Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ZPR (The Pancreas Regulome: From causality to prediction of non-coding mutations in human pancreatic diseases)

Teaser

Several human pancreatic diseases have been characterized, being diabetes the most common. Like in others, Genome Wide Association Studies (GWAS) have pointed to diabetes risk alleles located in non-coding cis-regulatory elements (CREs) of the genome. This suggests that...

Summary

Several human pancreatic diseases have been characterized, being diabetes the most common. Like in others, Genome Wide Association Studies (GWAS) have pointed to diabetes risk alleles located in non-coding cis-regulatory elements (CREs) of the genome. This suggests that diabetes might also be explained by disrupted regulation of gene’s transcription, however, it’s still unclear how mutations on CREs contribute to disease. The translation from the “non-coding code” to phenotype is an exciting and unexplored field that we are approaching in this project with the help of the zebrafish as a suitable animal model. We aim to uncover the implications of the disruption of pancreas CREs and how they contribute to diabetes in vivo, a disease that has been increasing dramatically in prevalence and is predicted to affect 592 million worldwide by 2035. Diabetes pathogenesis is partially related with insufficient insulin production by the pancreas and pancreatic islet dysfunction. Understanding the islet genome regulation, could provide valuable mechanistic insights, in particular improving our understanding of the heritable factors that contribute to the development of this disease. This will be crucial for the demonstration and prediction of diabetes causative genetic alleles.
In this project we are studying the transcriptional regulation of genes (the regulome) in zebrafish. The similarities between zebrafish and mammal pancreas and the evolutionary conservation of pancreas transcription factors make it an excellent model to approach and study diabetes. We are characterizing the zebrafish insulin producing beta-cell regulome, by determining the active CREs, in particular enhancers, and their bound transcription factors. We are also comparing this information with a similar dataset recently available for human beta-cells, to define functional equivalent sequences (orthologs) in these species. Some of these enhancers, which human functional orthologs overlap with diabetes risk alleles, will be mutated in the zebrafish genome to demonstrate their causative effect in the development of diabetes like phenotypes. Other zebrafish enhancers will be tested by loss-of-function assays to identify new diabetes associated CREs. In summary, with this project we will create a model system that will allow the identification of diabetes-associated CREs, which might have a great impact in clinical management of this epidemic disease.

Work performed

In the first period of the ZPR project we have generated and expanded zebrafish reporter lines for different endocrine cell types. At the same time we started to implement techniques of ATAC-seq and CHIP-seq to identify active enhancers in the whole pancreas and in isolated endocrine pancreas (ATAC-seq). Using Hi-ChIP to associate enhancers to genes, we were able to demonstrate that the cluster of putative enhancers detected are highly associated to transcripts expressed in different pancreatic cell types, including the endocrine pancreas. We also found clusters of transcription factor binding sites to be highly enriched in the identified putative enhancers.
Using the preliminary draft of zebrafish pancreas regulome, we have tested about 25 sequences for enhancer activity in zebrafish. 5 of these sequences have shown to have enhancer activity in transient transgenics and we are now generating stable transgenic lines. At the same time we have cloned 11 human sequences that have epigenetic marks for enhancer activity overlapping with risk alleles for Type 2 Diabetes and we are currently performing enhancers assays in zebrafish. These assays will be complemented with luciferase assays in cell lines.
We were able to identify an ectopic insulator integration in the zebrafish genome that causes a reduced number of differentiated beta cells. We have screened the vicinity of the insulator for functional enhancers and we were able to identify 2 enhancers that might be blocked by this insulator. Other enhancers will be targeted during the development of this project.
We have performed 4C-seq experiments to identify the gene that is under the control of the potentially blocked enhancers. In addition, preliminary 4C-seq experiments suggest that the 2 identified enhancers are being disconnected from the promoter of the identified gene. Also, this gene is downregulated in the insulator integration mutant background, a result confirmed by in situ hybridization. We have characterized this mutant background, showing that the reduction of the number of insulin expressing cells is accompanied by an increase in the number of alfa cells. We have also developed a CRISPR/Cas9 mutant for the identified gene that we are currently analyzing, and we have recapitulated the loss of beta cells phenotype by using morpholinos for gene knockdown. Furthermore, we cloned and are currently screening stable transgenic lines for 12 putative enhancers from the human equivalent locus, to identify the functional orthologs to the ones uncovered in zebrafish.

Final results

In summary, the ZPR project envision to use the zebrafish as a vertebrate model organism to clarify the impact that non-coding regulatory mutations have in the development of diabetes. To reach this, we will go from the description of the zebrafish pancreas regulome, to find functional ortholog CREs between zebrafish and humans and finally understand the phenotypic relevance to diabetes when impairing these CREs. Because a group of CREs from a genomic landscape control the expression of a gene, the impairment of only one CRE is not equivalent to the complete loss-of-function of the target gene. Indeed, regulatory mutations might result in phenotypes qualitatively different from the phenotypes obtained from a complete loss-of-function of the affected gene. Because we will be using genome wide tools, by the end of this project an atlas of the zebrafish pancreas regulome will be available. This information can be used in the future for a predictive analysis of human genomic regions that might be susceptible for diabetes risk alleles. In this project we will test this hypothesis for a restricted number of cases. The screening of risk alleles in patients by focusing on these specific regions of interest (diabetes-associated enhancers) instead of whole-genome screening would be much more effective and faster. Therefore, defining a regulatory map of diabetes-associated CREs will open new avenues on the early detection and clinical management of diabetes.