Cortical spreading depression differentially affects lysine methylation of H3 histone at neuroprotective genes and retrotransposon sequences.

Recently cortical spreading depression (CSD) has been hypothesized to involve epigenetic control of gene expression, by inducing an overall decrease of H3K4 and increase of H3K9 di-methylation. Here we evaluated the H3K4 and H3K9 di-methylation level at specific loci in rat brains 24 h after CSD induction. Analysis of two selected neuroprotective genes, iNOS and HIF-1α, showed marked increase in lysine 4 di-methylation and decrease in lysine 9 di-methylation of H3 histone. In addition, di-methylation of H3K4 increased moving toward 5' end of the genes in CSD-induced rat hemispheres. Such behavior may reflect an epigenetic molecular memory of actively transcribed genes. We extended our analysis on the H3K4 and H3K9 di-methylation levels of two long interspersed sequences (LINEs). We showed that CSD induction led to di-methylation decrease in lysine 4 and increase in lysine 9 of H3 histone, a trend which reflected the overall chromatin changes previously demonstrated. In conclusion, our data corroborate the hypothesis that epigenetic regulation of gene expression can be affected by CSD and that might be a pivotal molecular mechanism of CSD-induced preconditioning phenomenon which induces tolerance to a subsequent episode of ischemia. In such control, we evidenced two effects: i) a molecular memory of transcribed neuroprotective genes, ii) an epigenetic silencing of retrotransposable sequences.

Relevance of arginines in the mode of binding of H1 histones to DNA.

The mode of binding of sperm and somatic H1 histones to DNA has been investigated by analyzing the effect of their addition on the electrophoretic mobility of linear and circular plasmid molecules. Low concentrations of sperm histones do not appear to alter the electrophoretic mobility of DNA, whereas at increasing concentrations, an additional DNA band is observed near the migration origin. This band then becomes the only component at higher values. In contrast, somatic histones cause a gradual retardation in the mobility of the DNA band at low concentrations and aggregated structures are observed only at higher values. Experiments on the H1 globular domain obtained by limited proteolysis indicate that the mode of binding to DNA depends on the H1 globular domain. The arginine residues appear to be relevant for the different effects as indicated by experiments on sperm histone and on protamine with arginines deguanidinated to ornithines. The modified molecules influence DNA mobility like somatic H1s, indicating that the positive guanidino groups of arginines cannot be substituted by the positive amino groups of ornithines. Modifications of the amino groups of lysines show that these residues are necessary for the binding of H1 histones to DNA but they have no influence on the binding mode.

Epigenetic chromatin modifications in the cortical spreading depression.

Preconditioning with Cortical Spreading Depression induces a sort of tolerance to a subsequent episode of ischemia. The mechanism of this tolerance is not clear. We studied if such treatment induces epigenetic chromatin modifications on the hemispheres of rats preconditioned by Cortical Spreading Depression. The contralateral hemispheres were used as control. We determined the level of H3K4 and H3K9 methylation and the mRNA amounts for the two well known H3K4 methyltransferases (MLL and SET7) in rats 24 degrees h after the Cortical Spreading Depression induction. Western blotting experiments have been performed using three different types of primary antibodies against mono-, di- and tri-methyl H3K4 and primary antibody anti-dimethyl H3K9. In the same samples we checked if the H3 histones were replaced by the H3.3 histone variants that could be an additional marker of chromatin modifications. The level of mono- and di-methyl H3K4 was significantly lower in samples of the treated hemispheres than those of the contralateral hemispheres (40% and about 60%, respectively) while the level of tri-methylation remained unchanged. The level of di-methyl H3K9 was almost 60% higher in the treated hemispheres than the contralateral hemispheres. The treatment for Cortical Spreading Depression affected also the level of expression of H3K4 histone methyltransferase MLL and SET7 that decreased in the treated hemispheres in comparison to the control hemispheres (80% and 40%, respectively). The treatment for Cortical Spreading Depression instead had no effects on the overall amounts of mRNA for H3 and H3.3 histones. In conclusion epigenetic chromatin modifications are evident in rats 24 degrees h after the Cortical Spreading Depression induction.

H3K4 histone methylation in oral squamous cell carcinoma.

Methylation of specific lysine residues in histone tails has been proposed to function as a stable epigenetic marker that directs biological functions altering chromatin structure. Recent findings have implicated alteration in heterochromatin formation as a contributing factor in cancer development. In order to verify whether changes in the overall level of H3K4 histone methylation could be involved in oral squamous carcinoma, the levels of H3K4me1, me2 and me3 were measured in oral squamous carcinoma, leukoplakias and normal tissues. The levels of H3K4me2 and me3 were significantly different in oral squamous cell carcinoma in comparison with normal tissue: the level of H3K4me2 was increased while that of H3K4me3 decreased. No significant differences could be found between the two types of tissues in the level of H3K4me1. A similar trend was found in the leukoplakias that appeared more like the pathological than normal tissue. These results support the idea that alteration of chromatin structure could contribute to oncogenic potential.