mouse embryos, and adult neurogenesis. While the characteristics of these neurodevelopmental systems differ, we find interesting overlaps. Collectively, these new results obtained in ESC-derived NPCs significantly add to Zeb2 role as neurodevelopmental regulator as well as the causal gene in Mowat-Wilson Syndrome. Also, Zeb2 was found to map to loci mutated in other human congenital syndromes, making variant or disturbed levels of ZEB2 a candidate modifier principle in these.

Different E-box binding transcription factors, similar neuro-developmental defects: ZEB2 (Mowat-Wilson syndrome) and TCF4 (Pitt-Hopkins syndrome)

“…The N-terminal (NZF, with four fingers) and C-terminal cluster (CZF, with three fingers) enable ZEB proteins to bind with their two hands (and two zinc fingers in each) to DNA. Binding occurs to two separated CACCT sequences [63,64] , often also found as CACCTG known as E2-boxes, and in few cases also presenting as CACANNTG [8,52] (CANNTG being an E-box). This could make ZEB proteins putative competitors of bHLH TFs for the same E-type boxes, including for the TCF4-containing heterodimers that bind to the CANNTG E-box, with a preference for C or G in the N positions [65] .…”

“…Interestingly, the 5'-C( A / G )-CANNTG-3' motif has also been identified as a binding site for ZEB1 and, due to the high conservation of DNA-binding zinc fingers between ZEBs, been accepted for both ZEB1 and ZEB2 [8,63] . For such motif, the influence of methylation (of a 5'-CG) for binding of ZEBs has not been documented yet.…”