Effect of Demethylation on the Presence of DNA Methyltransferases in Complex with Histone Methylation Markers in Two Myeloma Cell Lines

Background: Enzymes that catalyze CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B) are also implicated in human developmental disorders and cancers, supporting the critical role of DNA methylation. Posttranslational modifications of histones influence the representation of DNMTs in protein complexes including specific histone markers. H3K4me3, H3K36me3 are histone markers of active gene transcription, while for example H3K9me3, H3K27me3 are histone markers of inactive chromatin associated with repressive transcription of the respective genes.

Objective: The aim of the study was to find whether demethylation of myeloma cells can affect 1) the formation of a complex of the repressive histone marker - H3K9me3 with DNMT3A and/or DNMT3B; and 2) association of the transcriptionally active histone marker H3K4me3 with DNMT1.

Material & Methods: Chromatin lysate was isolated from two myeloma cell lines - KMS12-BM and KMS12-PE, after their 72 hours treatment with 5-Azacytidine (AZA) and/or 5-Aza-2´-deoxycitidine (DAC). Precipitation was performed with antibodies against H3K9me3 and/or H3K4me3. The occurrence of DNMT3A and/or DNMT3B in precipitated complex with H3K9me3 was quantified by qPCR (SYBR Green I) with primers specific for both promoter or coding regions of the respective DNMT genes. The assesment of DNMT1 in complex precipitated with H3K4me3 was quantified by qPCR (SYBR Green I) with primers specific for DNMT1. For result calculation, the 2-delta Ct values of immunoprecipitated samples were normalized to the 2-delta Ct value of IgG respective antibodies, and then the obtained 2(-(delta delta Ct)) value of analyzed samples was normalized to the 2(-(delta delta Ct)) value of control untreated cells (DMSO).

Results: In KMS12-PE myeloma cells, we determined the 2(-(delta delta Ct)) values of DNMT3B in precipitated complex with H3K9me3 - 1.52 and 1.47, in cells treated with 0.5 µM AZA and 0.5 µM DAC, respectively. The obtained 2(-(delta delta Ct) values were higher than that of control untreated (DMSO) myeloma cells [with the of 2(-(delta delta Ct)) value of 0.85]. Increased occurrence of DNMT3A was found in H3K9me3 complex of all analyzed samples in comparison to untreated control cells [with the of 2(-(delta delta Ct)) value of 0.89]; the highest 2(-(delta delta Ct) values - 1.07 and 1.21 were determined after DAC treatment. The obtained 2(-(delta delta Ct)) values of DNMT1 from analyzed precipitated samples with H3K4me3 were lower than that of DMSO treated KMS12-PE cells [with the of 2(-(delta delta Ct)) value of 0.85]. In KMS12-BM, we did not detect any changes in the abundance of DNMT3B in precipitated complexes including H3K4me3.

Conclusion: In KMS12-PE, but not in KMS12-BM, both demethylation agents used, 5-Aza-2´-deoxycitidine and 5-Azacytidine, affected the appearance of DNMT3A and DNMT3B in a protein complex containing the repressive histone marker H3K9me3 with possible potential to inactivate gene transcription.