Testing the cis-regulatory of type 2 diabets associated non-coding sequences

• Autor(a) principal: Eufrásio, Ana Catarina Macedo
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/25405
• topic: Type 2 diabetes; Endocrine islet; β-cells; Genome wide association studies; Single nucleotide polymorphism; Enhancer; Transcription factor

Type 2 diabetes (T2D) affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. This condition is partially characterized by endocrine pancreatic islet dysfunction, leading to insufficient insulin production. By now, genome-wide association studies have shown that some single nucleotide polymorphisms (SNPs) are associated to T2D. Part of these variants are located in non-coding sequences with marks for enhancer activity, and some of them overlap with binding sites of transcription factors (TFs) known to be required for proper endocrine pancreas function. Enhancers are non-coding sequences that regulate the expression of their target genes by interacting with their promoters in cis. Our working hypothesis is that T2D associated SNPs might impair TF binding, affecting the enhancer activity of the sequence, ultimately translating into transcriptional changes of the downstream genes. At first, to approach this hypothesis, we have performed in vivo transgenesis assays in zebrafish to test if sequences overlapping with T2D associated loci were enhancers. We found that five out of ten tested sequences are endocrine pancreas enhancers. Secondly, we analyzed the impact of the risk associated variant in the enhancer activity. We found that in one out of three sequences, the enhancer activity was disrupted by the presence of a single nucleotide modification in a putative binding site for PDX1, an important transcription factor in pancreas development. We further analyzed this sequence by dividing it in fragments, testing them for endocrine enhancer activity. These results lead us to conclude that most likely the loss of the PDX1 binding site is accompanied by the gain of a repressor binding site that might contribute to the inactivation of the tested enhancer. As future approaches, we will test the enhancer activity of the selected sequences in human beta cell lines and perform Circularized Chromosome Conformation Capture (4C-seq) to identify the enhancer’s target genes. Overall this project will help to better understand the importance of non-coding variants in the development of T2D.