Title: Mechanisms of long-range gene regulation in craniofacial development and disease
Funder: Medical Research Council

Abstract: How information encoded within our genomes directs our development is fundamental to understanding how genetic alterations drive evolutionary change, normal-range variation and human developmental disorders. The face is an excellent system for investigating how changes in genotype impact morphology as it is one of the most divergent structures that forms during development and around one third of human congenital disorders are associated with craniofacial defects. Increasingly, mutations in the non-coding genome are implicated in Mendelian developmental disorders, and the majority of genetic variants contributing to complex traits are often outside of genes (e.g. cleft palate). These observations implicate perturbation of regulatory elements in driving phenotypic variation, for example enhancer elements normally activate target gene expression and their ablation can therefore detrimentally impact development (Long et al, Cell, 2016). The overarching aim of this research programme is to investigate mechanisms governing gene regulation across human craniofacial development and to uncover how non-coding mutations drive facial variation and disease. To investigate gene regulatory mechanisms during human facial development, we will leverage differentiation of facial progenitors (cranial neural crest cells, CNCCs) from human embryonic stem cells, and explore the connection to craniofacial form using animal models. Both intragenic and non-coding variants cause craniofacial dysmorphology at the SOX9 locus, and we previously characterised extreme long-range enhancers whose loss drives lower jaw defects as seen in patients (Long et al, Cell Stem Cell, 2020). Further dissection of this complex regulatory landscape will illuminate fundamental mechanisms of gene regulation in a disease-relevant context. Objective 1: Determine mechanisms of combinatorial gene regulation at disease-associated enhancer clusters Key developmental genes are often subject to complex regulation and understanding how multiple enhancers act in combination to drive developmental expression will be fundamental to interpreting how mutations impact target gene expression and developmental phenotypes. At the SOX9 locus we previously identified super-additive enhancer activity for two enhancer clusters. We will explore sequence determinants of this enhancer synergy using a high-throughput mutagenesis approach in CNCCs, and spatial reporter assays in zebrafish and mouse. Objective 2: Examine the role of chromosome topology in mediating long-range gene expression Distal enhancer clusters direct SOX9 expression at enormous genomic distances (1.45 and 1.25 Mb) in CNCCs. The genome-organising factor CTCF appears to facilitate long-range SOX9 enhancer function through an enhancer-associated element (Chen*, Long* et al, under preparation) warranting further examination of the role of local chromosomal folding in the regulation of gene activation. CTCF binding in different genomic and cellular contexts appears to have distinct roles in genome structure and gene expression. We will therefore explore mechanisms that influence CTCF function across the SOX9 regulatory domain, and examine whether CTCF-dependence is a common regulatory strategy utilised by other important craniofacial genes. An enhanced understanding how non-coding variation impacts human development will ultimately improve diagnosis and intervention options for patients, and has broad relevance for a wide range of genetic disorders.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CONGENITAL DISORDERS