Functional characterization of the ZEB2 regulatory landscape.

Reut Bar Yaacov,Einan Farhi,Tommy Kaplan,Reut Eshel,Fania Shemuluvich,Ramon Y Birnbaum

doi:10.1093/hmg/ddy440

Abstract

Zinc finger E-box–binding homeobox 2 (ZEB2) is a key developmental regulator of the central nervous system (CNS). Although the transcriptional regulation of ZEB2 is essential for CNS development, the elements that regulate ZEB2 expression have yet to be identified. Here, we identified a proximal regulatory region of ZEB2 and characterized transcriptional enhancers during neuronal development. Using chromatin immunoprecipitation sequencing for active (H3K27ac) and repressed (H3K27me3) chromatin regions in human neuronal progenitors, combined with an in vivo zebrafish enhancer assay, we functionally characterized 18 candidate enhancers in the ZEB2 locus. Eight enhancers drove expression patterns that were specific to distinct mid/hindbrain regions (ZEB2#e3 and 5), trigeminal-like ganglia (ZEB2#e6 and 7), notochord (ZEB2#e2, 4 and 12) and whole brain (ZEB2#e14). We further dissected the minimal sequences that drive enhancer-specific activity in the mid/hindbrain and notochord. Using a reporter assay in human cells, we showed an increased activity of the minimal notochord enhancer ZEB2#e2 in response to AP-1 and DLX1/2 expressions, while repressed activity of this enhancer was seen in response to ZEB2 and TFAP2 expressions. We showed that Dlx1 but not Zeb2 and Tfap2 occupies Zeb2#e2 enhancer sequence in the mouse notochord at embryonic day 11.5. Using CRISPR/Cas9 genome editing, we deleted the ZEB2#e2 region, leading to reduction of ZEB2 expression in human cells. We thus characterized distal transcriptional enhancers and trans-acting elements that govern regulation of ZEB2 expression during neuronal development. These findings pave the path toward future analysis of the role of ZEB2 regulatory elements in neurodevelopmental disorders, such as Mowat–Wilson syndrome.

Highlights

Zinc finger E-box–binding homeobox (ZEB2) is a key transcription factor that acts as a multifunctional regulator during nervous system development
To determine the regulatory region that modulates Zinc finger E-box–binding homeobox 2 (ZEB2) neuronal expression, we initially examined the organization of the genome into topologically associating domains (TADs) in the ZEB2 locus
The proximal region of this sub-TAD boundary is enriched in epigenetic marks of active enhancers (H3K4me1 and H3K27ac) in human brain tissues but not in H1-ESC, suggesting that this ZEB2 TAD contains clusters of regulatory elements that are required for neuronal activity

Summary

Introduction

Zinc finger E-box–binding homeobox (ZEB2) is a key transcription factor that acts as a multifunctional regulator during nervous system development. ZEB2 is expressed in the developing neural tube, as well as in neural crest cells, the hippocampus and the cerebral cortex [3,4]. Zeb knockout mice die around embryonic day (E) 9.5 and exhibit severe neural plate and neural crest defects from E8.5 [4,5]. Conditional Zeb knockout mice show open neural tube defects, delamination arrest of neural crest cells [4] and hypo-cellularity of enteric neurons, all of which are characteristic of Mowat– Wilson syndrome (MWS) [6]. MWS is a neurodevelopmental syndrome characterized by a combination of defects with variable penetrance, including seizures, that can be caused by de novo mutations in ZEB2 [7]. While more than 100 heterozygous ZEB2 haploinsufficiency mutations have been demonstrated in MWS, some phenotypic cases do not present ZEB2-coding region mutations, suggesting other genomic variants—perhaps in ZEB2 regulatory elements—might underlie the molecular basis of this syndrome [8]

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