Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and regeneration

Abstract

Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure. We carried out two forward genetic screens for regulators of endodermal organ development. In the first, we screened for altered morphology of developing digestive organs, while in the second we screed for the lack of terminally differentiated cell types in the pancreas and liver. From these screens, we identified two mutant alleles of zebrafish dnmt1. Both lesions are predicted to eliminate dnmt1 function; one is a missense mutation in the catalytic domain and the other is a nonsense mutation that eliminates the catalytic domain. In zebrafish dnmt1 mutants, the pancreas and liver form normally, but begin to degenerate after 84 hours post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis are halted in the absence of detectable Dnmt1. However, endocrine cells and ducts are largely spared. Surprisingly, dnmt1 mutants and dnmt1 morpholino-injected larvae show increased capacity for pancreatic beta cell regeneration in an inducible model of pancreatic beta cell ablation. Thus, our data suggest that Dnmt1 is dispensable for pancreatic duct or endocrine cell formation, but not for acinar cell survival. In addition, Dnmt1 may influence the differentiation of pancreatic beta cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate.

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

“…The S-phase arrest in uhrf1 mutants is phenocopied by dnmt1 mutation dnmt1 mutants resemble uhrf1 mutants in that they also fail to undergo hepatic outgrowth (Anderson et al, 2009), and they have similar severe defects in the lens (Tittle et al, 2011). As previously reported (Tittle et al, 2011), the level of DNA methylation in 5 dpf dnmt1 and uhrf1 mutants was equivalent (Fig.…”

“…Several lines of evidence support the conclusion that DNA hypomethylation is the cause of cell cycle arrest in uhrf1 mutants: (1) zebrafish dnmt1 mutants phenocopy the defects in the liver (Anderson et al, 2009;Sadler et al, 2007) and lens (Tittle et al., 2011); (G) Quantification of left liver lobe size in 10-17 larvae on 5 dpf. Top and bottom of box plot designate 75% and 25% of the population, respectively, separated by the median; bars represent 10th and 90th percentiles.…”

“…Embryos were sorted for GFP expression within 4 h of heat shock; only the brightest embryos were assessed for liver size on 5 dpf. Dnmt1 s904 mutants (Anderson et al, 2009) were obtained from M. Goll (Memorial Sloan-Kettering Cancer Center, New York, USA). Tg(fabp10:dsRed) fish (Dong et al, 2007) were obtained from D. Stainier.…”

DNA Methylation in Mammalian and Non-Mammalian Organisms

“…It chimes with the situation in Xenopus, where certain key roles of DNMT1 do not depend on it methylating DNA (Dunican et al, 2008;Stancheva et al, 2001). Genetic analysis reinforces this difference, as zebrafish mutants in uhrf1 and dnmt1 also have defects in lens development and maintenance and they are not embryonic lethal, despite being hypomethylated (Anderson et al, 2009;Tittle et al, 2011). This suggests that attributing developmental defects in these mutants to hypomethylation may be premature, as the biology may be more complex.…”

Cellular Plasticity and Heterogeneity in Pancreatic Regeneration and Malignancy

“…Interestingly, loss of Dnmt1 leads to enhanced regeneration of pancreatic beta cells after targeted ablation. These early studies underscored the complex roles of epigenetic regulation in development and regeneration [54] . In injury, the epigenetic regulator Bmi1, a member of the polycomb repressor complex 1 (Prc1), is upregulated in acinar cells and coordinates regeneration in the exocrine compartment.…”