BACKGROUND
the basic unit of chromatin in eukaryotes is the nucleosome, which is composed of ~ <dig> base pairs of dna wrapped around an octameric core of the histone molecules h2a, h2b, h <dig> and h <dig>  <cit> .
the amino-terminal tails of these histones are subject to various post-translational modifications such as methylation, acetylation, phosphorylation, ubiquitination and adp-ribosylation  <cit> .
various histone-modifying enzymes able to add or remove chromatin modifications, including histone acetyltransferases , histone deacetylases  and lysine methyltransferases, have been identified  <cit> .
in yeast, hats and hdacs have been found to alter global histone acetylation levels over large regions of chromatin  <cit> .
dna itself may be modified through the methylation of cytosine by dna methyltransferase   <cit> .
histone and dna modifications are dynamic and have wide-ranging and profound effects on many nuclear processes  <cit> .
epigenetic modifications dynamically alter chromatin structure and play an important role in the mitosis-dependent transition from decondensed interphase chromatin to condensed metaphase chromosome in eukaryotes.
phosphorylation of h <dig> is initiated in late g <dig>  and this modification spreads along the chromatin as it undergoes condensation through the end of mitosis in mammalian cells  <cit>  and plant cells  <cit> .
the latest reports show that histone h <dig> phosphorylation at serine  <dig>  is found in transcriptionally active regions such as the nucleolus  <cit> .
in various mammalian cell lines, h4k <dig> is deacetylated in metaphase in contrast to interphase  <cit> .
mono- and diacetylation of newly synthesized histone h <dig> molecules are dramatically decreased from g <dig> to m phase in hela s <dig> cells  <cit> .
in plants, the most intense h <dig> acetylation occurs during replication in both euchromatin and heterochromatin  <cit> .
the deacetylation of h <dig> during the interphase to metaphase transition has been observed to be associated with chromatin condensation in tobacco protoplasts  <cit>  as well as in barley  <cit> .
in tobacco protoplasts, histone h <dig> dimethylation at lysine  <dig>  and histone h <dig> dimethylation at lysine  <dig>  levels remain unchanged during interphase and mitosis  <cit> .
it has been reported that histone acetylation, histone methylation and dna methylation are correlated and combined to regulate heterochromatin assembly in arabidopsis  <cit> .
however, little is known about the relationships among dna methylation, histone methylation and acetylation during mitosis, from prophase to metaphase.
5-azacytidine , an analog of 5-cytosine, cannot be methylated and thereby inhibits dna  methylases, reducing the overall level of dna methylation in chromatin  <cit> .
trichostatin a  is an inhibitor of hdacs and can be used to increase histone acetylation in chromatin  <cit> .
tsa treatment of human fibroblasts for  <dig> hours was found to induce hyperacetylation of chromatin but did not prevent the progression of mitosis  <cit> .
however, in human primary fibroblasts, tsa treatment resulted in impaired chromosome compaction and sister chromatid separation  <cit> .
in addition, tsa treatment for  <dig> hours led to the accumulation of nuclei in metaphase and the appearance of abnormal anaphase events in tobacco protoplasts  <cit> .
these observations imply that histone acetylation plays a complex role in the progression of mitosis.
to investigate the mitosis-dependent cross-talk between histone h <dig> tetraacetylation , dna methylation and h3k <dig> dimethylation  as well as their influence on mitosis in maize, we examined chromatin conformational changes using a micrococcal nuclease  assay.
we also used specific antibody immunostaining to detect and compare h4ac, h3k9me <dig> and dna methylation patterns from prophase to metaphase following 5-ac or tsa treatment.
we also analyzed the effect of 5-ac and tsa on mitotic index.
