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)).
