5 Results
The Dnmt2 family of proteins consists of enzymes that display strong sequence similarities to DNA (cytosine-5)-methyltransferases (m5C MTases) of both prokaryotes and eukaryotes. Human DNMT2 was the first MTase-like protein from higher eukaryotes the structure of which has been solved and some characteristic features were first examined in vitro (Dong et al, 2001). The experiments had shown that investigation of both in vivo and in vitro functions of Dnmt2-like proteins is quite a challenging task, requiring a careful choice of model organism to deal with as well as a reasonable attentiveness in experimental design. Since the slime mold Dictyostelium discoideum has the DNMT2 homolog gene (dnmA) as the only member of the DNA methyltransferase family, shows some detectable level of genomic DNA methylation and provides easy and convenient gene manipulation techniques, it is perfectly suited for the investigation of Dnmt2 functions. Thus, let us start this work with the characterization of genomic locus ofdnmAgene inDictyostelium discoideum.
many clusters of co-expressed neighboring genes, ranging from 10 to 30 genes in size (Spellman
& Rubin, 2002). Furthermore, the human genome shows large regions in which most genes are expressed at high levels, alternating with regions that contain predominantly lowly expressed genes ((Caron et al, 2001), (Versteeg et al, 2003)). These observations strongly suggest that juxtaposition of genes in the linear genome can facilitate their coordinated regulation (Razin et al, 2007). Chromatin is a principal orchestrator of transcription. Neighboring genes can be packaged together into a single chromatin domain that may act as a regulatory unit ((Sproul et al, 2005), (Dillon, 2006) and (Talbert & Henikoff, 2006)). Therefore, on basis of the genetic map (Fey et al, 2009) and currently available Dictyostelium discoideum gene expression database (http://www.ailab.si/dictyExpress) which was constructed by Shaulsky and coworkers (Rot et al, 2009), we were able to make some suggestions on putative chromatin organization of the dnmA genomic locus and its possible connection with expression level of this gene.
Figure 5.1.1Scheme of the genomic locus ofdnmAgene on chromosome 5 of theDictyosteliumgenome.
Accordingly to the map of theDictyostelium genome, thednmAgene is located on chromosome 5 at the coordinates 1026957 to 1028573, Crick strand, based on Genome Browser (Figure 5.1.1, http://dictybase.org/gene/DDB_G0288047). We analyzed the neighbourhood of dnmA gene at this locus at the distances about 20 kbp left and right of dnmA and compared the expression
profiles available for this set of genes during development. The results of analysis are presented in the Figure 5.1.2 and Figure 5.1.3.
Figure 5.1.2 Scheme of the genomic region on left side of dnmA gene (upper panel). Lower panel represent developmental expression profiles for genes, adjacent to dnmA. The expression profiles of genes were high with comparison withdnmAprofile.
The results appeared to be quite interesting as we can clearly see that adjacent genes indeed had a tendency to cluster into the groups. One of these groups consists of four genes (including dnmA), extend about 10 kbp and show similar low expression profiles (less than 10 times difference in expression level). This region is surrounded by sets of genes with relatively high levels of expression (more than 10 times difference in expression level).
Figure 5.1.3 Scheme of the genomic region on right side of dnmA gene (upper panel). Lower panels represent developmental expression profiles for genes, adjacent to dnmA. The profiles of three genes closest todnmA show
relatively low and comparable levels of expression. Genes, lying next to these demonstrate relatively high levels of expression.
Unfortunately, most of the tested genes are not annotated in the current version of Dictyostelium database and a function of their products is not known. Pfam search (http://pfam.sanger.ac.uk) also did not show any significant matches with the known conserved domains. Nevertheless, it is tempting to suggest that the group of genes, which include dnmA and share a low expression profile, may well be representing the functional chromatin domain, probably organized into facultative heterochromatin structures. Further experimental evidences must be gathered to come to a valid conclusion.
5.2 Intracellular localization of GFP-tagged DnmA
To study the nuclear localization of DnmA, the expression vector pDneo2a-dnmA-GFP was created and transformed into Dictyosteliumcells to produce C-terminally GFP-tagged version of the protein (see Figure 3.7.3 in Materials/Plasmids). After fixation of cells using various protocols for immunofluorescence (see Methods), the cells were analyzed by fluorescence microscopy. The images revealed that GFP-tagged DnmA fusions can be found in interphase cells both in the cytoplasmic and nucleoplasmic compartments, although with some bias towards nuclei (Figure 5.2.1A). In some cells it appeared that DnmA-GFP partially co-localized with DAPI-stained DNA but this was not found for all cells and may depend on fixation procedures.
Nevertheless, another explanation of this observation could be that DnmA protein is actually functional at certain stages of interphase, perhaps during S-phase of cell cycle. The distribution of proteins within the cytoplasm seems to be rather homogeneous with some slight tendency of accumulation near centrosomes (Figure 5.2.1B).
Figure 5.2.1Localization of the DnmA-GFP fusion protein in fixedDictyosteliumDnmA KO cells. (A) Partial co-localization DnmA-GFP with DAPI stained DNA. (B) Slight enrichment of DnmA-GFP proteins in a regions marked by white arrows, presumably corresponding to centrosomes. White bars represent 10 micrometers.
It should be noted that the nucleoli which are usually more weakly stained by DAPI display a homogeneous distribution of DnmA during interphase in most cases. Nevertheless, some fixed cells in the interphase stage may show difference in protein distribution in the nucleoli and the
rest of the nuclei which could also represent the artifacts of fixation protocol (Figure 5.2.2A and B). This observation could demonstrate yet the redistribution of DnmA-GFP fusion during different stages of interphase. To further prove this assumption, additional experiments should be made with specific markers for G1, S and G2 stages of the cell cycle.