RESULTS
tsa and 5-ac both induce changes in interphase chromatin conformation
to determine the chromatin state in tsa- and 5-ac-treated cells, micrococcal nuclease  assays were carried out.
as shown in figure  <dig>  after digestion with mnase, treated and untreated nuclei both yielded the typical nucleosome ladder, with a repeat length of about  <dig> bp.
the chromatin from tsa-and 5-ac-treated nuclei was more sensitive to mnase digestion than chromatin from control nuclei , implying that tsa and 5-ac both induced chromatin decondensation.
tsa and 5-ac both affect h <dig> acetylation, h3k <dig> dimethylation and dna methylation
to assess the effects of tsa and 5-ac on levels of h4ac and h3k9me <dig>  western blots were performed with anti-h4ac and anti-h3k9me <dig> antibodies.
the results of the western blots showed that tsa caused a clear increase in global histone h <dig> acetylation  in maize root tips compared with controls .
a decrease in total h3k9me <dig> was also detected in the tsa-treated nuclei  compared with controls  .
interestingly, 5-ac also caused a global increase in the level of h4ac  and a decrease in the level of h3k9me <dig>  .
a dot-blot immunoassay was performed on genomic dna samples spotted on deae membranes to determine changes in overall dna methylation levels in tsa- and 5-ac-treated cells .
dna was spotted onto a deae membrane, and anti-5mec antibody was used to detect cytosine methylation in the different dna samples.
anti-5mec signals decreased after treatment with either tsa or 5-ac , even though figure 3b shows that the amount of each dna sample bound to the membrane was approximately equal.
the mean gray value for dna methylation after treatment with tsa or 5-ac was significantly reduced compared with the value of the control by student's t-test .
moreover, the mean gray value for dna content in the treated and untreated samples was not significantly different  .
our results indicate that tsa and 5-ac both induced a change at the level of histone h <dig> acetylation, h3k9me <dig> methylation and dna methylation, all of which are involved in chromatin decondensation in living maize root cells.
tsa and 5-ac both affect cell cycle progression through mitosis in maize root meristems
to investigate the effects of these drugs on mitotic progression, the mitotic indices from each treatment condition were compared and three independent biological replicates were carried out.
student's t-test show that tsa and 5-ac treatments caused a significant decrease  in the mitotic index .
after tsa- and 5-ac-treatment, the percentage of anaphase reduced from  <dig> % to  <dig> %  and from  <dig> % to  <dig> %  respectively  as well as the percentage of telophase significant decreased .
these data indicate that tsa or 5-ac treatment leads to mitotic arrest and, therefore, the histone deacetylation and dna methylation may play an important role in mitotic progression.
histone h <dig> is hyperacetylated in all stages of mitosis after treatment with either tsa or 5-ac
to investigate in detail how h <dig> hyperacetylation affects mitosis, we performed in situ chromatin immunostaining for both normal maize root tip cells and tsa-treated cells.
deacetylation of histone h <dig> usually occurs during mitosis following chromatin condensation, as seen in the control cells.
immunostaining of normal interphase nuclei with anti-h4ac antibody showed that acetylated histone h <dig> was dispersed within the nucleus but that the nucleoli showed much less acetylation .
in control cells, the deacetylation of histone h <dig> was observed at prophase, and very weak acetylation signals were observed during metaphase and anaphase .
at telophase, anti-tetra-acetyl-histone h <dig> signals reappeared .
figure 4a shows that tsa and 5-ac treatment resulted in the strong tetraacetylation of histone h <dig> during interphase and mitosis in maize root tip cells.
quantification of the signal intensity by measuring mean gray values showed that the acetylation of histone h <dig> was increased by approximately 10% to 50% after treatment with either tsa or 5-ac, compared with the control cells .
treatment with either tsa or 5-ac causes a decrease in global h3k9me <dig> during interphase and mitosis
immunostaining for h3k9me <dig> showed dispersed labeling in interphase nuclei and nuclei from all phases of mitosis in control cells .
the mean gray values of the h3k9me <dig> signals revealed a subtle decrease in h3k9me <dig> following chromatin condensation.
furthermore, following chromatin decondensation, there was a slight increase in h3k9me <dig> during mitosis.
compared with untreated cells, a decrease in h3k9me <dig> was observed in interphase and at all stages of mitosis after treatment with either tsa or 5-ac .
quantification of mean gray values showed that the h3k9me <dig> level was reduced by approximately 30% to 50% after tsa or 5-ac treatment .
tsa or 5-ac treatment causes a decrease in global dna methylation during interphase and mitosis
indirect immunostaining of 5-methylcytidine  showed weak and dispersed dna methylation within control nuclei.
