Dnmt1 Mutant Research Articles

DNA hypomethylation activates Cdk4/6 and Atr to induce DNA replication and cell cycle arrest to constrain liver outgrowth in zebrafish.

Coordinating epigenomic inheritance and cell cycle progression is essential for organogenesis. UHRF1 connects these functions during development by facilitating maintenance of DNA methylation and cell cycle progression. Here, we provide evidence resolving the paradoxical phenotype of uhrf1 mutant zebrafish embryos which have activation of pro-proliferative genes and increased number of hepatocytes in S-phase, but the liver fails to grow. We uncover decreased Cdkn2a/b and persistent Cdk4/6 activation as the mechanism driving uhrf1 mutant hepatocytes into S-phase. This induces replication stress, DNA damage and Atr activation. Palbociclib treatment of uhrf1 mutants prevented aberrant S-phase entry, reduced DNA damage, and rescued most cellular and developmental phenotypes, but it did not rescue DNA hypomethylation, transposon expression or the interferon response. Inhibiting Atr reduced DNA replication and increased liver size in uhrf1 mutants, suggesting that Atr activation leads to dormant origin firing and prevents hepatocyte proliferation. Cdkn2a/b was downregulated pro-proliferative genes were also induced in a Cdk4/6 dependent fashion in the liver of dnmt1 mutants, suggesting DNA hypomethylation as a mechanism of Cdk4/6 activation during development. This shows that the developmental defects caused by DNA hypomethylation are attributed to persistent Cdk4/6 activation, DNA replication stress, dormant origin firing and cell cycle inhibition.

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

DNMT1 mutant ants develop normally but have disrupted oogenesis

Although DNA methylation is an important gene regulatory mechanism in mammals, its function in arthropods remains poorly understood. Studies in eusocial insects have argued for its role in caste development by regulating gene expression and splicing. However, such findings are not always consistent across studies, and have therefore remained controversial. Here we use CRISPR/Cas9 to mutate the maintenance DNA methyltransferase DNMT1 in the clonal raider ant, Ooceraea biroi. Mutants have greatly reduced DNA methylation, but no obvious developmental phenotypes, demonstrating that, unlike mammals, ants can undergo normal development without DNMT1 or DNA methylation. Additionally, we find no evidence of DNA methylation regulating caste development. However, mutants are sterile, whereas in wild-type ants, DNMT1 is localized to the ovaries and maternally provisioned into nascent oocytes. This supports the idea that DNMT1 plays a crucial but unknown role in the insect germline.

Rngtt governs biliary-derived liver regeneration initiation by transcriptional regulation of mTORC1 and Dnmt1 in zebrafish.

In cases of end-stage liver diseases, the proliferation of existing hepatocytes is compromised, a feature of human chronic liver disease, in which most hepatocytes are dysfunctional. So far, liver transplantation represents the only curative therapeutic solution for advanced liver diseases, and the shortage of donor organs leads to high morbidity and mortality worldwide. The promising treatment is to prompt the biliary epithelial cells (BECs) transdifferentiation. However, the critical factors governing the initiation of BEC-derived liver regeneration are largely unknown. The zebrafish has advantages in large-scale genetic screens to identify the critical factors involved in liver regeneration. Here, we combined N-ethyl-N-nitrosourea screen, positional cloning, transgenic lines, antibody staining, and in situ hybridization methods and identified a liver regeneration defect mutant ( lrd ) using the zebrafish extensive liver injury model. Through positional cloning and genomic sequencing, we mapped the mutation site to rngtt . Loss of rngtt leads to the defects of BEC dedifferentiation, bipotential progenitor cell activation, and cell proliferation in the initiation stage of liver regeneration. The transdifferentiation from BECs to hepatocytes did not occur even at the late stage of liver regeneration. Mechanically, Rngtt transcriptionally regulates the attachment of mRNA cap to mTOR complex 1 (mTORC1) components and dnmt1 to maintain the activation of mTORC1 and DNA methylation in BECs after severe liver injury and prompt BEC to hepatocyte conversion. Furthermore, rptor and dnmt1 mutants displayed the same liver regeneration defects as rngtt mutation. In conclusion, our results suggest Rngtt is a new factor that initiates BEC-derived liver regeneration.

Epigenetic Protection of Vertebrate Lymphoid Progenitor Cells by Dnmt1.

