Novel splice variants associated with one of the zebrafish dnmt3genes
© Smith et al; licensee BioMed Central Ltd. 2005
Received: 08 June 2005
Accepted: 19 October 2005
Published: 19 October 2005
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.
We have examined two of the dnmt3 genes in zebrafish that are located in close proximity in the same linkage group and we find that the two genes are more similar to each other than they are to the other zebrafish dnmt3 genes. We have found evidence for the existence of several different splice variants and alternative splice sites associated with one of the two genes and have examined the relative expression of these genes/variants in a number of zebrafish developmental stages and tissues.
The similarity of the dnmt3-1 and dnmt3-2 genes suggests that they arose due to a relatively recent gene duplication event. The presence of alternative splice and start sites, reminiscent of what is seen with the human DNMT3s, demonstrates strong parallels between the control/function of these genes across vertebrate species. The dynamic expression levels of these genes/variants suggest that they may well play a role in early development and this is particularly true for dnmt3-2-1 and dnmt3-1. dnmt3-2-1 is the predominantly expressed form prior to zygotic gene activation whereas dnmt3-1 predominates post zygotic gene activation suggesting a distinct developmental role for each.
The epigenetic modification of DNA by the addition of a methyl group to the 5 position of cytosine is an important mechanism for control of gene expression in vertebrates. This is particularly true during development where DNA methylation is thought to have a role in genome imprinting [1, 2], X inactivation  and lineage determination . Methylation has been most intensely studied in mammals where the levels have been shown to be quite dynamic during early development, decreasing soon after fertilization and increasing again by the gastrula stage [5, 6]. The importance of this de-methylation/re-methylation cycle to the developmental process has been clearly demonstrated by perturbations of that methylation that generally leads to embryonic lethality [7, 8]. Given the importance of methylation in sustaining normal early developmental processes, the enzymes that add and maintain that methylation are of significant interest. The dnmt3 family of methyltransferases that are thought to be important in de novo methylation (that is the addition of methyl groups to previously unmethylated sequences) are of particular interest in this context. There are three members of this family in mammals; two have catalytic activity, Dnmt3a and Dnmt3b; and the third, Dnmt3L, is important as a cofactor, particularly for the methylation of imprinted loci . Functionally, however, the dnmt3 family is not limited to just three products because both the Dmnt3a and b transcripts can be alternatively spliced to generate a number of different RNAs. Dnmt3a has two splice variants differing in the 5' region whereas dnmt3b has a number of possible splicing products . These variations in the dnmt3 proteins may allow for a greater diversity in the function and/or targets of these enzymes.
Methylation in zebrafish has recently been the focus of a number of reports, and methylation has been found to be dynamic during its early development . Also, as in mammals, blocking re-methylation in zebrafish results in abnormal development and death .
The zebrafish actually has at least twice the mammalian number of dnmt3 genes; six have been submitted to databases so far (GenBank numbers AB196914, AB196915, AB196916, AB196917, AB196918, AB196919) . The significance of the increase in dnmt3 gene copy number in zebrafish is unknown.
We have isolated and analysed a number of the zebrafish dnmt3 gene sequences and have identified two dnmt3 sequences that are located very close together in a single linkage group. The very close proximity of the two sequences provides an interesting opportunity to examine how the expression of these genes is controlled since one copy has a very limited upstream promoter region relative to the other.
Results and Discussion
Recent additions to the sequence databases included two zebrafish sequences that appear to correspond to the same two genes and were named dnmt3 and dnmt5 respectively (GenBank numbers AB196914, AB196916). Our sequencing data corroborate the sequences submitted to the databases except for a few minor variations in regions with triplet repeats which may be an artefact of polymerase slippage in cloning or represent real triplet repeat differences that exist in the gene.
The high homology between dnmt3-1 and dnmt3-2 relative to other zebrafish dnmt3's, as well as their close proximity, suggests that these genes represent a duplication event. Postlethwait et al.  provides support for a model where two polyploidization events occurred in a common ancestor of zebrafish and mammals. However, there are often additional multigene members in zebrafish. Postlethwait et al.  argues that either chromosome duplication or another tetraploidization event in the zebrafish lineage is the most likely mechanisms by which these additional members arose. The tight clustering seen here, however, suggests that, at least in this instance, tandem gene duplication has occurred.
