Disruption of neural crest enhancer landscapes as an etiological mechanism for human neurocristopathies

The embryonic development of the human facial features is a highly complex mechanism which requires very exact spatial and temporal regulation of gene expression during neural crest (NC) development. NC cells (NCC) are a transient embryonic cell type with wide differentiation potential that contributes to the formation and morphogenesis of multiple tissues and organs, including many parts of the face. Just like any other cell type, NCC possess a characteristic set of enhancers that, by controlling the expression of specific genes, define cellular identity. Impairment of this regulation can lead to craniofacial malformations, such as orofacial cleft (OFC), which are frequently referred to as neurocristopathies and that represent a heavy burden on both the affected individuals and society. Understanding how genetic or structural disruption of enhancer activity during NC development can lead to human neurocristopathies is the central goal of this work. In the long term, the gained knowledge should serve to enable early detection and show potential therapeutic approaches. Here we investigate the pathomechanism of both syndromic (i.e. Branchiooculofacial Syndrome (BOFS)) and non-syndromic (i.e. OFC) neurocristopathies, by combining in vitro and in vivo NC developmental models with genetic engineering approaches and multiple genomic methods. First, we describe a unique patient with BOFS, who, in contrast to previously reported cases, does not present a heterozygous mutations within TFAP2A, a NC master regulator. Instead, the patient carries a de novo heterozygous 89 Mb inversion in which one of the breakpoints is located 40 kb downstream of TFAP2A. We first showed that this inversion separates TFAP2A from enhancers that are located within the same large topologically associating domain (TAD) and that are essential for TFAP2A expression in NCC. Importantly, using patient-specific human induced pluripotent stem cells (hiPSC) and a robust in vitro differentiation system towards NCC, we then showed that the inversion causes a loss of physical interactions between the inverted TFAP2A allele and its cognate enhancers, leading to TFAP2A monoallelic and haploinsufficient expression in human NCC. Overall, this first part provides a powerful approach to investigate the pathological mechanisms of structural variants predicted to disrupt 3D genome organization of gene regulatory landscapes and that, due to various reasons (i.e. limited access to relevant patient material, differences in gene dosage sensitivity between mice and humans, difficulties in recapitulating certain structural variants), cannot be properly evaluated in vivo. Second, we combined previously generated hNCC enhancer maps with OFC risk-loci identified through genome-wide association studies (GWAS) and, as a result, we revealed a highly conserved enhancer (i.e. Enh2p24.2) as a potential candidate harboring genetic variants involved in OFC. GWAS link common single nucleotide polymorphisms (SNPs) with quantitative traits and complex disorders. However, most disease-associated SNPs occur in non-coding regions of the human genome and consequently, the etiological relevance of these genetic variants cannot be easily connected to a gene. Nevertheless, accumulating evidences suggest that these disease-associated SNPs may contribute to human disease susceptibility by altering enhancers. Interestingly, SNPs associated with OFC are overrepresented in NCC enhancers. Therefore, we hypothesize that SNPs associated with OFC contribute to the etiology of the disorder by altering NCC enhancers and, consequently, the expression of relevant genes. Using Enh2p24.2 as a bait in circularized chromosome conformation capture sequencing (4C-seq) experiments, we identified two distally located genes, MYCN and DDX1, as its potential targets. Using in vitro and in vivo NCC developmental models, we then demonstrated that both genes are essential for normal facial development. While MYCN was not a surprising candidate to be involved in the etiology of OFC, the identification of DDX1 as a novel regulator of facial development might provide new insights into the molecular processes (e.g. transcription-coupled DNA repair) implicated in OFC and, potentially, other human neurocristopathies (e.g. neuroblastoma).