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. 2011 Apr;31(4):343-9.
doi: 10.1007/s10059-011-0044-4. Epub 2011 Feb 2.

Deacetylation and Methylation at Histone H3 Lysine 9 (H3K9) Coordinate Chromosome Condensation During Cell Cycle Progression

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Free PMC article

Deacetylation and Methylation at Histone H3 Lysine 9 (H3K9) Coordinate Chromosome Condensation During Cell Cycle Progression

Jin-Ah Park et al. Mol Cells. .
Free PMC article

Abstract

Interphasic chromatin condenses into the chromosomes in order to facilitate the correct segregation of genetic information. It has been previously reported that the phosphorylation and methylation of the N-terminal tail of histone H3 are responsible for chromosome condensation. In this study, we demonstrate that the deacetylation and methylation of histone H3 lysine 9 (H3K9) are required for proper chromosome condensation. We confirmed that H3K9ac levels were reduced, whereas H3K9me3 levels were increased in mitotic cells, via immunofluorescence and Western blot analysis. Nocodazole treatment induced G2/M arrest but co-treatment with TSA, an HDAC inhibitor, delayed cell cycle progression. However, the HMTase inhibitor, AdoX, had no effect on nocodazole-induced G2/M arrest, thereby indicating that sequential modifications of H3K9 are required for proper chromosome condensation. The expression of SUV39H1 and SETDB1, H3K9me3-responsible HMTases, are specifically increased along with H3K9me3 in nocodazole-arrested buoyant cells, which suggests that the increased expression of those proteins is an important step in chromosome condensation. H3K9me3 was highly concentrated in the vertical chromosomal axis during prophase and prometaphase. Collectively, the results of this study indicate that sequential modifications at H3K9 are associated with correct chromosome condensation, and that H3K9me3 may be relevant to the condensation of chromosome length.

Figures

Fig. 1.
Fig. 1.. Changes of histone modifications during cell cycle progression. (A) Histone modifications (red) were determined by immunostaining in culturing A549 cells. DNA was DAPI-stained for assumptions of mitotic stage. Acetylation of H3K9 (H3K9ac) was reduced in the mitotic cells, but trimethyation of H3K9 (H3K9me3) was increased in the mitotic cells. However, monomethylation of H3K9 (H3K9me1) and dimethylation of H3K9 (H3K9me2) were not changed under identical conditions. P-H3S10 is shown as a mitotic control. White arrow head; mitotic cells. (B) Western blot analyses were conducted using synchronized HeLa cells. H3K9me3 and H3K9ac expressions were altered in the nocodazole and colcemid treatment groups, but not in the serum star-vation group.
Fig. 2.
Fig. 2.. Delay of cell cycle progression in deacetylation inhibition, not in methylation inhibition. (A) Nocodazole treatment induced G2/M phase arrest in A549 cells. TSA treatment increased G2/M phase. However, combination treatment of TSA and nocodazole evidenced a delayed G2/M phase. Each cell population was measured and expressed as a number. (B) AdoX was treated for HMTase inhibition. AdoX had no effect on nocodazole-induced G2/M arrest. (C) Western blot analyses were conducted after TSA treatment in HeLa cells. H3K9ac levels were increased in the TSA treatment group.
Fig. 3.
Fig. 3.. Increased expression of H3K9me3-responsible HMT-ases. Nocodazole was applied for 12 h, and the at-tached cells and buoyant cells, respectively, were harvested. (A) Western blotting was conducted with various antibodies including SUV39H1, SETDB1, and G9a. Cyclin B was employed as a marker for G2/M phase. (B) Western blot analyses were conducted in order to detect histone modifications. H3K9me3 was increased in the buoyant cells, whereas nei-ther H3K9me1 or H3K9me2 were altered.
Fig. 4.
Fig. 4.. Histone modification profiles during mitotic stages. Histone modifications were determined by immunostaining in cultured A549 cells. The histone modifications evidenced distinct patterns relative to interphase cells. H3K9me1 and H3K9me2 were temporarily decreased in mitotic entry, but restored at anaphase. H3K9me3 levels were increased during mitotic stages. As a control, p-H3S10 was also increased at prophase and metaphase, and reduced after anaphase.
Fig. 5.
Fig. 5.. Analyses of histone modifications at the chromosome levels. To extract mitotic chromosomes, A549 cells were arrested by colcemid and spread onto the slides with hypotonic solution, followed by immunostaining with the indicated histone antibodies. Histone modifications were detected in the chromosome arms, where-as H3K9me3 was detected at the vertical axis of the chromosome during prophase and prometaphase.

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