SummaryDNA methylation is a universal epigenetic mechanism involved in regulation of gene expression and genome stability. The DNA maintenance methylase DNMT1 ensures that DNA methylation patterns are faithfully transmitted to daughter cells during cell division. Because loss of DNMT1 is lethal, a pan-organismic analysis of DNMT1 function is lacking. We identified new recessive dnmt1 alleles in medaka and zebrafish and, guided by the structures of mutant proteins, generated a recessive variant of mouse Dnmt1. Each of the three missense mutations studied here distorts the catalytic pocket and reduces enzymatic activity. Because all three DNMT1 mutant animals are viable, it was possible to examine their phenotypes throughout life. The consequences of genome-wide hypomethylation of DNA of somatic tissues in the Dnmt1 mutants are surprisingly mild but consistently affect the development of the lymphoid lineage. Our findings indicate that developing lymphocytes in vertebrates are sensitive to perturbations of DNA maintenance methylation.

Epigenetic factors Dnmt1 and Uhrf1 coordinate intestinal development

Intestinal tract development is a coordinated process involving signaling among the progenitors and developing cells from all three germ layers. Development of endoderm-derived intestinal epithelium has been shown to depend on epigenetic modifications, but whether that is also the case for intestinal tract cell types from other germ layers remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced numbers of ectoderm-derived enteric neurons and severe disruption of mesoderm-derived intestinal smooth muscle. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron precursors and cell-non-autonomously in surrounding intestinal cells, consistent with what is known about signaling interactions between these cell types that promote one another’s development. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neurons and smooth muscle progenitors. dnmt1 mutants have fewer enteric neurons and disrupted intestinal smooth muscle compared to wildtypes. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important to coordinate intestinal tract development, provides the first demonstration that these genes influence development of the ENS, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.

Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1 + cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.

In vivo replacement of damaged bladder urothelium by Wolffian duct epithelial cells

The bladder's remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.

Loss of DNA methylation in zebrafish embryos activates retrotransposons to trigger antiviral signaling.

Complex cytoplasmic nucleotide-sensing mechanisms can recognize foreign DNA based on a lack of methylation and initiate an immune response to clear the infection. Zebrafish embryos with global DNA hypomethylation caused by mutations in the ubiquitin-like with PHD and ring finger domains 1 (uhrf1) or DNA methyltransferase 1 (dnmt1) genes exhibit a robust interferon induction characteristic of the first line of defense against viral infection. We found that this interferon induction occurred in non-immune cells and examined whether intracellular viral sensing pathways in these cells were the trigger. RNA-seq analysis of uhrf1 and dnmt1 mutants revealed widespread induction of Class I retrotransposons and activation of cytoplasmic DNA viral sensors. Attenuating Sting, phosphorylated Tbk1 and, importantly, blocking reverse transcriptase activity suppressed the expression of interferon genes in uhrf1 mutants. Thus, activation of transposons in cells with global DNA hypomethylation mimics a viral infection by activating cytoplasmic DNA sensors. This suggests that antiviral pathways serve as surveillance of cells that have derepressed intragenomic parasites due to DNA hypomethylation.

Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells.

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate their roles and genomic targets. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets.

DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish.

BackgroundDNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored.MethodsAn N-ethyl-N-nitrosourea (ENU)-based genetic screening was performed to identify putative mutants with defects in definitive HSPCs during hematopoiesis in zebrafish. The expression of hematopoietic markers was analyzed via whole mount in situ hybridization assay (WISH). Positional cloning approach was carried out to identify the gene responsible for the defective definitive hematopoiesis in the mutants. Analyses of the mechanism were conducted by morpholino-mediated gene knockdown, mRNA injection rescue assays, anti-phosphorylated histone H3 (pH3) immunostaining and TUNEL assay, quantitative real-time PCR, and bisulfite sequencing analysis.ResultsA heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced in dnmt1 which resulted in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha (C/ebpa) was upregulated, which correlated with hypomethylation of CpG islands in the regulation regions of cebpa gene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in the dnmt1 mutants. Finally, dnmt1 and cebpa double null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered by dnmt1 mutation were C/ebpa dependent.ConclusionsDnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0115-7) contains supplementary material, which is available to authorized users.

The DNA Methyltransferase Dnmt1 Directly Interacts with the SET and RING Finger-associated (SRA) Domain of the Multifunctional Protein Uhrf1 to Facilitate Accession of the Catalytic Center to Hemi-methylated DNA

Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.

Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.