as the cells progressed into prophase and metaphase, the levels of 5-cytosine methylation after chromatin condensation during mitosis were gradually increased, and anti-5-mec signals began to decrease again at telophase .
tsa or 5-ac caused global dna hypomethylation in both interphase and mitotic cells when the root tips were treated for  <dig> hours .
the histogram of mean gray values shows that dna methylation was reduced by 10% to 20% after treatment with either tsa or 5-ac .
discussion
dynamic changes in global h4ac, h3k9me <dig> and dna methylation regulate chromatin state and affect mitotic progression in maize root tips
in our study, h4ac, h3k9me <dig> and dna methylation showed cyclical alternations associated with chromatin condensation and decondensation during mitosis in maize root tip cells.
both tsa and 5-ac treatment induced  chromatin decondensation,  a reduction in the proportion of nuclei undergoing anaphase and telophase and  an increase in h4ac, a decrease in h3k9me <dig> and a decrease in dna methylation during mitosis in maize root tips.
posttranslational modifications contribute to driving changes in chromatin conformation and compaction  <cit> .
h4k16ac has been found to inhibit the formation of compact 30-nanometer-like fibers as well as fiber-fiber interactions in vitro  <cit> .
hdacs can function as part of chromatin-remodeling complexes  <cit> .
therefore, the inhibition of hdacs may influence the function of chromatin-remodeling complexes and subsequently affect chromatin condensation.
5-ac was reported to induce dna hypomethylation, thus causing human chromosome decondensation  <cit> .
dna methylation prevents the histone octamer from interacting with an otherwise high-affinity positioning sequence  <cit>  and thereby influences nucleosome formation  <cit> .
mitosis is a unique cell cycle phase in which duplicated chromosomes are highly condensed.
during mitosis, dynamic changes in histone h <dig> acetylation accompanied by chromatin condensation/decondensation have been reported in mammalian, tobacco and barley cells  <cit> .
in maize root tips, histone h <dig> deacetylation and reestablishing was also observed during mitosis.
this mitosis-specific change in histone acetylation may be conserved in both mammalian and plant cells.
in mammalian cells, h3k9me <dig> dramatically increases in g <dig>  reaching a maximum at metaphase, and then rapidly declines during entry into the next interphase  <cit> .
however, in tobacco protoplasts, there were no detectable changes in h3k9me <dig> and h3k4me <dig> throughout the cell cycle  <cit> .
our results show that, in maize root tips, cells entering mitosis with hypoacetylated histone h <dig> exhibit an altered chromatin conformation associated with h3k9me <dig> hypomethylation and dna hypermethylation.
interestingly, changes in dna methylation during mitosis were observed in maize root tips.
the maize chromomethylase zea methyltransferase  <dig>  has de novo activity and is responsible for the establishment of cpnpg methylation patterns  <cit> .
meanwhile, rna-directed dna methylation and demethylation provide plants with a versatile system that facilitates epigenetic plasticity during development and environmental stress responses  <cit> .
the above mentioned pathways might both be involved in mitosis-specific dna hypermethylation in maize.
in this study, we found that h <dig> hyperacetylation and dna hypomethylation led to cell cycle arrest at metaphase associated with h3k9me <dig> hypomethylation in maize.
zmrpd <dig>  a maize rpd3-type histone deacetylase, can interact with the maize retinoblastoma-related  protein, an important regulator of cell cycle progression  <cit> .
tsa, which inhibits the activity of histone deacetylases, might influence the reaction between zmrpd <dig> and zmrbr <dig> and alter cell cycle progression.
h <dig> hyperacetylation arrests cells at metaphase or anaphase and causes abnormal anaphase in tobacco protoplasts  <cit> .
in human cells, histone hyperacetylation can arrest cells in g2/m phase, prevent sister chromatin separation and cause chromosome segregation defects  <cit> .
the hdac3-akap95/ha95-aurora b pathway regulating mitosis has been characterized in mammalian cells  <cit> , and it is known that histone deacetylation is required for progression through mitosis in both plants and mammals.
complete inactivation of dnmt <dig> led to dna hypomethylation and mitotic catastrophe in human cancer cells  <cit> .
