Expression of the dnmt3 genes in zebrafish development: similarity to Dnmt3a and Dnmt3b

The zebrafish differs from mammals in that they have six dnmt3 genes as opposed to the two that can produce a catalytically active protein in mammals. Zebrafish also do not show evidence of genomic imprinting and lack the Dnmt3l gene necessary to that process in mammals. As such, they offer a unique opportunity to compare the two genetic situations in order to define the roles of the multiple genes in developmental gene methylation. To this end, we have analyzed the developmental expression of the six dnmt3 genes in zebrafish and find that they fall into two distinct patterns. The expression patterns of the dnmt6 and dnmt8 genes, which more closely resemble the mammalian Dnmt3a gene in sequence, also show an expression pattern that is more similar to the expression of Dnmt3a rather than Dnmt3b. Conversely, the other four dnmt3 genes in zebrafish (dnmt3, dnmt4, dnmt5, and dnmt7) show an expression pattern that is more similar to Dnmt3b. The dnmt6 and dnmt8 genes are also expressed in the adult zebrafish and in the brain in particular. In situ expression analyses show that the dnmt6 and/or dnmt8 genes also show tissue-specific differences in expression with those two genes being more ubiquitously expressed in the developing zebrafish than the other dnmt3 genes. Although differences in dnmt3 function may exist between mammals and fish, our results showing similar expression patterns between the genes in fish and mammals suggest that the six dnmt3 genes in the zebrafish may be analogous to the two Dnmt3 genes in mammals.     

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.     

Novel splice variants associated with one of the zebrafish <Emphasis Type="Italic">dnmt3</Emphasis>genes

Background  

DNA methylation and the methyltransferases are known to be important in vertebrate development and this may be particularly true for the Dnmt3 family of enzymes because they are thought to be the de novo methyltransferases. Mammals have three Dnmt3 genes; Dnmt3a, Dnmt3b, and Dnmt3L, two of which encode active enzymes and one of which produces an inactive but necessary cofactor. However, due to multiple promoter use and alternative splicing there are actually a number of dnmt3 isoforms present. Six different dnmt3 genes have recently been identified in zebrafish.     

Epigenetic regulation of left–right asymmetry by DNA methylation

DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left–right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 (dnmt1) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates.     

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.     

Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against γ-radiation-induced epigenetic modifications

Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that γ-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6 Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C₁) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (P < 0.01), while hepatic homocysteine levels decreased (P < 0.01) compared to NCD fed and irradiated animals. Although hepatic folate level increased significantly in MSD fed animals (P < 0.01), it showed a decrease in response to high doses of γ-irradiation. Under these conditions, a marked suppression of S-adenosylmethionine (SAM) levels occurred in NCD fed and irradiated animals, suggesting reduced conversion of homocysteine to SAM. Concomitant with decline in liver SAM Pool, activities of DNA methyltransferase (Dnmt, that methylates DNA) and methionine synthase (MSase, that regenerates methionine from homocysteine) were both decreased in NCD fed and irradiated mice. However, in MSD fed and irradiated mice, they were increased. These results strongly indicated that increased levels of dnmt and MSase may enhance C₁ flux towards DNA methylation reactions in MSD fed animals. These results were confirmed and further substantiated by measuring genomic DNA methylation levels, which were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals (P < 0.01). In conclusion, our results suggest that maintenance of genomic DNA methylation under γ-radiation stress might be a very dynamic, progressive diet dependent process that could involve increased one-carbon flux through various C₁ metabolites.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Background

The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.

Methods and Principal Findings

Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.

Conclusions

The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.     

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.     

Impact of the DNA methyltransferases expression on the methylation status of apoptosis-associated genes in glioblastoma multiforme

Disruption of apoptosis is considered as an important factor aiding tumorigenesis, and aberrant DNA methylation of apoptosis-associated genes could be an important and significant mechanism through which tumor cells avoid apoptosis. However, little is known about (1) the impact of methylation status of apoptosis-associated genes on the presence of apoptosis evasion phenotype in glioma; and (2) the molecular mechanism governing the aberrant methylation of apoptosis-associated genes in glioma. By analyzing human glioma biopsies, we first show that low level of apoptosis in tumor is correlated with aberrant methylation of the bcl-2, bax and XAF-1 genes, but not with the aberrant methylation of the bcl-w, survivin, TMS1, caspase-8 and HRK genes. Our work also indicates that the expression levels of DNA methyltransferase 1 (Dnmt1), Dnmt3b and Dnmt1/Dnmt3a coregulate the methylation status of survivin, TMS1 and caspase-8, whereas no correlation was observed between the expression level of Dnmts and the methylation status of the bcl-w, bcl-2, bax, XAF-1 and HRK genes. Thus, these results indicate that the epigenetic regulation of some apoptosis-regulated genes could dictate whether glioma harbors the apoptosis evasion phenotype, and provide some bases to the identification of the methylation machineries of apoptosis-associated genes for which the Dnmt expression acts as a limiting factor.     

