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Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors [1]

['Ruth M. Williams', 'Stowers Institute For Medical Research', 'Kansas City', 'Missouri', 'United States Of America', 'University Of Oxford', 'Mrc Weatherall Institute Of Molecular Medicine', 'Radcliffe Department Of Medicine', 'Oxford', 'United Kingdom']

Date: 2024-10

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

Funding: This work was supported by the Wellcome Trust (215615/Z/19/Z) and institutional support from the Stowers Institute for Medical Research to TSS. Human embryonic multiome datasets were generated for a different study at the University of Oxford by DMF and funded by CRUK Doctoral Training Programme (BST00230-H400.01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we identify multiple novel enhancers driving Chd7 expression in developing chick embryos. We validate enhancer activity in vivo and functionally determine key upstream transcription factors mediating Chd7 enhancer activity in the neural crest. Using human embryonic chromatin accessibility data, we find that neural crest-specific CHD7 enhancers are highly conserved. Furthermore, we demonstrate that human enhancers are active in developing chick embryos, suggesting that the tissue-specific regulatory mechanisms enhancing Chd7 in the neural crest are conserved between chicken and human. Our findings provide a potential mechanism to explain the aetiology of CHARGE syndrome where the Chd7 gene itself is unperturbed. Finally, we demonstrate the upstream regulation of a chromatin-remodeller by tissue-specific transcription factors as an important mechanism to ensure enhanced chromatin remodelling activity in the developing neural crest.

Previous studies have shown that CHD7 function is essential for proper neural crest development and migration. In cell models of human neural crest cells, CHD7 occupies distal regulatory elements for neural crest transcription factors SOX9 and TWIST1 [ 26 ]. In mice, Chd7 heterozygotes present with CHARGE syndrome-like features [ 27 – 29 ] and trunk neural crest cells require Chd7 to maintain their multipotency [ 30 , 31 ]. In Xenopus, Chd7 mutant embryos have reduced Sox9, Twist1, and Snai2 expression and display CHARGE syndrome-like features [ 26 ]. Recent work in the chick neural crest, employing weighted gene co-expression network analysis (WGCNA) analysis [ 32 ], showed that Chd7 expression strongly correlated with expression of neural crest regulators (Sox5, Sox9, Zeb2, and NeuroD4), other chromatin remodellers (Kdm1B, Kdm2A, Kdm3B, Kdm7A) and Semaphorins (Sema3A, Sema 3E, Sema4D, Sema6D) which have previously been shown to be regulated by Chd7 in mice [ 33 ]. Collectively, these findings provide strong evidence for positioning Chd7 within the neural crest gene regulatory network. However, the regulatory mechanisms governing Chd7 up-regulation in the neural crest have not been investigated. Since CHD7 is a component of the general cellular epigenetic machinery and is broadly expressed in a multitude of embryonic tissues, it could be assumed that its expression is governed by basal, proximal enhancers; however, the clinical features of CHARGE syndrome clearly suggest CHD7 has tissue and developmental stage-specific roles [ 3 ].

CHARGE syndrome (OMIM 214800) individuals present with ocular Coloboma, Heart malformations, choanal Atresia, Retardation of growth, Genital hypoplasia, and Ear abnormalities. Over 500 different pathogenic variants of CHD7 have been described, accounting for >90% of CHARGE syndrome cases [ 18 , 19 ]. However, in routine clinical screening, mutations in Chd7 are only detected in 32% to 41% of suspected CHARGE cases [ 18 ]. Pathogenic variants have been reported throughout the CHD7 gene body, indicating premature termination of the protein is significantly detrimental to CHD7 function [ 18 – 22 ]. CHD7 influences gene regulation [ 18 , 23 ] by catalysing nucleosome repositioning in an ATP-dependent manner [ 24 ]. In keeping with this regulatory role, CHD7 associates with distal regulatory sites carrying H3K4me1 chromatin modifications characteristic of poised enhancers in mouse embryonic stem cells [ 25 ].