our findings indicate that dna methylation plays an important role in mitotic progression in maize root tips.
histone h <dig> hyperacetylation correlates with a decrease in h3k9me <dig> and dna methylation
during both interphase and mitosis, we observed that inhibition of hdacs by tsa caused hyperacetylation of histone h <dig> and hypomethylation of h3k <dig> during mitosis in maize root tips.
this observation suggests that the acetylation of chromatin proteins may affect histone methylation, either directly or indirectly.
earlier studies proposed that the methylation of lysine residues in histone tails functions as a static and irreversible epigenetic mark directing specific chromatin-mediated processes due to the absence of the corresponding histone demethylases  <cit> .
however, two histone lysine demethylases, lsd <dig> and jmjc, have recently been identified to catalyze the removal of methyl groups on histone lysine residues  <cit> .
the discovery of histone demethylases provides evidence for the dynamic nature of histone methylation.
our results raise the possibility that hyperacetylation of histone h <dig> recruits histone demethylases, resulting in the demethylation of h3k <dig>  histone hypoacetylation is often required to prepare the histone template for histone methyltransferases  that act at different lysine residues  <cit> .
we have determined that tsa mediates a reduction in the level of dna methylation and an increase in histone acetylation in interphase and mitotic cells of maize root tips.
both dna methylation and hypoacetylation of core histones are frequently associated with the repression of gene expression  <cit> .
analysis of dna methylation demonstrated that tsa could cause selective loss of methylation in neurospora  <cit> .
some reports have suggested that histone h <dig> hyperacetylation affects dna methylation levels  <cit> .
hats and hdacs have been reported to alter global histone acetylation levels in yeast  <cit> .
although we cannot rule out the possibility that some gene sites did not lose their methylation, the tsa-mediated loss of global dna methylation in interphase and mitotic cells that we observed in our experiments supports the conclusion that acetylation of histones can control dna methylation  <cit> .
dna hypomethylation leads to an increase in h4ac and a decrease in h3k9me2
the cytosine analog and dna methylase inhibitor, 5-ac, is responsible for reducing dna methylation.
genome-wide loss of dna methylation was observed, and h4ac levels were increased in interphase and mitotic cells during maize root tip growth after 5-ac treatment, implying that dna hypomethylation can also directly or indirectly affect histone acetylation.
methylated dna sequences associated with epigenetic silencing in animals have also been found to be associated with hypoacetylated histones  <cit> .
it is well established that dna methylation can lead to the recruitment of hdacs  <cit> .
the observation of interchangeable effects of 5-ac and tsa indicates a mutually reinforcing relationship between histone acetylation and dna methylation in control of cellular processes.
h3k9me <dig> was also reduced during interphase and throughout mitosis after 5-ac treatment.
there are several possible explanations for the histone hypomethylation effect.
dna hypomethylation may first cause histone hyperacetylation.
subsequently, as discussed above, hyperacetylation of histones would recruit histone demethylases that remove methyl groups on histone lysine residues  <cit> .
alternatively, dna methylation may directly affect the extent of histone methylation.
dna methylation has been reported to be tightly correlated with histone h <dig> methylation  <cit> .
soppe et al.
<cit>  demonstrated that dna methylation controls the methylation of h3k <dig> and heterochromatin assembly in arabidopsis, and this conclusion was supported by tariq et al.
<cit> .
CONCLUSIONS
therefore, we propose that a mutually reinforcing cross-talk exists between histone acetylation, dna methylation and h3k9me <dig> .
histone h <dig> hyperacetylation may cause a loss of global dna methylation, and dna hypomethylation may mediate histone h <dig> acetylation.
histone h <dig> hyperacetylation and dna hypomethylation may mediate decreased h3k9me <dig> levels, but it is also possible that a reduction in h3k9me <dig> may feed back onto the extent of h4ac and dna methylation.
this model needs to be further modified or refined and supplemented using histone methylation mutants or inhibitors and other experimental systems.
more studies will be required to discover the cross-talk connections between modifications of histones and dna and between these modifications and cellular functions.

