C.3 Dnmt3a/b2 dependent de novo DNA methylation in the context of chromatin. 62
C.3.5 In vitro methylation of DNA and mono-nucleosomes by Dnmt3a and Dnmt3b2. 67
In vitro methylation of nucleosomes by the maintenance and de novo DNA methyltransferases have already been under investigation, although the results were rather inconsistent (Robertson et al. 2004; Gowher et al. 2005b) (Takeshima et al. 2006).
Two DNA templates (MF79/80 342bp; MF124/125 147bp, (E.2.13.1) harboring an altered 601 nucleosome positioning sequence were amplified by PCR (pGA-BN601 Mr Gene; E.1.8) and assembled into nucleosomes (E.2.10.2) (Figure 37, A). In vitro DNA methyltransferase assays (E.2.11.1) with 100nM of Dnmt3a and Dnmt3b2, 480nM SAM (1.5µCi) and 480nM CpG sites of either the free DNA or the nucleosomal DNA were performed. ‘Naked’ DNA and accessible DNA in the linker region of mono-nucleosomes were equally well methylated, whereas the 147bp nucleosome was almost not methylated at all (Figure 37, B). Although the overall methylation efficiency of Dnmt3a and Dnmt3b2 differed (different protein preparations), methylation of the nucleosome (MF124/125 nuc) was decreased 35-fold and 27-fold compared to ‘naked’ DNA for Dnmt3a and Dnmt3b2 respectively (as determined from the 40min timepoint).
Figure 37 Radioactive DNA methylation assay with DNA and mono-nucleosomes
A. DNA fragments and mono-nucleosomes used for Dnmt specific radioactive methylation assay. The MF79/80 (342bp) and MF124/125 (147bp) DNA fragments that bear a modified 601 nucleosome positioning sequence (from pGA BN601 Mr Gene) were PCR amplified as described (E.2.13.1) and fully assembled with Drosophila histones by salt dialysis (E.2.10.2). Analysis on a 5% PAA gel in 0.4x TBE with ethidium bromide staining.
Molecular weight marker is indicated B. Radioactive DNA methylation assay was performed (E.2.11.1) in the presence of 480nM SAM (1.5µCi), 100nM N-His Dnmt3a (Sf21 cells) and C-His Dnmt3b2 (E. coli), 480nM CpG sites of the indicated templates. At the given time points samples were withdrawn, stopped and treated as described (E.2.11.1). Radioactivity (cpm) was determined in a scintillation counter and plotted against the individual samples for each time point.
C.3.6 Bisulfite analysis of in vitro methylated DNA and mono-nucleosomes
In order to study in detail in vitro DNA methylation of Dnmt3a and Dnmt3b2 towards mono-nucleosomes, the PCR-fragment MF79/80 (342bp, 27 CpG sites, amplified on pGA BN601 Mr Gene E.1.8) was reconstituted with Drosophila histones by the method of salt dialysis (E.2.10.2) (Figure 38, A). The assumed position of the nucleosome was verified by restriction enzyme digesting analysis (Figure 38, B).Figure 38 Reconstitution and mapping of the nucleosome binding site
A. The MF79/80 (342bp) DNA fragment that bears a modified 601 nucleosome positioning sequence (from pGA BN601 Mr Gene) was PCR amplified as described (E.2.13.1) and assembled in different ratios with Drosophila histones by salt dialysis (E.2.10.2) in the presence of competitor DNA (pCpGL basic, 250ng). Analysis on a 5%
PAA gel in 0.4x TBE with ethidium bromide staining. Molecular weight marker is indicated. Arrow denotes nucleosomal template used for non-radioactive in vitro DNA methylation assay B. Although the high-affinity 601 nucleosome positioning sequence is present, its location was mapped. Partially assembled nucleosomes were digested with the indicated restriction enzymes (RE) and either directly (I.), or after proteinaseK digest and ethanol precipitation (Xref) (II.) loaded onto a 5% PAA gel. StyI cuts within the proposed nucleosomal region. In I.
the 342bp DNA fragment is cut and shifted to smaller sizes (* denotes the theoretic position). After proteinaseK treatment, the DNA fragment (*) that had been protected from RE digest appears. Smaller fragments are no longer visible due to inefficient ethanol precipitation.