The shortest of these variants, dnmt3-2-1, corresponds to the dnmt5 sequence in the database. The two novel variants reported here differ in size from that sequence by 187 (dnmt3-2-2) and 265 (dnmt3-2-2b) base pairs. These variants are actually associated with the gene having the most restricted promoter region. A schematic of the three products is shown in figure 5.
There are several interesting aspects of these dnmt3-2 variants. To begin with, although the splicing difference between variant dnmt3-2-2 and dnmt3-2-2b appears to involve the same 3' splice junction it has a different 5' splice junction, meaning that one of those splice sites is located within the exon of the other variant. However, both of the junctions still abide by the GT/AG rule for splice junctions.
The second interesting aspect of these splice variants is that all of them are 5' to the initiator AUG. Therefore, none of them actually affect the amino acid sequence. This suggests that either the splicing differences are trivial or they play a regulatory role in the translation or localization or some other aspect of the various splice variants. The latter possibility is a more reasonable assumption since, parsimoniously, it seems unreasonable to assume that this RNA would be alternatively spliced in a variety of ways for no biologically relevant reason. This situation is not unique to zebrafish dnmt3 genes. Similar splice variants in the 5'untranslated region have also been reported for human DNMT3s .
We have isolated and analysed several of the dnmt3 genes from the zebrafish. In this report we have focused on two of the genes that are located in close proximity in a single linkage group and we find that the two genes are considerably more similar to each other than they are to the other zebrafish dnmt3 genes. This suggests that they arose as a result of a relatively recent gene duplication event. We have also found evidence for the existence of several different splice variants and alternative splice sites associated with one of the two genes, reminiscent of what is seen with the human DNMT3s. Expression analyses of these genes and variants demonstrate that are dynamic during development with distinct patterns that suggest they are independently controlled and, possibly, have different functions in development.
PCR products were fractionated on 0.8% agarose gels, visualized with ethidium bromide, excised from the gel and cloned into pCR 2.1 vectors (TOPO TA cloning kit, Invitrogen Inc. Carlsbad, CA). The cloned products were then purified (Wizard®Plus Minipreps, Promega Inc. Madison, WI), and sequenced (Cortec DNA Service Laboratories Inc., Kingston, ON).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to determine the relative expression levels of gene 1, gene 2, and its variants in tissues. Total RNA from zebrafish ovarian tissue, 1–2 cell embryos, 64 cell embryos, 6 hour embryos, muscle tissue, and brain tissue was isolated as described above, and the integrity checked by ethidium bromide staining. The RNA was then reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen Inc. Carlsbad, CA) using primers specific for the various genes and variants. GSP2 was used for first strand cDNA synthesis of Gene 1 in conjunction with GSP3, generating a predicted amplicon of 521 bp. Primer GSP4 designed to anneal to all three gene 2 variants was used with GSP5 for dnmt3-2-1 to produce a 420 bp amplicon and with GSP6 to produce two amplicons of 597 bp and 675 bp from gene 2 variants dnmt3-2-2, and dnmt3-2b. In addition, RT-PCR was conducted to generate a 440 bp amplicon with GSP7 and GSP8, primers specific for a constitutively expressed gene, max . PCR reactions were set up as described by the manufacturer, except that 2 ul of cDNA template were used for each reaction. PCR conditions were designed to ensure that all amplifications were within the logarithmic phase. Those conditions were; 94°C for 1 min, 25 cycles of 94°C 30 sec, 59°C for 30 sec, 72°C for 1 min, and a 72°C for1 min final extension for all primer sets except max which was only amplified for 14 cycles. Controls lacking RT were run for each RNA sample.
RT-PCR products were separated on a 1.5 % agarose gel, transferred to nylon membrane (Roche, Indianapolis, IN) and visualized by hybridization with a biotin labeled sequence designed to hybridize to gene 1, gene 2, and the variants (North2South Biotin labeling kit, Pierce Biotechnology Inc. Rockford, Il). Densitometric analysis of autoradiographs was performed to determine the relative expression levels of the genes and their isoforms at the above mentioned zebrafish developmental stages and tissues. Samples could be compared on different blots by using a control sample present on each autoradiograph, and samples were calibrated using the endogenous control max.
Zebrafish care and feeding was performed essentially as described by Westerfield . All experimentation was done with the approval of the Canadian Council on Animal Care.
The authors wish to thank Denise Flint for her editorial assistance. This work was supported by grants from the Natural Sciences and Engineering Research council of Canada.
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