 the basic unit of chromatin in eukaryotes is the nucleosome, which is composed of ~ base pairs of dna wrapped around an octameric core of the histone molecules h2a, h2b, h and h. the amino-terminal tails of these histones are subject to various post-translational modifications such as methylation, acetylation, phosphorylation, ubiquitination and adp-ribosylation. various histone-modifying enzymes able to add or remove chromatin modifications, including histone acetyltransferases, histone deacetylases and lysine methyltransferases, have been identified. in yeast, hats and hdacs have been found to alter global histone acetylation levels over large regions of chromatin. dna itself may be modified through the methylation of cytosine by dna methyltransferase. histone and dna modifications are dynamic and have wide-ranging and profound effects on many nuclear processes. epigenetic modifications dynamically alter chromatin structure and play an important role in the mitosis-dependent transition from decondensed interphase chromatin to condensed metaphase chromosome in eukaryotes. phosphorylation of h is initiated in late g and this modification spreads along the chromatin as it undergoes condensation through the end of mitosis in mammalian cells and plant cells. the latest reports show that histone h phosphorylation at serine is found in transcriptionally active regions such as the nucleolus. in various mammalian cell lines, h4k is deacetylated in metaphase in contrast to interphase. mono- and diacetylation of newly synthesized histone h molecules are dramatically decreased from g to m phase in hela s cells. in plants, the most intense h acetylation occurs during replication in both euchromatin and heterochromatin. the deacetylation of h during the interphase to metaphase transition has been observed to be associated with chromatin condensation in tobacco protoplasts as well as in barley. in tobacco protoplasts, histone h dimethylation at lysine and histone h dimethylation at lysine levels remain unchanged during interphase and mitosis. it has been reported that histone acetylation, histone methylation and dna methylation are correlated and combined to regulate heterochromatin assembly in arabidopsis. however, little is known about the relationships among dna methylation, histone methylation and acetylation during mitosis, from prophase to metaphase. 5-azacytidine, an analog of 5-cytosine, cannot be methylated and thereby inhibits dna methylases, reducing the overall level of dna methylation in chromatin. trichostatin a is an inhibitor of hdacs and can be used to increase histone acetylation in chromatin. tsa treatment of human fibroblasts for hours was found to induce hyperacetylation of chromatin but did not prevent the progression of mitosis. however, in human primary fibroblasts, tsa treatment resulted in impaired chromosome compaction and sister chromatid separation. in addition, tsa treatment for hours led to the accumulation of nuclei in metaphase and the appearance of abnormal anaphase events in tobacco protoplasts. these observations imply that histone acetylation plays a complex role in the progression of mitosis. to investigate the mitosis-dependent cross-talk between histone h tetraacetylation, dna methylation and h3k dimethylation as well as their influence on mitosis in maize, we examined chromatin conformational changes using a micrococcal nuclease assay. we also used specific antibody immunostaining to detect and compare h4ac, h3k9me and dna methylation patterns from prophase to metaphase following 5-ac or tsa treatment. we also analyzed the effect of 5-ac and tsa on mitotic index.  tsa and 5-ac both induce changes in interphase chromatin conformation to determine the chromatin state in tsa- and 5-ac-treated cells, micrococcal nuclease assays were carried out. as shown in figure after digestion with mnase, treated and untreated nuclei both yielded the typical nucleosome ladder, with a repeat length of about bp. the chromatin from tsa-and 5-ac-treated nuclei was more sensitive to mnase digestion than chromatin from control nuclei, implying that tsa and 5-ac both induced chromatin decondensation. tsa and 5-ac both affect h acetylation, h3k dimethylation and dna methylation to assess the effects of tsa and 5-ac on levels of h4ac and h3k9me western blots were performed with anti-h4ac and anti-h3k9me antibodies. the results of the western blots showed that tsa caused a clear increase in global histone h acetylation in maize root tips compared with controls. a decrease in total h3k9me was also detected in the tsa-treated nuclei compared with controls. interestingly, 5-ac also caused a global increase in the level of h4ac and a decrease in the level of h3k9me. a dot-blot immunoassay was performed on genomic dna samples spotted on deae membranes to determine changes in overall dna methylation levels in tsa- and 5-ac-treated cells. dna was spotted onto a deae membrane, and anti-5mec antibody was used to detect cytosine methylation in the different dna samples. anti-5mec signals