Ubiquitination/deubiquitination and acetylation/deacetylation: Making DNMT1 stability more coordinated

In mammals, DNA methylation plays a crucial role in the regulation of gene expression, telomere length, cell differentiation, X chromosome inactivation, genomic imprinting and tumorigenesis1. DNA methylation patterns are established de novo by DNA methyltransferases (DNMTs) 3a and 3b, whereas DNMT1 maintains the parent-specific methylation from parental cells to their progeny2. After DNA replication, the new DNA strand is unmethylated. Thus with the mother methylated strand, the DNA is hemimethylated. The protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) recognizes and binds to the hemimethylated sites by its SRA domain. Then DNMT1 is recruited to the sites by the same domain, thereby it methylates the newly synthesized DNA strand3, 4. Increased DNMT1 abundance has been seen in many human cancers, and the roles of DNMT1 in tumorigenesis have been shown by some genetic knockout and RNA interference-mediated knockdown studies5, 6. Seeing that the mRNA abundance of DNMT1 contributes less to DNMT1 abundance, the stability of DNMT1 protein therefore plays an important role in human cancers7, 8. Ubiquitin-proteasome pathway is significant in the stability of DNMT18, but ubiquitin-mediated protein degradation can be enhanced or attenuated by some modifications like acetylation/deacetylation, protein methylation/demethylation, phosphorylation and S-nitrosylation9, 10, 11. Esteve et al demonstrated that SET7-mediated lysine methylation of DNMT1 decreased DNMT1 level by ubiquitin-mediated degradation10. Furthermore, an early study12 showed that HDAC inhibitors could induce degradation of DNMT1. These suggest that DNMT1-associated proteins may play key roles in DNMT1 stability. In a recent issue of Science Signaling, Du et al reported13 that some DNMT1-associated proteins can ubiquitinate/deubiquitinate and acetylate/deacetylate DNMT1. These modifications make the regulation of DNMT1 stability more coordinated (Figure 1). Figure 1 A model of posttranslational regulation of DNMT1 stability13. DNMT1 physically interacts with HAUSP, Tip60, UHRF1, HDAC1, and PCNA on chromatin. After the completion of DNA methylation in S phase, HAUSP dissociates from DNMT1 to enable DNMT1 degradation. ... They identified deubiquitinase HAUSP (herpesvirus-associated ubiquitin specific protease) as a DNMT1-associated protein. Knockout or knockdown of HAUSP increased DNMT1 ubiquitination and reduced its abundance, then they demonstrated the direct deubiquitination of DNMT1 in vitro by purified HAUSP recombinant proteins. They also found that overexpression of DNMT1-associated E3 ligase UHRF1 led to increased ubiquitination of DNMT1 and decreased abundance of DNMT1 mutant lacking the HAUSP interaction domain, but not the full-length protein. These results show the coordination between ubiquitination of DNMT1 by UHRF1 and deubiquitination by HAUSP. Furthermore, they found that knockdown of HDAC1 increased DNMT1 acetylation, and reduced DNMT1 abundance. Additionally, acetyltransferase Tip60 which was found to acetylate DNMT1 promoted its ubiquitination, then destabilized it. At last, Tip60 and HAUSP were found to regulate DNMT1 protein stability during the cell cycle. The following clinical samples of human colon cancer also revealed the correlation between the abundance of HAUSP and the abundance of DNMT1. Drugs targeting epigenetic modifications have been edging toward anticancer therapies. DNMT1 also provides a candidate anti-cancer target14. Although global genomic DNA hypomethylation and tumor suppressor genes hypermethylation have been frequently observed in different human cancers, this methylation change is not caused simply by increased levels of DNMT115. This paper shows that DNMT1, HAUSP, UHRF1, Tip60, HDAC1, and PCNA (proliferating cell nuclear antigen) can interact with each other, thus they regulate DNMT1 stability and activity by a more coordinated way. Consequently, the current or future epigenetic drugs such as 5-aza CdR (DNMT1 inhibitor), HDAC inhibitors, HAUSP inhibitors, and UHRF1 inhibitors may have a potential combination for effective cancer therapy. And equally, the drug interactions and side effects should be taken into account, as one of the inhibitors may affect the other targets.

Impact of Genomic Methylation on Radiation Sensitivity of Colorectal Carcinoma

To investigate the influence of demethylation with 5-aza-cytidine (AZA) on radiation sensitivity and to define the intrinsic radiation sensitivity of methylation deficient colorectal carcinoma cells. Radiation sensitizing effects of AZA were investigated in four colorectal carcinoma cell lines (HCT116, SW480, L174 T, Co115), defining influence of AZA on proliferation, clonogenic survival, and cell cycling with or without ionizing radiation. The methylation status for cancer or DNA damage response-related genes silenced by promoter methylation was determined. The effect of deletion of the potential target genes (DNMT1, DNMT3b, and double mutants) on radiation sensitivity was analyzed. AZA showed radiation sensitizing properties at >or=1 micromol/l, a concentration that does not interfere with the cell cycle by itself, in all four tested cell lines with a sensitivity-enhancing ratio (SER) of 1.6 to 2.1 (confidence interval [CI] 0.9-3.3). AZA successfully demethylated promoters of p16 and hMLH1, genes associated with ionizing radiation response. Prolonged exposure to low-dose AZA resulted in sustained radiosensitivity if associated with persistent genomic hypomethylation after recovery from AZA. Compared with maternal HCT116 cells, DNMT3b-defcient deficient cells were more sensitive to radiation with a SER of 2.0 (CI 0.9-2.1; p = 0.03), and DNMT3b/DNMT1-/- double-deficient cells showed a SER of 1.6 (CI 0.5-2.7; p = 0.09). AZA-induced genomic hypomethylation results in enhanced radiation sensitivity in colorectal carcinoma. The mediators leading to sensitization remain unknown. Defining the specific factors associated with radiation sensitization after genomic demethylation may open the way to better targeting for the purpose of radiation sensitization.