Increased Expression of DNA Methyltransferase 3a in Obese Adipose Tissue: Studies With Transgenic Mice

Epigenetic mechanisms are likely to be involved in the development of obesity. This study was designed to examine the role of a DNA methyltransferase (Dnmt3a), in obese adipose tissue. The gene expression of Dnmts was examined by quantitative real‐time PCR analysis. Transgenic mice overexpressing Dnmt3a in the adipose tissue driven by the aP2 promoter were created (Dnmt3a mice). DNA methylation of downregulated genes was examined using bisulfite DNA methylation analysis. Dnmt3a mice were fed a methyl‐supplemented or high‐fat diet, and subjected to body weight measurement and gene expression analysis of the adipose tissue. Expression of Dnmt3a was markedly upregulated in the adipose tissue of obese mice. The complementary DNA (cDNA) microarray analysis of Dnmt3a mice revealed a slight decrease in the gene expression of secreted frizzled‐related protein 1 (SFRP1) and marked increase in that of interferon responsive factor 9 (IRF9). In the SFRP1 promoter, DNA methylation was not markedly increased in Dnmt3a mice relative to wild‐type mice. In experiments with a high‐fat diet or methyl‐supplemented diet, body weight did not differ significantly with the genotypes. Gene expression levels of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) were higher in Dnmt3a mice than in wild‐type mice on a high‐fat diet. This study suggests that increased expression of Dnmt3a in the adipose tissue may contribute to obesity‐related inflammation. The data highlight the potential role of Dnmt3a in the adult tissue as well as in the developing embryo and cancer.     

Temporal and spatial expression of the four Igf ligands and two Igf type 1 receptors in zebrafish during early embryonic development

The insulin-like growth factor (Igf) family is an evolutionarily conserved system essential for normal growth and development in vertebrates. Unlike mammals, four distinct Igf ligands (Igf1, Igf2a, Igf2b and Igf3) and two Igf type 1 receptors (Igf1ra and Igf1rb) are present in zebrafish. However, the localization of these multiple ligands and receptors especially the recently discovered igf3 during early development of zebrafish is poorly understood. In this study, detailed expression patterns of these components of the Igf system during embryogenesis of zebrafish were analyzed. It was found that igf1 is specifically expressed in the trigeminal ganglia region from 18 hpf to 72 hpf, while igf2a is restricted to the caudal regions of the notochord from 14 hpf to 18 hpf as well as in the midbrain, dorsal hind brain and otic vesicle at 24 hpf. On the other hand, igf2a is highly expressed in the midbrain and pharyngeal arch region at 48 hpf, followed by its appearance in the liver and brain at 72 hpf, while igf2b is restricted to the floor plate and hypochord from 12 hpf to 18 hpf, and strong expression is also detected in the midbrain and dorsal hind brain at 24 hpf. The teleost specific igf3 is highly expressed in the pharyngeal arch region before 24 hpf, but is then restricted to the sternohyoideus after 48 hpf. The receptor subtype igf1ra is ubiquitously expressed before 24 hpf but is confined to the brain at 72 hpf. However, igf1rb is widely expressed before 10 hpf, but is more confined to the brain region at 24 hpf and 72 hpf. This dynamic temporal-spatial expression during embryogenesis of zebrafish, together with the unique and overlapping expression patterns of the Igf ligands and receptors suggest the coordination of the divergent functions of the Igf system during early development in zebrafish.     

Circadian expression of DNA methylation and demethylation genes in zebrafish gonads

This research aimed at investigating the light synchronization and endogenous origin of daily expression rhythms of eight key genes involved in epigenetic mechanisms (DNA methylation and demethylation) in zebrafish gonads. To this end, 84 zebrafish were distributed into six tanks, each one containing 14 fish (7 males and 7 females). Animals were subjected to 12 h light:12 h dark cycles (LD, lights on at ZT0 h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20 days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4 h during a 24 h cycle (ZT2, 6, 10, 14, 18, and 22 h). The results revealed that most of the epigenetic genes investigated exhibited a significant daily rhythm. DNA methylation genes (dnmt4, dnmt5, dnmt7) exhibited a daily rhythm of expression with a nocturnal acrophase (ZT14:01~ZT22:17 h), except for dnmt7 in males (ZT2:25 h). Similarly, all DNA demethylation genes (tet2, tdg, mb4, gadd45aa, and apobec2b) revealed the existence of statistically significant daily rhythms, except for gadd45aa in females. In females, tdg, mb4, and apobec2b presented a nocturnal peak (ZT14:20 ~ ZT22:04 h), whereas the tet2 acrophase was diurnal (ZT4:02 h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26~ZT21:31 h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02 h, respectively). To determine the endogenous nature of gene expression rhythms, another experiment was performed: 12 groups of 14 fish (7 males and 7 females) were kept in complete darkness (DD) and sampled every 4 h during a 48 h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 h). Under DD, most of the genes (7 out of 8) presented circadian rhythmicity with different endogenous periodicities (tau), suggesting that the epigenetic mechanisms of DNA methylation and demethylation in the gonads follow an internal control, functioning as part of the translation network linking the environment into somatic signals in fish reproduction.     

Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration

We used the 500-bp Xenopus ef1-alpha promoter and the 2-kb zebrafish histone 2A.F/Z promoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)nt line, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-alpha:EGFP)nt line, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, and msxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of the ef1-alpha:EGFP transgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that the ef1-alpha:EGFP transgene is also re-expressed during adult retinal regeneration. Specifically, the ef1-alpha:EGFP transgene colabels with PCNA in the Müller glia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-alpha:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.