Cross-Talk Between Epigenetic Modifications During Mitosis In Maize

Chromatin epigenetic modificaiongs includes histone post-translational modifications (PTMs) and DNA methylation. The core histones, H2A, H2B, H3, and H4, and the H1 family of linker histones are subjected to various PTMs such as methylation, acetylation, phosphorylation, and ubiquitination. DNA itself may be modified by methylation of cytidine. Genetic and immunochemical analyses focusing on the function of individual histone PTMs and DNA methylation have revealed that they play key roles in controlling the accessibility of DNA to regulatory factors and complexes responsible for transcription, replication, repair and cell cycle process through direct effects on nucleosome stability and chromatin compaction and by recruiting other proteins which affect these properties. Currently, the cross-talk among epigenetic modifications during mitosis in maize is poorly understood. Exploring the roles of epigenetic information during mitosis in maize will lay the foundation for elucidating the mechanisms of cell differentiation and development of higher plants.In the present study, trichostatin A (TSA), an inhibitor of HDACs, which can induce increase histone acetylation of chromatin and 5-azacytidine (5-AC), an analog of 5-cytosine, which can not be methylated and inhibit DNA (5-cytosine) methylases to reduce the level of overall DNA methylation of chromatin were used to treat maize root tip cells. Flow cytometric analysis showed that TSA treatment arrested cells at G2 phase, while 5-AC treatment arrested cells at G1 phase. MNase assay showed that treatment with 5-AC or TSA caused the chromatin decondensation in maize root tip cells. The immunostaing assay showed that in control maize root-tip cells, histone H4 was deacetylated, H3K9me2 was demethylated and DNA was hypermethylated from prophase to metaphase. And from metaphase to telophase, the inverse progression of epigenetic modifications changes was associated with chromatin condensation. In the TSA treated cells, immunostaing assay showed that an increase of acetylated histone H4 accompanied with a decrease in global H3K9 dimethylation and DNA methylation during mitosis compared to control maize root tip cells. In the 5-AC treated maize root tip cells, immunostaing assay showed that 5-AC mediated an increase of H4 actylation and a decrease of H3K9me2 and DNA methylation compared to the control maize root tip cells. Western-blotting and DNA methylation dot blotting confirmed these cytological observations. Moreover, the mitotic index was significantly decreased and cells were arrested at metaphase after TSA and 5-AC treated.The above results indicated that histone acetylation might directly or indirectly affect DNA and histone methylation and a decrease in DNA methylation could causes a reduction in H3K9 dimethylation and an increase in H4 acetylation. And TSA and 5-AC treatment that caused the epigenetic modification changes resulted in the cells atrrested at metaphase during mitosis, suggesting that H4 deacetylation and DNA and H3K9 methylation might contain necessary information for mitosis in maize root tips.According to the effect of 5-AC and TSA on H4 acetylation, DNA methylation and H3K9 dimethylation, a mutual re-enhancing cross-talk relationship between histone acetylation, DNA methylation and H3K9me2 is proposed. Histone H4 hyperacetylation may cause a global loss of DNA methylation, and DNA hypomethylation may mediate histone H4 acetylation. Not only may histone H4 hyperacetylation and DNA hypomethylation mediate decreased H3K9me2, but also it is possible that a reduction of H3K9me2 may feed back on the extent of H4ac and DNA methylation.