The effect of Argonaute A on DNA methylation

3.7.1 Expression of DIRS-1 and Skipper in AgnA knock down mutants

The transcription of the retrotransposon Skipper was activated when DNA methylation was inhibited in AgnA knock down cell lines, but the expression of DIRS-1 was not affected by a loss of DNA methylation (Fig. 2-39). These results were similar to those obtained for knock out mutants of the only DNA methyltransferase DnmA in Dictyostelium (Kuhlmann et al. 2005). Assumed that this discrepancy maybe caused by the different organization and the different strategies of transposition of the elements (Kuhlmann et al. 2005). The inactive transcription of DIRS-1 in AgnA mutant is maybe due to the transposition of the elements.

DIRS-1 has methylated C-residues on the inverted terminal repeats, while Skipper has unmethylated direct terminal repeats. Furthermore, there are many incomplete copies of DIRS-1 in the genome and the element frequently transposes into its own copies, while Skipper has almost complete and separate copies in the genome (Glockner et al. 2001).

3.7.2 Decreased DNA methylation in AgnA knock down mutants

In Arabidopsis, AGO4 controls histone and non-CpG DNA methylation, and accumulation of locus-specific siRNA (Zilberman 2003). The deletion of AGO4 decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation. In addition, the mutant blocked the accumulation of siRNA that correspond to the retroelement AtSN1. In Dictyostelium a decreased DNA methylation was observed in AgnA knock down mutant as well. The bisulfite sequencing showed that the AgnA knock down mutants decreased the methylation of asymmetric C-residues in DIRS-1 loci and Skipper RT, and completely abolished C-methylation on both mvpB and telA genes. Interestingly, two C-residues on DIRS-1 and one on telA were consistently not detectable. The loss of C-residues

consistently appeared in every sequencing data for DIRS-1 and telA. We cannot explain the reason why these C-residues were lost in sequencing data. The difference of methylation pattern between AgnA knock down and DnmA knock out mutants might be explained by their different silencing level: the dnmA is interrupted in the knock out, which leads to the loss of DNA methylation, while the residual AgnA cannot be completely excluded in the knock down, which leads to the inhibition of DNA methylation not the complete loss.

All tested methylated genes showed reduced or no DNA methylation in AgnA knock down cell lines. This demonstrates that AgnA influences methylation as AGO4 does in Arabidopsis (Zilberman et al. 2003). However AGO4 in Arabidopsis affect symmetric DNA methyaltion that is mediated by CMT3, a different class of methyltransferase, than by the only Dnmt2-like enzyme that is present in the Dictyostelium genome. Moreover, DnmA functions on mostly asymmetric DNA methylation in Dictyostelium.

The loss of DNA methylation was observed in DnmA knock outs (Kuhlmann et al 2005). However, from the directed yeast two-hybrid experiments it could be concluded that there was no direct interaction between AgnA and DnmA. Therefore AgnA does not affect DNA methylation through DnmA directly, but rather through some other factors, possibly SahA and SamS, the two enzymes involved in the S-Adenosyl-methionin biosynthesis pathway that were identified as PPWa interaction partners in the Yeast two-hybrid screen.

L-methionine ¹ S-Adenosyl-methionine

2

S-Adenosyl-L-Homocysteine

3

L-Homocysteine

Fig. 3-1 Metabolism of SAM. 1. SamS, 2 methyltransferase, 3 SahA

SamS functions as methionine adenosyl transferase in the synthesis of S-Adenosyl-methionine. The second enzyme, SahA, hydrolyses the reaction product

of RNA and DNA methyl transferases, S-Adenosyl-L-Homocysteine, to L- Homocysteine (Fig.3-1).

The strong down regulation of SamS on both RNA and cDNA levels detected by Northern blot and RT-PCR might be the main reason for lack of DNA methylation in the AgnA knock down mutants. SamS functions in providing the methyl group donors for methyltransferase, the decreased SamS might result in less substrates for reaction, which, as a result, would reduce DNA methylation.

The slight down regulation of SahA in the AgnA knock down mutant may be the second reason for decreased DNA methylation. The Arabidopsis HOG1 gene coding for SahA is required for DNA methylation-dependent gene silencing (Rocha et al.

2005). It is shown in this paper that mutations of HOG1 relieve transcriptional gene silencing and result in genome-wide demethylation, and the mutated plants show reduced SAH hydrolase activity. Moffatt and Weretilnyk (2001) explained that SahA plays a role in the removal of SAH (S-Adenosyl-L-Homocysteine), the by-product of the transmethylation reactions where SAM (S-Adenosyl-methionine) is the group donor. SAH is a strong inhibitor of SAM-dependent methyltransferases, and it appears that the relative levels of SAH and SAM are critical in the methylation of DNA, proteins, pectins and other small molecules in the cell.

Then we speculate that AgnA may influence DNA methylation by its effects on expression of SamS and SahA not by its direct effects on DnmA. The knock down of AgnA downregulated the expression of SamS and SahA in the pathway from DNA to RNA (shown by RT-PCR and Northern blot), then this maybe lead to low expression levels of SamS and SahA in AgnA mutants. The decreased SamS may lead to less methyl donor for DNA methyltransferase, and the slightly decreased SahA might lead to accumulation of SAH, and consequently the accumulated SAH may inhibit the activity of methyltransferase. This speculation is different from AGO4 in Arabidopsis, which directly affects CMT3 (Zilberman 2003). However, AgnA also

could comply the similar mechanism as in Arabidopsis by which the protein is directly involved in DNA methylation machinery.

The slightly down regulated SahA maybe another reason for slow growth of the AgnA knock down mutants, which is similar to the result shown by Moffatt and Weretilnyk (2001). They reported that an Arabidopsis line with a point mutation of SAH hydrolase 1 showed reduced growth. It is unclear whether this problem is caused by the SAH hydrolase deficiency directly or mediated by genome hypomethylation indirectly (Moffatt and Weretilnyk 2001). To investigate these possibilities, a knock down of SahA in Dictyostelium cells might be used to analyze the growth behavior

The involvement of AgnA in DNA methylation and RNAi indicated a connection between RNAi and DNA methylation, i.e. gene regulatory processes on the transcriptional and post-transcriptional level in Dictyostelium. While accumulation of siRNA in the AgnA knock down mutants was not investigated, the role of AGO4 in siRNA accumulation is known in Arabidopsis (Zilberman et al., 2003). Since siRNAs against DIRS-1 were found to cover essentially the entire DIRS-1 sequence, all methylation sites in the short segment that were sequenced by the bisulfite method had a corresponding siRNA (Kuhlmann et al. 2005). It is worthy to investigate if AgnA has a function in accumulation of siRNA derived from DIRS-1, which are produced by the Dicers and RNA dependent RNA polymerases (Martens et al. 2002b). The produced siRNA might direct DNA methylation in cells.

In Dictyostelium cells, the coding sequencing rather than the LTR are methylated in Skipper. This may indicate that DNA methylation causes chromatin remodeling over the entire retroelement and thus blocks the accessibility for the transcription machinery (Kuhlmann et al. 2005). As known in yeast, Ago1 is required for silencing of pericentric chromatin (Ekwall 2004); the AGO4 in Arabidopsis is linked to transposon siRNAs as well as DNA and histone methylation (Zilberman et al.

2003) The inhibition of DNA methylation of Skipper in AgnA knock down mutants indicates that AgnA in Dictyostelium is linked to chromatin remodeling as Ago1 in yeast and AGO4 in Arabidopsis. This suggests that RNAi, DNA methylation and histone modification regulate gene expression simultaneously in Dictyostelium.