Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.

Hannah K Long,Alexander T Adams,Kazuya Ikeda,Mervenaz Koska,Tomek Swigut,Yiran E Liu,Marco Osterwalder,Axel Visel,Joanna Wysocka,Tim Mohun,Karissa Hansen,Matthew H Porteus,Ruth M Williams,Jim R Hughes,Robert Aho,Licia Selleri,Diane E Dickel,Douglas R Higgs,James O J Davies ,Neiha Arora ,Tatjana Sauka‐Spengler ,Ian Welsh

doi:10.1016/j.stem.2020.09.001

Abstract

SummaryNon-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

Highlights

Distal regulatory sequences called enhancers control gene transcription at a distance and play a critical role in directing developmental gene expression patterns (Long et al, 2016)
Three Clusters of Candidate Human Cranial Neural Crest Enhancers Overlap Sequences Lost in Pierre Robin sequence (PRS) Many large non-coding deletions identified in PRS patients map to the SOX9 locus but are mostly non-overlapping, suggesting the presence of multiple regulatory elements with nonredundant functions whose loss leads to similar phenotypic outcomes (Amarillo et al, 2013; Benko et al, 2009; Gordon et al, 2014)
To identify candidate human CNCCs (hCNCCs) enhancer elements that map within regions of the SOX9 gene desert lost in PRS patients, we used chromatin immunoprecipitation sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) datasets from in-vitro-derived hCNCCs (Prescott et al, 2015; this study; Figure 1A)

Summary

Introduction

Distal regulatory sequences called enhancers control gene transcription at a distance and play a critical role in directing developmental gene expression patterns (Long et al, 2016). Non-coding mutations are increasingly being implicated in human disease (Franke et al, 2016; Laugsch et al, 2019; Lupian ̃ ez et al, 2015), and, in particular, perturbations of enhancers have been documented as being causative because of their effects on gene regulation during development (Spitz, 2016). Mutations of protein-coding sequences often affect multiple tissues in which a given gene is active, mutations in non-coding regulatory regions can selectively perturb target gene expression in specific tissue contexts.

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Conclusion