decreased after treatment with either tsa or 5-ac, even though figure 3b shows that the amount of each dna sample bound to the membrane was approximately equal. the mean gray value for dna methylation after treatment with tsa or 5-ac was significantly reduced compared with the value of the control by student's t-test. moreover, the mean gray value for dna content in the treated and untreated samples was not significantly different. our results indicate that tsa and 5-ac both induced a change at the level of histone h acetylation, h3k9me methylation and dna methylation, all of which are involved in chromatin decondensation in living maize root cells. tsa and 5-ac both affect cell cycle progression through mitosis in maize root meristems to investigate the effects of these drugs on mitotic progression, the mitotic indices from each treatment condition were compared and three independent biological replicates were carried out. student's t-test show that tsa and 5-ac treatments caused a significant decrease in the mitotic index. after tsa- and 5-ac-treatment, the percentage of anaphase reduced from % to % and from % to % respectively as well as the percentage of telophase significant decreased. these data indicate that tsa or 5-ac treatment leads to mitotic arrest and, therefore, the histone deacetylation and dna methylation may play an important role in mitotic progression. histone h is hyperacetylated in all stages of mitosis after treatment with either tsa or 5-ac to investigate in detail how h hyperacetylation affects mitosis, we performed in situ chromatin immunostaining for both normal maize root tip cells and tsa-treated cells. deacetylation of histone h usually occurs during mitosis following chromatin condensation, as seen in the control cells. immunostaining of normal interphase nuclei with anti-h4ac antibody showed that acetylated histone h was dispersed within the nucleus but that the nucleoli showed much less acetylation. in control cells, the deacetylation of histone h was observed at prophase, and very weak acetylation signals were observed during metaphase and anaphase. at telophase, anti-tetra-acetyl-histone h signals reappeared. figure 4a shows that tsa and 5-ac treatment resulted in the strong tetraacetylation of histone h during interphase and mitosis in maize root tip cells. quantification of the signal intensity by measuring mean gray values showed that the acetylation of histone h was increased by approximately 10% to 50% after treatment with either tsa or 5-ac, compared with the control cells. treatment with either tsa or 5-ac causes a decrease in global h3k9me during interphase and mitosis immunostaining for h3k9me showed dispersed labeling in interphase nuclei and nuclei from all phases of mitosis in control cells. the mean gray values of the h3k9me signals revealed a subtle decrease in h3k9me following chromatin condensation. furthermore, following chromatin decondensation, there was a slight increase in h3k9me during mitosis. compared with untreated cells, a decrease in h3k9me was observed in interphase and at all stages of mitosis after treatment with either tsa or 5-ac. quantification of mean gray values showed that the h3k9me level was reduced by approximately 30% to 50% after tsa or 5-ac treatment. tsa or 5-ac treatment causes a decrease in global dna methylation during interphase and mitosis indirect immunostaining of 5-methylcytidine showed weak and dispersed dna methylation within control nuclei. as the cells progressed into prophase and metaphase, the levels of 5-cytosine methylation after chromatin condensation during mitosis were gradually increased, and anti-5-mec signals began to decrease again at telophase. tsa or 5-ac caused global dna hypomethylation in both interphase and mitotic cells when the root tips were treated for hours. the histogram of mean gray values shows that dna methylation was reduced by 10% to 20% after treatment with either tsa or 5-ac. discussion dynamic changes in global h4ac, h3k9me and dna methylation regulate chromatin state and affect mitotic progression in maize root tips in our study, h4ac, h3k9me and dna methylation showed cyclical alternations associated with chromatin condensation and decondensation during mitosis in maize root tip cells. both tsa and 5-ac treatment induced chromatin decondensation, a reduction in the proportion of nuclei undergoing anaphase and telophase and an increase in h4ac, a decrease in h3k9me and a decrease in dna methylation during mitosis in maize root tips. posttranslational modifications contribute to driving changes in chromatin conformation and compaction. h4k16ac has been found to inhibit the formation of compact 30-nanometer-like fibers as well as fiber-fiber interactions in vitro. hdacs can function as part of chromatin-remodeling complexes. therefore, the inhibition of hdacs may influence the function of chromatin-remodeling complexes and subsequently affect chromatin condensation. 