Figure 5.2.2Localization of the DnmA-GFP fusion protein in nuclei of fixed DictyosteliumDnmA KO cells. (A) Local increase in green fluorescent signal in the regions of nuclei corresponding to nucleoli. In this case DAPI-staining may reflect the beginning of process of chromosome condensation and removal (active or passive) of DnmA-GFP fusion from nuclei. Such cells also demonstrate absence of the co-localization of DnmA-GFP and DAPI-Stained DNA. (B) Local decrease of DnmA-GFP signal in the nucleoli. This case represents the cells where partial co-localization of DnmA-GFP fusion with DAPI-stained DNA can still be observed and which stay likely in earlier phases of interphase, presumably S or early G2. White bars represent 10 micrometers.
The expression of full-sized GFP tagged DnmA could also be confirmed by western blotting with α-GFP antibodies. As expected the observed size of DnmA-GFP fusion is about 77 kDa which corresponds well to theoretical estimation of 72 kDa (Figure 5.2.3).
Additional evidences for the role of DnmA in the interphase of cell cycle rather than in the mitosis were gathered in the further examination of the DnmA-GFP localization during mitosis.
Cells at different mitotic stages in asynchronously grownDictyosteliumculture were found using DAPI staining as well as the antibodies directed against α-Tubulin, which stain the centrosomes and the mitotic spindle.
Figure 5.2.39% SDS-PAGE (left panel) and Western blot analysis (right panel) of cell extracts from DnmA-GFP expressing Dictyostelium DnmA KO cell line. Prestained protein marker (Fermentas, #SM1811) was used to determine the size of DnmA-GFP fusion protein. The theoretically calculated size for DnmA-GFP was 72 kDa.
Additional minor band (*) below DnmA-GFP protein may correspond to a degradation product.
The mitotic cells show mainly homogeneous distribution of DnmA-GFP fusion within cells both in cytoplasmic and nucleoplasmic compartments. There appears to be no characteristic features but the observation that during the mitotic cycle the bulk of DnmA-GFP proteins had a tendency to leak out from nuclei into cytoplasm (Figure 5.2.4). This observation can be clearly seen from prophase till at least anaphase, and it is perhaps the result of partial distortion of the nuclear envelope during these phases of mitosis.
Figure 5.2.4Localization of DnmA-GFP fusion proteins during mitotic cycle in fixed cells. Cells at different stages of mitosis were identified by DAPI staining and immunostaining with α-Tubulin antibodies. Most of mitotic phases were detected. White bars represent 10 micrometers.
We did not find clear telophase cells or cells undergo cytokinesis on our preparations, though we often observed cells with two nuclei. These represent the result of a failure in cytokinesis that is frequently found in axenically growing cell cultures. From the overall data we assume that
DnmA-GFP start accumulating in nuclei during telophase or shortly after this phase, most probably after reconstruction of intact nuclear envelopes (Figure 5.2.5).
Figure 5.2.5 Localization of DnmA-GFP in the Dictyostelium cells carrying two nuclei due to cytokinesis error.
DnmA-GFP fusion proteins accumulate within nuclei during or shortly after telophase. White bar represent 10 micrometers.
On the other hand, the dynamics of DnmA-GFP distribution during mitosis could indirectly support the suggestion that DnmA may be functional in the nuclei of interphase cells and has no specific function during mitosis. Diffusion of DnmA into the cytosol during mitotic phases may also provide means to allow for cytoplasmic functions of the protein, although additional experiments should be performed to support this hypothesis.
DNA methyltransferases, including Dnmt2-like proteins, are reported to be present in the nuclear matrix of almost all higher eukaryotes. Recently, it was shown that the Dnmt2 homolog Ehmeth inEntamoeba histolyiticais a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (Banerjee et al, 2005). Similar data was provided for Dnmt2-like m5C methyltransferase in Drosophila melanogaster, where this protein was found to be associated with the nuclear matrix, mainly during mitosis (Schaefer et al, 2008). To test the possibility that DnmA protein in Dictyosteliumis also involved in nuclear matrix structures, we performed subcellular fractionation to show the distribution of DnmA-GFP and DnmA-TAP fusion proteins within subcellular compartments. Figure 5.2.6 shows the principal of the experiment, resulting SDS-PAGE gels and western blots with antibodies against GFP and TAP to visualize DnmA-GFP and DnmA-TAP proteins in different subfractions. Indeed, significant amounts of DnmA proteins were found in the nuclear matrix and core filament fractions. This
bound DnmA was not removed by extensive washing steps and, therefore, represents a protein strongly embedded into corresponding structures.
Figure 5.2.6 (A) Outline of the procedures employed for preparation of cellular and nuclear subfractions. The various cellular and nuclear subfractions isolated are shown in the boxes in the diagram. The details of the method are given in the Methods. Nuclear matrix represents a 2.0 M NaCl extract of NM plus intermediatefilaments (NM-IF). Subfractions designated as 1, 2, 3, 4 and 5. (B) 8-9% SDS-PAGE gels of prepared fractions and corresponding
western blot analyses with antibodies against GFP and TAP. Loaded % of Lysate volume represent volume of loaded sample adjusted to total volume of cell lysate. Prestained protein marker (Fermentas, #SM1811) was used as the size reference.