The neural crest is a transient and migratory embryonic progenitor population that contributes to a remarkable range of neural and mesenchymal tissues in the vertebrate body. Neural-neural crest derivatives include facial ganglia, and both neurons and glia of the peripheral and enteric nervous systems. Mesenchymal neural crest derivatives include craniofacial cartilage and bone, as well as smooth muscle of facial blood vessels, striated muscle forming the cardiac outflow tract and septal, and the majority of the body’s pigment cells. Consequently, errors in neural crest patterning, migration, and differentiation result in a wide range of congenital birth anomalies collectively termed neurocristopathies. Neurocristopathies account for almost one third of all birth defects [ 1 ]. These include CHARGE syndrome, which affects the eye, heart, and facial structures [ 2 , 3 ]; Hirschsprung’s disease, characterised by the loss of neural crest-derived enteric ganglia [ 4 , 5 ]; Waardenburg syndrome, characterised by deafness, pigmentation, and craniofacial defects [ 6 ]; and Treacher Collins syndrome presenting with craniofacial defects [ 7 , 8 ]. Neurocristopathies can be caused by pathogenic variants of master neural crest regulators, for example, SOX10 in Hirschsprung’s disease and Waardenburg syndrome [ 9 – 13 ] or PAX3 in Waardenburg syndrome [ 14 – 16 ]. However, mutations in genes encoding general cellular machinery can also result in neurocristopathies as demonstrated in Treacher Collins syndrome caused by pathogenic variants of RNA polymerase I [ 8 ] and CHARGE syndrome where heterozygous mutations in the chromatin remodeller CHD7 (chromodomain helicase DNA binding protein 7) are detected [ 17 ].

Results

Chd7 expression during early chick development Chd7 was previously shown to be enriched in bulk RNA-seq data from chick cranial neural crest cells [32] where it was co-expressed with other neural crest genes and clustered with pre-migratory neural crest markers https://livedataoxford.shinyapps.io/Chick_NC_GRN-TSS-Lab/. Here, we used fluorescent in situ hybridisation (hybridisation chain reaction, HCR) [34] to resolve spatiotemporal Chd7 expression in developing chicken embryos. Chd7 was first detected at HH8 [35] within the cranial neural tube and pre-migratory neural crest cells as indicated by colocalisation with the neural crest marker Sox10 and at lower levels in the surrounding neuroectoderm (Fig 1A). Chd7 transcripts continued to overlap with Sox10 in delaminating and migrating neural crest cells at HH9/10 and were also detected in the neuroectoderm, forebrain, and neural tube at the vagal level from HH9 through HH15 (Fig 1A and 1B). At later stages (HH13—HH15) Chd7 was more broadly expressed, with transcripts distributed across the head regions (midbrain and hindbrain) including the trigeminal ganglia, developing face mesenchyme, eye, and otic vesicle as well as some vagal neural crest cells. In addition, at HH15, pharyngeal arches and dorsal root ganglion cells were also Chd7 positive (Fig 1B). Quantification of the HCR signal from Chd7 transcripts revealed approximately 85% of DAPI positive cells expressed Chd7, compared to 10% of cells that were Chd7 and Sox10 double positive (Fig 1C). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Chd7 expression during early chick development. (A, B) In vivo Chd7 expression (green) determined using HCR. Sox10 (magenta) is used as a neural crest marker. (A) Chd7 is expressed in neural crest cells and throughout the neural tube, as well as the surrounding ectoderm and underlying mesoderm at HH8-10. (B) Chd7 is more broadly expressed at HH13-HH15 including head and hindbrain structures. From HH13 through HH15 Chd7 expression is detected in the otic vesicle (ov), trigeminal ganglia (tg) eye, and vagal neural crest (vNC). At HH15 Chd7 is also expressed in the pharyngeal arches 1–4 (pa1-4) and dorsal root ganglion (drg). (C) Quantification of HCR signal from Chd7 and Sox10 transcripts in stage HH10 chick embryos (n = 4). (D) UMAP representation of 5,669 single cells resolved into 12 clusters of based on shared transcriptional identities. (E) Feature plots of Chd7 and Sox10 expression across scRNA-seq clusters. (F) Violin plots of selected chromatin remodellers and transcription factors expressed across scRNA-seq clusters. HCR, hybridisation chain reaction. https://doi.org/10.1371/journal.pbio.3002786.g001 To gauge the dynamics of Chd7 expression in individual cells in the entire embryo, we performed single-cell RNA-seq using the 10X Genomics 3’ scRNA-seq platform. We dissected the anterior half of 10 HH10 chick embryos (Fig 1D) and obtained 5,669 single-cell transcriptomes, resolved into 12 clusters (Fig 1E). Chd7 expression was detected in all clusters (Fig 1F), comparable with other chromatin remodellers (Figs 1G and S1A) and in contrast to cluster-specific expression of bona fide transcription factors (Figs 1F, 1H, and S1B). Crucially, this suggests the inherent differences in the regulatory mechanisms controlling the dynamic expression of tissue-specific transcription factors and more broadly expressed chromatin regulators.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002786

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