5-ac was reported to induce dna hypomethylation, thus causing human chromosome decondensation. dna methylation prevents the histone octamer from interacting with an otherwise high-affinity positioning sequence and thereby influences nucleosome formation. mitosis is a unique cell cycle phase in which duplicated chromosomes are highly condensed. during mitosis, dynamic changes in histone h acetylation accompanied by chromatin condensation/decondensation have been reported in mammalian, tobacco and barley cells. in maize root tips, histone h deacetylation and reestablishing was also observed during mitosis. this mitosis-specific change in histone acetylation may be conserved in both mammalian and plant cells. in mammalian cells, h3k9me dramatically increases in g reaching a maximum at metaphase, and then rapidly declines during entry into the next interphase. however, in tobacco protoplasts, there were no detectable changes in h3k9me and h3k4me throughout the cell cycle. our results show that, in maize root tips, cells entering mitosis with hypoacetylated histone h exhibit an altered chromatin conformation associated with h3k9me hypomethylation and dna hypermethylation. interestingly, changes in dna methylation during mitosis were observed in maize root tips. the maize chromomethylase zea methyltransferase has de novo activity and is responsible for the establishment of cpnpg methylation patterns. meanwhile, rna-directed dna methylation and demethylation provide plants with a versatile system that facilitates epigenetic plasticity during development and environmental stress responses. the above mentioned pathways might both be involved in mitosis-specific dna hypermethylation in maize. in this study, we found that h hyperacetylation and dna hypomethylation led to cell cycle arrest at metaphase associated with h3k9me hypomethylation in maize. zmrpd a maize rpd3-type histone deacetylase, can interact with the maize retinoblastoma-related protein, an important regulator of cell cycle progression. tsa, which inhibits the activity of histone deacetylases, might influence the reaction between zmrpd and zmrbr and alter cell cycle progression. h hyperacetylation arrests cells at metaphase or anaphase and causes abnormal anaphase in tobacco protoplasts. in human cells, histone hyperacetylation can arrest cells in g2/m phase, prevent sister chromatin separation and cause chromosome segregation defects. the hdac3-akap95/ha95-aurora b pathway regulating mitosis has been characterized in mammalian cells, and it is known that histone deacetylation is required for progression through mitosis in both plants and mammals. complete inactivation of dnmt led to dna hypomethylation and mitotic catastrophe in human cancer cells. our findings indicate that dna methylation plays an important role in mitotic progression in maize root tips. histone h hyperacetylation correlates with a decrease in h3k9me and dna methylation during both interphase and mitosis, we observed that inhibition of hdacs by tsa caused hyperacetylation of histone h and hypomethylation of h3k during mitosis in maize root tips. this observation suggests that the acetylation of chromatin proteins may affect histone methylation, either directly or indirectly. earlier studies proposed that the methylation of lysine residues in histone tails functions as a static and irreversible epigenetic mark directing specific chromatin-mediated processes due to the absence of the corresponding histone demethylases. however, two histone lysine demethylases, lsd and jmjc, have recently been identified to catalyze the removal of methyl groups on histone lysine residues. the discovery of histone demethylases provides evidence for the dynamic nature of histone methylation. our results raise the possibility that hyperacetylation of histone h recruits histone demethylases, resulting in the demethylation of h3k histone hypoacetylation is often required to prepare the histone template for histone methyltransferases that act at different lysine residues. we have determined that tsa mediates a reduction in the level of dna methylation and an increase in histone acetylation in interphase and mitotic cells of maize root tips. both dna methylation and hypoacetylation of core histones are frequently associated with the repression of gene expression. analysis of dna methylation demonstrated that tsa could cause selective loss of methylation in neurospora. some reports have suggested that histone h hyperacetylation affects dna methylation levels. hats and hdacs have been reported to alter global histone acetylation levels in yeast. although we cannot rule out the possibility that some gene sites did not lose their methylation, the tsa-mediated loss of global dna methylation in interphase and mitotic cells that we observed in our experiments supports the conclusion that acetylation of histones can control dna methylation. dna hypomethylation leads to an increase in h4ac and a decrease in h3k9me2 the cytosine analog and dna methylase inhibitor, 5-ac, is responsible for reducing dna methylation. genome-wide loss of dna methylation was observed, and h4ac levels were increased