Periodic Reporting for period 4 - ZPR (The Pancreas Regulome: From causality to prediction of non-coding mutations in human pancreatic diseases)
Periodo di rendicontazione: 2020-12-01 al 2021-12-31
Several human pancreatic diseases have been characterized, being diabetes the most common. Genome Wide Association Studies (GWAS) have uncovered many 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 the ZPR project, with the help of the zebrafish as a suitable animal model. The ZPR project aims 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 pancreas 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 approached the transcriptional regulation of genes in the zebrafish pancreas (the pancreas regulome). The similarities between zebrafish and mammal pancreas and the evolutionary conservation of pancreas transcription factors (TFs) make it an excellent model to approach and study diabetes. Overall, the main objectives of the ZPR project where reached. By combining chromatin profiling and interaction points with in vivo reporter assays in zebrafish, the ZPR project uncovered functionally equivalent human enhancers, helping to study, demonstrate and predict pancreatic disease-relevant enhancers.
In this project we approached the transcriptional regulation of genes in the zebrafish pancreas (the pancreas regulome). The similarities between zebrafish and mammal pancreas and the evolutionary conservation of pancreas transcription factors (TFs) make it an excellent model to approach and study diabetes. Overall, the main objectives of the ZPR project where reached. By combining chromatin profiling and interaction points with in vivo reporter assays in zebrafish, the ZPR project uncovered functionally equivalent human enhancers, helping to study, demonstrate and predict pancreatic disease-relevant enhancers.
In the ZPR project we have performed ATAC-seq and ChIP-seq to identify active enhancers in the zebrafish whole pancreas, and in isolated endocrine cell types (ATAC-seq). Using Hi-ChIP we associated enhancers to genes. Using the zebrafish pancreas regulome, we tested several sequences for enhancer activity validating them as pancreatic enhancers. We further confirmed these results performing mutagenesis for these enhancers, observing a downregulation of the respective target genes. One of such cases was in the regulatory landscape of ptf1a. In humans, this locus is known to contain an enhancer that when deleted causes pancreatic agenesis. Using the zebrafish pancreas regulome we found an equivalent ptf1a pancreatic enhancer, which deletion (doi:10.1016/j.xpro.2020.100208) generates pancreatic agenesis, as its human counterpart. We further identified a zebrafish pancreatic enhancer in the regulatory landscape of the tumor suppressor gene arid1ab. We demonstrated the existence of a human functional equivalent enhancer, which deletion results in a decreased expression of ARID1A, suggesting a potential role in the susceptibility to pancreatic cancer. These results suggest the existence of human/zebrafish functional orthologue CREs, helping to translate our results to human health (Bordeira-Carriço et al; accepted in principle, Nature Communications. Also:doi:10.1101/2020.04.27.064220; Duque et al(2021)FEBS Journal(doi:10.1111/febs.16075)).
Cloning several human sequences that have epigenetic marks for enhancer activity overlapping with risk alleles for Type 2 Diabetes(T2D), we have performed enhancers assays in zebrafish, showing that many of these sequences are endocrine pancreatic enhancers. For some of these enhancers, single nucleotide polymorphisms (SNP) associated to T2D can result in their dysregulation. Also, we found single nucleotide modifications able to ablate completely the activity of the human endocrine enhancer. One example was the rs13266634 SNP that locates in a SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, which is the canonical explanation for T2D risk association. However, other type 2 diabetes-associated SNPs that truncate SLC30A8 confer protection from this disease, contradicting this explanation. We show that rs13266634 boosts the activity of an overlapping enhancer, suggesting an SLC30A8 gain of function as the cause for the increased risk for the disease (Eufrasio et al(2020) Diabetes (doi:10.2337/db19-1049)).
In addition, we show that the loss-of-function of nog2 in zebrafish impairs beta-cell differentiation, suggesting that Nog2, a known Bmp inhibitor, might counteract the antagonistic role of Bmp in beta-cell differentiation. This pancreatic function derives from the expression of nog2 in the notochord, that is induced by at least one notochord enhancer and its loss-of-function is sufficient to impair beta-cell differentiation. Tracing Nog2 diffusion, we show that it co-localizes with pancreas progenitor cells. Finally, we found a notochord enhancer in the landscape of human and mice Nog genes. In summary, this work shows that the disruption of a nog2 notochord enhancer impairs endocrine pancreas development, resulting in a pancreatic disease associated phenotype. Despite lack of sequence conservation between mammal and zebrafish enhancers, we show potential equivalent functional roles, further consolidating the main findings of the ZPR project(Amorim et al(2020) Cell Reports (doi:10.1016/j.celrep.2020.107862)).
Cloning several human sequences that have epigenetic marks for enhancer activity overlapping with risk alleles for Type 2 Diabetes(T2D), we have performed enhancers assays in zebrafish, showing that many of these sequences are endocrine pancreatic enhancers. For some of these enhancers, single nucleotide polymorphisms (SNP) associated to T2D can result in their dysregulation. Also, we found single nucleotide modifications able to ablate completely the activity of the human endocrine enhancer. One example was the rs13266634 SNP that locates in a SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, which is the canonical explanation for T2D risk association. However, other type 2 diabetes-associated SNPs that truncate SLC30A8 confer protection from this disease, contradicting this explanation. We show that rs13266634 boosts the activity of an overlapping enhancer, suggesting an SLC30A8 gain of function as the cause for the increased risk for the disease (Eufrasio et al(2020) Diabetes (doi:10.2337/db19-1049)).
In addition, we show that the loss-of-function of nog2 in zebrafish impairs beta-cell differentiation, suggesting that Nog2, a known Bmp inhibitor, might counteract the antagonistic role of Bmp in beta-cell differentiation. This pancreatic function derives from the expression of nog2 in the notochord, that is induced by at least one notochord enhancer and its loss-of-function is sufficient to impair beta-cell differentiation. Tracing Nog2 diffusion, we show that it co-localizes with pancreas progenitor cells. Finally, we found a notochord enhancer in the landscape of human and mice Nog genes. In summary, this work shows that the disruption of a nog2 notochord enhancer impairs endocrine pancreas development, resulting in a pancreatic disease associated phenotype. Despite lack of sequence conservation between mammal and zebrafish enhancers, we show potential equivalent functional roles, further consolidating the main findings of the ZPR project(Amorim et al(2020) Cell Reports (doi:10.1016/j.celrep.2020.107862)).
The ZPR project established a proof-of-principle demonstrating that it is possible to identify zebrafish and human functional equivalent enhancers by analyzing chromatin profiles, bypassing the challenge of lack of sequence conservation. The ZPR project also demonstrated that in vivo functional assays in these enhancers can clarify the role of cis-regulatory mutations in the development of human pancreatic diseases, having also the potential to identify new disease associated enhancers. This is a great breakthrough, since the vast majority of alleles associated by GWAS to human diseases are located in the non-coding genome, many overlapping with putative enhancers. Studies focused in specific locus can now be performed to understand their specific contribution to the increased risk for T2D development, using datasets of chromatin profiling from zebrafish pancreatic cells and pipelines developed in the ZPR.