Analysis of non-­coding functional elements of gene in zebrafish and Epigenetic studies in a human developmental disorder

Project 1: Cis-regulatory regions such as enhancers are associated with regulating transcription of target genes. Many of these regulatory regions are distally located over 1kb away from their target promoter, and many studies been conducted to identify how enhancer and promoter regions are brought into proximity to allow interaction and initiation of transcription. Such research has identified protein complexes such as SAGA and ATCA, which bind to specific enhancers and promoters bringing them into close proximity. This study focuses on one unique subunit of the ATAC complex, zzz3. By knocking down expression of zzz3 in zebrafish by morpholino injection, the role zzz3 plays in development and phenotype can be determined. It was observed during this study that zzz3 knockdown resulted in a bent tail and ventralisation of embryos, suggesting a potential role for this protein in the development of the floor plate and notochord. Project 2: Genomic imprinting has long been implicated as a mechanism of growth regulation. Though most genes are expressed by both alleles, around 100 genes have been classified as imprinted and therefore one allele is preferentially expressed depending on the epigenetic profile of the maternal or paternal allele, resulting in a balance between growth promoting and growth repressing genes. Defects in the epigenetic profiles or simply over expression of one of these imprinted genes, results in developmental disorders such as Prader-Willi/Angelman syndrome (Ch15q11) and Beckwith-Wiedemann (Ch11p15)/Silver Russell syndrome (SRS) depending which allele is affected. This study focused on SRS, a disorder associated with pre- and postnatal growth retardation, investigating whether epimutations at six imprinted loci other than 11p15 had any affect on the disease. By utilising a novel methyl quantative PCR assay the methylation state of PLAGL1, IGF2R, GRB10, SNRPN, PEG3 and GNAS was determined in 40 SRS patients and 26 non-SRS laboratory controls and identified potential epimutations in 30% of the SRS individuals at PLAGL1, IGF2R and GRB10