in interphase and mitotic cells during maize root tip growth after 5-ac treatment, implying that dna hypomethylation can also directly or indirectly affect histone acetylation. methylated dna sequences associated with epigenetic silencing in animals have also been found to be associated with hypoacetylated histones. it is well established that dna methylation can lead to the recruitment of hdacs. the observation of interchangeable effects of 5-ac and tsa indicates a mutually reinforcing relationship between histone acetylation and dna methylation in control of cellular processes. h3k9me was also reduced during interphase and throughout mitosis after 5-ac treatment. there are several possible explanations for the histone hypomethylation effect. dna hypomethylation may first cause histone hyperacetylation. subsequently, as discussed above, hyperacetylation of histones would recruit histone demethylases that remove methyl groups on histone lysine residues. alternatively, dna methylation may directly affect the extent of histone methylation. dna methylation has been reported to be tightly correlated with histone h methylation. soppe et al. demonstrated that dna methylation controls the methylation of h3k and heterochromatin assembly in arabidopsis, and this conclusion was supported by tariq et al..  therefore, we propose that a mutually reinforcing cross-talk exists between histone acetylation, dna methylation and h3k9me. histone h hyperacetylation may cause a loss of global dna methylation, and dna hypomethylation may mediate histone h acetylation. histone h hyperacetylation and dna hypomethylation may mediate decreased h3k9me levels, but it is also possible that a reduction in h3k9me may feed back onto the extent of h4ac and dna methylation. this model needs to be further modified or refined and supplemented using histone methylation mutants or inhibitors and other experimental systems. more studies will be required to discover the cross-talk connections between modifications of histones and dna and between these modifications and cellular functions. 
these observations suggest that histone acetylation may affect dna and histone methylation during mitosis. the interchangeable effects of 5-azacytidine and trichostatin a on h acetylation, dna methylation and h3k dimethylation indicate a mutually reinforcing action between histone acetylation, dna methylation and histone methylation with respect to chromatin modification. modifications of dna and histones in various combinations are correlated with many cellular processes. treatment with trichostatin a and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that h deacetylation and dna and h3k methylation may contain the necessary information for triggering mitosis in maize root tips. treatment with trichostatin a, which inhibits histone deacetylases, resulted in increased histone h acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global h3k dimethylation and dna methylation during mitosis in maize root tip cells. treatment with 5-azacytidine, a cytosine analog that reduces dna methylation, caused chromatin decondensation and mediated an increase in h acetylation, in addition to reduced dna methylation and h3k dimethylation during interphase and mitosis. these results suggest that decreased dna methylation causes a reduction in h3k dimethylation and an increase in h acetylation. in this study, we investigated the possible relationship between histone h tetraacetylation, dna methylation and histone h dimethylation at lysine during mitosis in maize root meristems.
 modifications of dna and histones in various combinations are correlated with many cellular processes. in this study, we investigated the possible relationship between histone h tetraacetylation, dna methylation and histone h dimethylation at lysine during mitosis in maize root meristems.  treatment with trichostatin a, which inhibits histone deacetylases, resulted in increased histone h acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global h3k dimethylation and dna methylation during mitosis in maize root tip cells. these observations suggest that histone acetylation may affect dna and histone methylation during mitosis. treatment with 5-azacytidine, a cytosine analog that reduces dna methylation, caused chromatin decondensation and mediated an increase in h acetylation, in addition to reduced dna methylation and h3k dimethylation during interphase and mitosis. these results suggest that decreased dna methylation causes a reduction in h3k dimethylation and an increase in h acetylation.  the interchangeable effects of 5-azacytidine and trichostatin a on h acetylation, dna methylation and h3k dimethylation indicate a mutually reinforcing action between histone acetylation, dna methylation and histone methylation with respect to chromatin modification. treatment with trichostatin a and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that h deacetylation and dna and h3k methylation may contain the necessary information for triggering mitosis in maize root tips. 