Identification of a region of the DNMT1 methyltransferase that regulates the maintenance of genomic imprints

Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.

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

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 h post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis is 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.

Transcriptional Silencing and Reactivation in Transgenic Zebrafish

Epigenetic regulation of transcriptional silencing is essential for normal development. Despite its importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in developing vertebrates. We describe a transgenic approach that allows monitoring of an epigenetically regulated fluorescent reporter in developing zebrafish and their progeny. Using a self-reporting Gal4-VP16 gene/enhancer trap vector, we isolated tissue-specific drivers that regulate expression of the green fluorescent protein (GFP) gene through a multicopy, upstream activator sequence (UAS). Transgenic larvae initially exhibit robust fluorescence (GFP(high)); however, in subsequent generations, gfp expression is mosaic (GFP(low)) or entirely absent (GFP(off)), despite continued Gal4-VP16 activity. We find that transcriptional repression is heritable and correlated with methylation of the multicopy UAS. Silenced transgenes can be reactivated by increasing Gal4-VP16 levels or in DNA methyltransferase-1 (dnmt1) mutants. Strikingly, in dnmt1 homozygous mutants, reactivation of gfp expression occurs in a reproducible subset of cells, raising the possibility of different sensitivities or alternative silencing mechanisms in discrete cell populations. The results demonstrate the power of the zebrafish system for in vivo monitoring of epigenetic processes using a genetic approach.

DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation

DNA methylation is a major epigenetic factor regulating genome reprogramming, cell differentiation and developmental gene expression. To understand the role of DNA methylation in central nervous system (CNS) neurons, we generated conditional Dnmt1 mutant mice that possess approximately 90% hypomethylated cortical and hippocampal cells in the dorsal forebrain from E13.5 on. The mutant mice were viable with a normal lifespan, but displayed severe neuronal cell death between E14.5 and three weeks postnatally. Accompanied with the striking cortical and hippocampal degeneration, adult mutant mice exhibited neurobehavioral defects in learning and memory in adulthood. Unexpectedly, a fraction of Dnmt1(-/-) cortical neurons survived throughout postnatal development, so that the residual cortex in mutant mice contained 20-30% of hypomethylated neurons across the lifespan. Hypomethylated excitatory neurons exhibited multiple defects in postnatal maturation including abnormal dendritic arborization and impaired neuronal excitability. The mutant phenotypes are coupled with deregulation of those genes involved in neuronal layer-specification, cell death and the function of ion channels. Our results suggest that DNA methylation, through its role in modulating neuronal gene expression, plays multiple roles in regulating cell survival and neuronal maturation in the CNS.

Parental Effect of DNA (Cytosine-5) Methyltransferase 1 on Grandparental-Origin-Dependent Transmission Ratio Distortion in Mouse Crosses and Human Families

Transmission ratio distortion (TRD) is a deviation from the expected Mendelian 1:1 ratio of alleles transmitted from parents to offspring and may arise by different mechanisms. Earlier we described a grandparental-origin-dependent sex-of-offspring-specific TRD of maternal chromosome 12 alleles closely linked to an imprinted region and hypothesized that it resulted from imprint resetting errors in the maternal germline. Here, we report that the genotype of the parents for loss-of-function mutations in the Dnmt1 gene influences the transmission of grandparental chromosome 12 alleles. More specifically, maternal Dnmt1 mutations restore Mendelian transmission ratios of chromosome 12 alleles. Transmission of maternal alleles depends upon the presence of the Dnmt1 mutation in the mother rather than upon the Dnmt1 genotype of the offspring. Paternal transmission mirrors the maternal one: live-born offspring of wild-type fathers display 1:1 transmission ratios, whereas offspring of heterozygous Dnmt1 mutant fathers tend to inherit grandpaternal alleles. Analysis of allelic transmission in the homologous region of human chromosome 14q32 detected preferential transmission of alleles from the paternal grandfather to grandsons. Thus, parental Dnmt1 is a modifier of transmission of alleles at an unlinked chromosomal region and perhaps has a role in the genesis of TRD.