Non-radioactive DNA methylation reactions with Dnmt3a, Dnamt3b2 and the bacterial methyltransferase M.SssI as control were performed as described (E.2.11.1) and methylated DNA subjected to bisulfite treatment (E.2.13.3). Bisulfite converted DNA was PCR amplified using two different primer pairs for the sense and the anti-sense strand respectively. PCR products were cloned into pGEM-T-EASY vector (Promega) and transformed into chemically competent Xl1 Blue E. coli cells following Blue/White selection on Bluo-Gal/ampicillin LB- plates. DNA was isolated and sent for sequencing. Analysis of bisulfite converted DNA was done with BiQ ANALYZER software (http://biq-analyzer.bioinf.mpi-inf.mpg.de/) that had been provided by C. Bock (Bock et al. 2005).
Figure 39 Bisulfite analysis of nucleosomal methylation by M.SssI, Dnmt3a and Dnmt3b2 The DNA methylation reaction with Dnmt3a/b was carried out in a reaction volume of 40µl in DNA methyltransferase buffer (20mM Hepes, pH 7.6, 1mM EDTA, 1mM DTT). Dnmt3a and Dnmt3b2 were used at a concentration of 600nM and 200nM, DNA or nuclesomal DNA (MF79/80 on pGA BN601 Mr Gene, 27 CpG sites) between 12.5 and 20nM CpG sites, BSA at 0.2µg/µl and SAM (Sigma A7007, 10mM) at 250µM. M.SssI was used as control in a separate reaction with 1x NEB buffer 2 and 4U M.SssI. The reactions were mixed well and incubated for 1 hour at 37°C. Another 250µM SAM were added following incubation for 1-1,5 hours. This step was repeated twice. The reactions were heat-inactivated at 65°C for 20min. and 40µl of the reaction were directly subjected to bisulfite conversion (E.2.13.3). After bisulfite conversiton, the (+) strand and the (-) strand were PCR amplified with the primer pairs MF89/90 and MF112/113 respectively. PCR products were cloned into the pGEM-T-EASY vector (Promega) following transformation into Xl1Blue E.coli cells. Isolated DNA from positive clones selected by Blue/White screening were sent for sequencing. Evaluation of the data was described elsewhere (E.2.13.6). M.SssI DNA(+) 22clones, M.SssI DNA(-) 13clones, M.SssI nuc (+) 19clones, M.SssI nuc (-) 11clones, D3b2 DNA (+) 31clones , D3b2 DNA (-) 37 clones, D3b2 25nuc (+) 25clones, D3b2nuc (-) 22clones, D3A DNA (+) 23clones, D3A nuc (+) 22clones. Black bars denote nucleosomal, light grey bars represent DNA templates.
The bacterial methyltransferase M.SssI (NEB) efficiently methylated both the upper and lower DNA strands whereas the DNA protected by the nucleosome was almost not accessible for methylation (Figure 39). Dnmt3b2 methylated the sense and anti-sense DNA strands, but to a lower extent compared to M.SssI and with an apparent sequence specificity, since some CpG sites were more frequently methylated than others. Analysis of the (+) and
the (-) strands was intended to see whether the DNA strand facing outwards of the nucleosome could be accessible for DNA methylation. As seen for M.SssI, Dnmt3b2 was also not able to methylate the DNA within the nucleosomal region except for some CpG sites situated at the entry/exit sites of the nucleosome indicating that nucleosomes represent a barrier for de novo methylation and the need for other mechanisms such as moving of histones or disassembly of histones to allow for DNA methylation.
The reaction efficiency of Dnmt3a was extremely low, for both the naked DNA and the nucleosomal template, although the picture of methylation, as for Dnmt3b2 and in comparison to the radioactive data (C.3.5), can be envisioned. After having recapitulated enzymatic prerequisites of Dnmt3a, it seemed that the amount of DNA/nucleosmal template with 20nM CpG sites was extremely below the KM value of 250nM-2500 CpG sites as reported in the literature (Aoki et al. 2001; Gowher and Jeltsch 2001; Yokochi and Robertson 2002; Suetake et al. 2003). Latest results by C. Neuhäuser showed an increase of DNA methylation by Dnmt3a in a reaction setup with 600nM CpG sites to meet the KM value requirements.
As a summary, Dnmt3a and Dnmt3b2 are able to methylate DNA and linker DNA, but hardly within the nucleosome suggesting the need for enzymatic activities like chromatin remodeling enzymes moving the histone octamer and thus providing accessibility.