Cis-regulatory similarities in the zebrafish and human pancreas uncover potential disease-related enhancers

Abstract

Introductory paragraphThe pancreas is a central organ for human diseases that have a dramatic societal burden, such as pancreatic cancer and diabetes 1,2 . Non-coding cis-regulatory elements (CREs) of DNA control gene expression 3,4 , being required for proper pancreas function. Most disease-associated alleles 5,6 are noncoding, often overlapping with CREs 5 , suggesting that alterations in these regulatory sequences contribute to human pancreatic diseases by impairing gene expression. However, functional testing of CREs in vivo is not fully explored. Here we analysed histone modifications, transcription, chromatin accessibility and interactions, to identify zebrafish pancreas CREs and their human functional equivalents, uncovering disease-associated sequences across species. We found a human pancreatic enhancer whose deletion impairs the tumour suppressor gene ARID1A expression, conferring a potential tumour suppressor role to this non-coding sequence. Additionally, we identified a zebrafish ptf1a distal enhancer which deletion generates pancreatic agenesis, demonstrating the causality of this condition in humans 7 and the interspecies functional equivalency of enhancers. ResultsThe pancreas of zebrafish, a vertebrate model suitable for genetic manipulation 8 , shares many similarities with the human pancreas, including transcription factors (TFs) operating in similar genetic networks of pancreas development and function 9,10 . We observed that these similarities are extensible to organ structure ( Fig.1a) and cellular composition (SupplementaryFig.1), suggesting that shared genetic networks might operate through equivalent sets of CREs in both species.To identify CREs active in the zebrafish adult pancreas, we performed ChIP-seq for H3K27ac 11 , a key histone modification associated to active enhancers and ATAC-seq 12 , to identify regions of open chromatin. We have also performed HiChIP 13 against H3K4me3 14 to determine active promoters interacting with the uncovered enhancers ( Fig.1b). We found 14753 putative active enhancers, mostly in intergenic regions (57.8%), and 23298 putative active promoters corresponding to 9848 genes ( Fig.1c; SupplementaryTable1-3). To identify a subset of pancreas enhancers with higher tissue-specificity, we asked which of them are inactive in whole embryos (dome to 48hpf), finding that 7115 (48.2%) are active only in the differentiated adult pancreas (PsE; Fig.1d; SupplementaryTable4) while the remaining 7638 (51.8%) are also active during embryonic development (DevE). Interestingly, DevE presented 4 clusters (C1-4) with different activity dynamics during development (Fig.1d; SupplementaryFig.2; SupplementaryTable5).Pancreatic enhancers should activate the expression of genes in the pancreas. To test this, we identified the nearest genes to each putative pancreas enhancer 15 , observing that genes nearby PsE are enriched for exocrine pancreas expression (p<4.27e-9; SupplementaryFig.3a; SupplementaryTable6-7), detected by in situ hybridization. These results contrast with DevE, ...

Ptf1a function and transcriptional cis‐regulation, a cornerstone in vertebrate pancreas development

“…However, the 3'-EA is not strictly required for the induction of Ptf1a expression, at least in the pancreas [78], and the existence of other downstream enhancers, active early in development and inactive in adult tissues, may indicate that additional regulatory elements are required to establish the Ptf1a levels needed for the activation of 5'-AR (Figure 6). In support of this theory, in humans, the distal h3'-DpE shows enhancer activity in early pancreatic progenitors and its deletion affects very early events of pancreas development, causing pancreatic agenesis [15], a phenotype recapitulated in zebrafish by the deletion of z3'-DpE [83]. These results suggest that these CREs may aid 3'-EA to establish initial proper Ptf1a levels to activate the 5'-AR mediated autofeedback loop.…”

“…This article is protected by copyright. All rights reserved agenesis [83]. Interestingly, h3'-DpE is active in human PPs but inactive in adult exocrine pancreatic cell lines [15], while z3'-DpE shows activity in zebrafish MPCs but remains active in the adult zebrafish exocrine pancreas [83].…”

“…Like in mouse, multiple sequences downstream of the zebrafish ptf1a gene display enhancer activity in the developing nervous system [78] (Fig. 5A) but, more recently, a zebrafish enhancer crucial for pancreas development was uncovered, 39kb downstream of the ptf1a TSS [83]. This ptf1a distal pancreatic enhancer (z3′‐DpE; Fig.…”

“…5A) is active in MPCs and differentiated acinar and duct cells. Similar to the 5′‐AR, z3′‐DpE‐driven reporter expression is weaker in progenitors and stronger in differentiated exocrine cells and the sequence interacts with the ptf1a promoter, suggesting that the z3′‐DpE also regulates ptf1a expression directly [83]. However, unlike the z5′‐AR, the z3′‐DpE does not contain PTF1 consensus binding sequences.…”

“…Despite showing no sequence conservation with the human enhancer (Fig. 5A), z3′‐DpE is likely functionally equivalent to h3′‐DpE, with deletion of z3′‐DpE resulting in pancreatic agenesis [83]. Interestingly, h3′‐DpE is active in human MPCs but inactive in adult exocrine pancreatic cell lines [15], while z3′‐DpE shows activity in zebrafish MPCs but remains active in the adult zebrafish exocrine pancreas [83].…”