DNA methylation in Dictyostelium discoideum

DNA methyltransferases of type Dnmt2 are a highly conserved protein family with enigmatic function. The aim of this work was to characterize DnmA, the Dnmt2 methyltransferase in Dictyostelium discoideum, and further to investigate its implication in DNA methylation and transcriptional gene silencing. The genome of the social amoeba Dictyostelium encodes DnmA as the sole DNA methyltransferase. The enzyme bears all ten characteristic DNA methyltransferase motifs in its catalytic domain. The DnmA mRNA was found by RT-PCR to be expressed during vegetative growth and down regulated during development. Investigations using fluorescence microscopy showed that both DnmA-myc and DnmA-GFP fusions predominantly localised to the nucleus. The function of DnmA remained initially unclear, but later experiment revealed that the enzyme is an active DNA methyltransferase responsible for all DNA (cytosine) methylation in Dictyostelium. Neither in gel retardation assays, nor by the yeast two hybrid system, clues on the functionality of DnmA could be obtained. However, immunological detection of the methylation mark with an α - 5mC antibody gave initial evidence that the DNA of Dictyostelium was methylated. Furthermore, addition of 5-aza-cytidine as demethylating agent to the Dictyostelium medium and subsequent in vitro incubation of the DNA isolated from these cells with recombinant DnmA showed that the enzyme binds slightly better to this target DNA. In order to investigate further the function of the protein, a gene knock-out for dnmA was generated. The gene was successfully disrupted by homologous recombination, the knock-out strain, however, did not show any obvious phenotype under normal laboratory conditions. To identify specific target sequences for DNA methylation, a microarray analysis was carried out. Setting a threshold of at least 1.5 fold for differences in the strength of gene expression, several such genes in the knock-out strain were chosen for further investigation. Among the up-regulated genes were the ESTs representing the gag and the RT genes respectively of the retrotransposon skipper. In addition Northern blot analysis confirmed the up-regulation of skipper in the DnmA knock-out strain. Bisufite treatment and sequencing of specific DNA stretches from skipper revealed that DnmA is responsible for methylation of mostly asymmetric cytosines. Together with skipper, DIRS-1 retrotransposon was found later also to be methylated but was not present on the microarray. Furthermore, skipper transcription was also up-regulated in strains that had genes disrupted encoding components of the RNA interference pathway. In contrast, DIRS 1 expression was not affected by a loss of DnmA but was strongly increased in the strain that had the RNA directed RNA polymerase gene rrpC disrupted. Strains generated by propagating the usual wild type Ax2 and the DnmA knock-out cells over 16 rounds in development were analyzed for transposon activity. Northern blot analysis revealed activation for skipper expression, but not for DIRS-1. A large number of siRNAs were found to be correspondent to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA methylation. In contrast, no siRNAs corresponding to the standard skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene regulation in Dictyostelium and that different, partially overlapping mechanisms control transposon silencing for skipper and DIRS-1. To elucidate the mechanism of targeting the protein to particular genes in the Dictyostelium genome, some more genes which were up-regulated in the DnmA knock-out strain were analyzed by bisulfite sequencing. The chosen genes are involved in the multidrug response in other species, but their function in Dictyostelium is uncertain. Bisulfite data showed that two of these genes were methylated at asymmetrical C-residues in the wild type, but not in DnmA knock-out cells. This suggested that DNA methylation in Dictyostelium is involved not only in transposon regulation but also in transcriptional silencing of specific genes.

Funktion und Biogenese kleiner RNAs in Dictyostelium discoideum

RNA mediated gene silencing pathways are highly conserved among eukaryotes and they have been well investigated in animals and in plants. Longer dsRNA molecules trigger the silencing pathways: RNase III proteins and their dsRNA binding protein (dsRBP) partners recognize those molecules as a substrate and process 21 nucleotide long microRNAs (miRNAs) or small interfering RNAs (siRNAs). Some organisms encode RNA dependent RNA polymerases (RdRPs), which are able to expand the pool of existing siRNAs. Argonaute proteins are able to bind small regulatory RNAs and are subsequently recruited to target mRNAs by base complementary. This leads in turn to transcriptional or posttranscriptional silencing of respective genes. The Dictyostelium discoideum genome encodes two Dicer homologues (DrnA and DrnB), five Argonaute proteins (AgnA to AgnE) and three RdRPs (RrpA to RrpC). In addition, the amoeba is known to express miRNAs and siRNAs, while the latter derive mainly from the DIRS-1 retrotransposon. One part of this work focused on the miRNA biogenesis pathway of D. discoideum. It was shown that the dsRNA binding protein RbdB is a necessary component for miRNA processing in the amoeba. There were no mature miRNAs detectable by Northern blot analysis in rbdB- strains, which is also true for drnB mutants. Moreover, primary miRNA-transcripts (pri-miRNAs) accumulated in rbdB- and drnB- strains. Fluorescence microscopy studies showed a nuclear localization of RbdB. RbdB accumulated in distinct perinucleolar foci. These were reminiscent of plant dicing bodies that contain essential protein components for miRNA processing. It is well known that RNase III enzymes and dsRBPs work together during miRNA processing in higher eukaryotes. This work demonstrated that the same is true for members of the amoebozoa supergroup. In Arabidopsis the nuclear zinc finger protein Serrate (SE) is also necessary for miRNA processing. The D. discoideum homologue SrtA, however, is not relevant which has been shown by the analysis of the respective knockdown strain. MiRNAs are known to be differentially expressed in several RNAi knockout strains. The accumulation of miRNAs in agnA- strains and a strong decrease in rbdB- strains were criteria that could thus be successfully used (among others) to identify and validate new miRNAs candidates by Illumina®-RNA sequencing. In another part of this study, the silencing and amplification of the DIRS-1 retrotransposons was analyzed in more detail. It was already known that DIRS-1 transcripts and extrachromosomal DIRS-1 DNA molecules accumulated in agnA- strains. This phenotype was correlated with the loss of endogenous DIRS-1 siRNAs in the knockout strain. By deep sequencing analysis of small RNAs from the AX2 wild type and the agnA- strain, the strong decrease of endogenous DIRS-1 siRNAs in the mutant strain (accounting for 70 %) could be confirmed. Further analysis of the data revealed an unequal distribution of DIRS-1 derived siRNAs along the retroelement in the wild type strain, since only very few of them matched the inverted terminal repeats (ITRs) and the 5’- half of the first open reading frame (ORF). Besides, sense and antisense siRNAs were asymmetrically distributed, as well. By using different reporter constructs it was shown indirectly that AgnA is necessary for the RrpC mediated production of secondary DIRS-1 siRNAs. These analyses also demonstrated an amplification of siRNAs in 5’- and in 3’-direction. Further analysis of the agnA- strain revealed that not only DIRS-1 sense transcripts but also ORF2 and ORF3 encoded proteins were enriched. In contrast, the ORF1 encoded protein GAG was equally expressed in the mutant and the wild type. This might reflect the unequal distribution of endogenous DIRS-1 siRNAs along the retrotransposon. Southern Blot and PCR-analyses showed that extrachromosomal DIRS-1 DNA molecules are present in the cytoplasm of angA- strains and that they are complementary to sense transcripts of intact DIRS-1 elements. Thus, the extrachromosomal DIRS-1 intermediates are likely incomplete cDNA molecules generated by the DIRS-1 encoded reverse transcriptase. One could hypothesize that virus like particles (VLPs) are the places of DIRS-1 cDNA synthesis. At least, DIRS-1 GAG proteins interact and fluorescence microscopy studies showed that they localize in distinct cytoplasmic foci which accumulate in close proximity to the nuclei.