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Title: CHD7 in CHARGE : the implications of neural crest specific regulation of chromatin remodeller CHD7 in CHARGE syndrome
Author: Taylor, Özlem Günes
ISNI:       0000 0004 8502 8044
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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The neural crest is a multipotent embryonic progenitor population. Differentiated neural crest cells contribute to a remarkable range of tissues and structures including the peripheral nervous system, bone and cartilage of the head, cardiac septum and the major vessels of the heart. CHARGE syndrome (OMIM 214800) is a sporadic, dominant disorder characterised by a multitude of congenital aberrations; ocular coloboma, heart malformations, atresia of the choanae, retardation of growth, genital hypoplasia and ear abnormalities. CHARGE syndrome is considered a neurocristopathy as the perturbed structures are significantly patterned by, or contain contributions from, the neural crest. The majority of CHARGE patients carry heterozygous mutations in chromatin remodeller CHD7. Neural crest cells lacking CHD7 have reduced expression levels of key migration markers and migrate aberrantly both in vitro and in vivo. Therefore, CHARGE syndrome could be attributed to CHD7 deficient neural crest cell migration and implicates CHD7 as a critical neural crest factor. The neural crest has been extensively studied and a gene regulatory network detailing the hierarchical interactions between key genes compiled. Enhancers are cis-regulatory sites used by upstream transcription factors to promote downstream gene transcription. As such, enhancers define the hierarchical relationships between upstream transcription factor inputs and downstream gene outputs. Consequently, enhancers have been used to establish architecture of gene regulatory networks, including the neural crest gene regulatory network. To integrate CHD7 within the known neural crest gene regulatory network and establish its role within neural crest development, neural crest-specific enhancers of CHD7 and the factors utilizing them must be identified. Compellingly, 40% of CHARGE patients lack mutations within the exons of CHD7. Mutations in critical CHD7 cis-regulatory elements such as neural crest specific enhancers could impact CHD7 expression, accounting for these unexplained CHARGE syndrome cases. Ten putative cranial neural crest specific cis-regulatory sites for CHD7 were identified by differential DNA accessibility profiling. Of the ten putative CHD7 enhancers tested by in vivo fluorescent reporter activity, seven demonstrated enhancing capacity and were deemed novel CHD7 enhancers. An advanced molecular toolkit was developed to further characterise these novel CHD7 enhancers, and potentially allow integration into the cranial neural crest gene regulatory network. To accommodate the limited number of cranial neural crest cells per chicken embryo, the classical neural crest model system, transcription factor biotagging was translated for small cell number chromatin immunoprecipitation. Additionally, CRISPRCas9 based genome- and epigenome- editing tools were generated to test the novel enhancer placement within the network. A novel enhancer with strong cranial neural crest activity, Chd7_a, was bound by critical neural crest factors FoxD3 and Sox10. Mutations of predicted Sox10 binding sites within the novel enhancer reduced enhancing activity, as did the targeting of endogenous Sox10 by CRISPR-Cas9 genome editing. Epigenome editing of Chd7_a by repressive dCas9-KRAB activity reduced Chd7 expression levels and confirmed Chd7_a regulation of Chd7 in the chicken cranial neural crest. Functional conservation of the novel chicken Chd7 enhancer Chd7_a in humans was demonstrated by confirming enhancing activity in the equivalent CHD7_a region. Consequently, we propose chromatin remodeller Chd7 is critically regulated in the neural crest by tissue-specific transcription factors Sox10 and FoxD3 driven enhancer elements, and that perturbation of this regulation can account for the aetiology of CHARGE syndrome.
Supervisor: Sauka-Spengler, Tatjana Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available