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

Abstract

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the rereplicating and arrested hepatocytes. Importantly, the DNA rereplication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis.

Zebrafish: An Important Tool for Liver Disease Research

“…The epigenetic regulator encoded by uhrf1 was found to regulate embryonic liver growth and regeneration, based on the finding that the liver of uhrf1 +/− adults did not fully regrow after resection. 139 This gene is also required for hepatocyte proliferation in embryos 139,141 and, as discussed previously, overexpression of UHRF1 in zebrafish hepatocytes led to development of HCC. 106 UHRF1 therefore appears to regulate hepatocyte proliferation in response to physiological (development and regeneration) and carcinogenic stimuli.…”

UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development

“…Protein was isolated from embryos collected in 2 µL of protein lysis buffer (50 mM Tris pH 6.8, 2% SDS, 5% Glycerol, 1% 2-Mercaptoethanol) containing protease inhibitor cocktail per embryo and homogenized by sonication as described (Jacob et al ., 2015). Soluble protein extract volumes from 6 hpf embryos equivalent to 2 embryos were loaded and run on 8% – 12% SDS-PAGE gels, transferred to nitrocellulose or PVDF membrane and blotted with antibodies to Myc-UHRF1 (1:1000 anti-Myc, Sigma-Aldrich), Dnmt1 (1:2000, Santa Cruz), and α-Tubulin (1:4000, Developmental Studies Hybridoma Bank).…”

“…UHRF1 then recruits DNMT1 to methylate newly synthesized cytosine residues of the daughter strand during DNA replication (Arita et al, 2008; Avvakumov et al, 2008; Bostick et al, 2007; Hashimoto et al, 2008; Qian et al, 2008; Sharif et al, 2007). In all cases studied, loss of either UHRF1 (Feng et al, 2010) or DNMT1 (Jackson-Grusby et al, 2001; Jacob et al, 2015) result in global DNA hypomethylation. In addition, UHRF1 is also essential for DNMT1 specificity, activity, and protein stability.…”

“…Loss of UHRF1 or DNMT1 is catastrophic for embryos, as null mutation in either gene causes early embryonic lethality (Bostick et al, 2007; Jacob et al, 2015; Lei et al, 1996; Muto et al, 2002; Sadler et al, 2007; Sharif et al, 2007) and knockdown of both maternal and zygotic Uhrf1 in zebrafish embryos causes death prior to gastrulation (Chu et al, 2012). However, hypomorphic mutations cause post-natal defects and cancer in mice (Gaudet et al, 2004) and multi-systemic defects resulting in lethality late in zebrafish development (Anderson et al, 2009; Jacob et al, 2015; Sadler et al, 2007; Tittle et al, 2011). Therefore, while it is clear that epigenetics underlies nearly every aspect of embryogenesis, whether the requirement for Uhrf1 and Dnmt1 during development can be solely ascribed to their role in DNA methylation has not been addressed.…”

The lure of zebrafish in liver research: regulation of hepatic growth in development and regeneration

“…Some zebrafish mutants such as def [46,47], elys [48,49], ssrp1a [50] and bms1l [51] exhibit liver hypoplasia due to nucleolar defects that culminate in replication stress or a DNA damage response. In similar work, Sadler and colleagues have determined that uhrf1 mutants developed a small-for-size liver during development due to DNA hypomethylation, which leads to cell cycle arrest and the induction of apoptosis[52-55]. Several genes that are highly enriched in the developing liver such as matrix metalloproteinase mmp23b [56], as well as the secretory pathway components leg1 [57,58] and sec13 [59] are required for hepatocyte proliferation.…”