Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers

The importance of non-coding regions for the development of diseases have been increasingly recognized, although still largely unknown. Combining several high-throughput next-generation sequencing techniques in zebrafish we were able to find potential disease-relevant enhancers.

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The pancreas is an organ in which diseases with a major burden occur, such as diabetes or pancreatic cancer. Diabetes is a long-term condition with major impact on the quality of life of individuals, with increased risks for chronic complications and with high costs for society.1,2 It is among the top 10 causes of death in adults worldwide.1 Pancreatic cancer is an extremely aggressive disease, diagnosed at an advanced stage in most cases, whose incidence is increasing, and which is projected to become the second-leading cause of cancer-related mortality by 2030.3 A deep understanding of the gene regulation in the genetic networks that are at work in pancreas is crucial to better understand and treat these diseases.

DNA, other than genes, is composed of the so-called “non-coding regions” that are still mostly unknown but play a crucial role in the gene regulation of different tissues. This is what we are interested in finding out: which of these unknown regions are important and can affect gene expression at a point of causing disease in pancreas. We are specifically interested in enhancers, non-coding regions where transcription factors bind, being responsible for driving the expression of genes. Without them the genes they regulate are silenced. Obtaining normal pancreatic tissue from humans, the ideal sample for our research, is very difficult. Therefore, we used zebrafish to help us reach that purpose, since the genetic and physiological similarities of zebrafish and human pancreas4,5 are enough to establish parallels between the two species. With zebrafish we were able to obtain enough pancreatic material to apply multiple techniques such as ATAC-seq, H3K27ac ChIP-seq, 4C-seq and HiChIP-seq, through which we obtained a very thorough dataset on the regulatory networks working in pancreas. We then selected some of these regions and tested in vivo whether they were actually active in zebrafish pancreas, and were able to validate several pancreatic enhancers. We compared our data with similar human datasets and we were able to observe a similar pattern of H3K27ac in the regulatory regions of human genes, as well as similar transcription factor motifs which allowed us to establish a bridge between the regulome of both species, and to further identify the enhancers in human holding an equivalent role.

We identified one enhancer of arid1a, a gene that in humans is involved in several cancers, including pancreatic.6 We then identified the equivalent region on the human genome and deleted it in a human pancreatic cell line. We observed a reduction in the expression of the ARID1A gene, showing that this enhancer regulates the expression of this gene in human, which suggests that mutations in this non-coding region may increase the risk for pancreatic cancer. Another enhancer we selected regulated the expression of ptf1a, despite its distal localization from the gene. Ptf1a is a central gene necessary for the development of the pancreas, its absence causing pancreatic agenesis.7 In a previous study, the deletion of a distal enhancer of PTF1A was shown to cause pancreatic agenesis in humans.8 We deleted an equivalent distal enhancer of ptf1a in zebrafish and observed a severe effect on pancreatic development. The similarity between the phenotypes of the two species after the deletion of that enhancer gives us confidence in establishing the causality of this condition in vivo.

With this work, by using an animal model amenable to genetic manipulation, we established a correlation between the pancreatic regulatory networks in zebrafish and in human. With the combination of techniques we used, we could find and validate enhancers active in zebrafish pancreas, and identify the functionally equivalent regions in human. With this approach we established a bridge between the non-coding genome of both species, allowing the identification and study of non-coding regions potentially involved in human disease.

By Joana Teixeira and Renata Bordeira-Carriço, for the authors.

Photo credits: Marta Duque

  1. Saeedi, P. et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9 th edition. Diabetes Res Clin Pract. 157:107843 (2019).
  2. Lascar, N. et al. Type 2 diabetes in adolescents and young adults. Lancet Diabetes Endocrinol. 6(1):69-80 (2018).
  3. Park, W. et al. Pancreatic Cancer: A Review. JAMA. 326(9):851-862 (2021).
  4. Kinkel, M. D. & Prince, V. E. On the diabetic menu: Zebrafish as a model for pancreas development and function. Bioessays 31, 139–152 (2009).
  5. Prince, V. E., Anderson, R. M. & Dalgin, G. Zebrafish Pancreas Development and Regeneration: Fishing for Diabetes Therapies. Curr. Top. Dev. Biol. 124, 235–276 (2017).
  6. Jones, S. et al. Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types. Hum. Mutat. 33, 100–103 (2012).
  7. Sellick, G. S. et al. Mutations in PTF1A cause pancreatic and cerebellar agenesis. Nat Genet. 36(12):1301-5 (2004).
  8. Weedon, M. N. et al. Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis. Nat. Genet 46, 61–64 (2014).

Renata Bordeira-Carriço

Postdoctoral researcher, IBMC - I3S