Histone H4 methyltransferase activity is cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely follows the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. To expand the observation made in mammalian cells that H4 Lys 20 methylation decreases in S-phase and peaks at mitosis, immunofluorescence studies were performed in Drosophila embryos. A recent report shows that the H4 Lys 20-methyl modification is present in Drosophila and is essential for Drosophila development and viability (Nishioka, 2002b). In addition, the embryos provide an excellent model to study H4 Lys 20 methylation during the cell cycle since they rapidly and repeatedly shift from S-phase to mitosis -- this can be easily determined by DAPI staining. Consistent with the above findings, H4 Lys 20 methylation is clearly detected on chromosomes during both metaphase and anaphase, whereas staining during S-phase results in a faint signal even upon overexposure. It is hypothesized that the faint signal during S-phase most likely reflects a combination of dilution of the modification by histone deposition as well as the decrease in chromatin condensation, which could contribute to a dispersion of the signal resulting in a decreased ability to detect the modification. Regardless, these data confirm that H4 Lys 20 methylation is decreased during S-phase and increased specifically during mitosis (Rice, 2002).
To gain insights into the function of H4-K20 methylation, the highly specific methyl H4-K20-specific antibodies were used to analyze the distribution of methylated H4-K20 on Drosophila polytene chromosomes and mouse embryonic fibroblasts (MEFs). Methylation of histone H4-K20 on polytene chromosomes coincides with condensed chromosomal regions, including chromocentric heterochromatin and numerous bands on the euchromatic arms. Competition experiments were performed to verify that the observed staining pattern was specific for methyl H4-K20. In these experiments, the staining was completely removed with peptides that contained the H4 tail methylated at lysine 20, but not with unmodified peptides or by peptides that contained the H3 tail methylated at lysine 4 or lysine 9. Moreover, costaining of polytene chromosomes with antibodies raised in different organisms, a rabbit polyclonal and a mouse monoclonal antibody to methyl H4-K20, shows complete overlap (Nishioka, 2002b).
Having a monoclonal antibody allowed for a direct comparison of the distribution of methylated H4-K20 with that of other modifications that occur on the H3 and H4 tails, using existing rabbit polyclonal antibodies. Comparison of the distribution of methyl H4-K20 and methyl H3-K9 on polytene chromosomes established that the H4-K20 methylation pattern is distinct from the predominantly chromocentric pattern of methylated H3-K9, a modification that has been associated with constitutive heterochromatin in various species (Nishioka, 2002b).
The distribution of the methyl H4-K20 was analyzed with respect to transcriptionally active or competent genes. To accomplish this, the staining pattern on Drosophila polytene chromosomes obtained with the polyclonal antibody to methyl H4-K20 was compared to that observed with monoclonal antibody to the transcription-engaged form of RNA polymerase II. This analysis demonstrated nonoverlapping patterns for each of the antibodies in the entire chromosomes, except at regions that were not fully spread; this led to the conclusion that methylated H4-K20 was very low or absent from transcriptionally competent regions. Similar results were obtained when the staining pattern of the antibody to methyl H4-K20 was compared to that of the transcriptionally active form of RNA polymerase II at heat shock loci under heat shock (transcriptionally permissive) conditions (Nishioka, 2002b).
To further analyze the association of the methyl H4-K20 mark with transcriptionally silent chromatin, the methyl H4-K20 staining pattern was compared to that obtained with antibodies specific to methyl H3-K4, a mark that has been correlated with transcriptionally competent genes in higher eukaryotes. Consistent with the results with RNAPII staining, nonoverlapping patterns for methyl H3-K4 and methyl H4-K20 were observed in the entire polytene chromosomes, except at regions that were not fully spread. Thus, it is concluded that the methylated H4-K20 modification marks transcriptionally silent chromatin (Nishioka, 2002b).
Previous studies have established that interplay occurs between acetylation and methylation of residues within the H3 and H4 histone tails (Rea, 2000, Wang, 2001a, 2001b; Nishioka, 2002a). The H4 tail can be acetylated at lysines 5, 8, 12, and 16; acetylation of lysines 5 and 12 correlates with histone deposition of newly synthesized histones in Drosophila and human cells (Sobel, 1995). Acetylation of lysine 16 is observed in the transcriptionally hyperactive male X chromosome in Drosophila (Turner, 1992) and is enriched in transcriptionally active chromatin in human cells (Johnson, 1998). In light of the results suggesting that methyl H4-K20 is associated with transcriptionally silent chromatin, tests were performed to see whether methyl H4-K20 affects acetylation of H4-K16. Given that hyperacetylated H4-K16 is a hallmark of the hyperactive Drosophila male X chromosome (Turner, 1992), the presence of methylated H4-K20 and acetylated H4-K16 on this chromosome was analyzed. Significant staining with antibody specific to acetylated H4-K16 is detected on the Drosophila male X chromosome and not on the female X. In contrast, low levels of methylated H4-K20 are detected on the male X chromosome, which is comparable to that of female. Thus, there is an inverse correlation in the number and intensity of bands containing methyl H4-K20 and acetyl H4-K16. These data suggest a negative interplay between methylation of H4-K20 and acetylation of H4-K16 (Nishioka, 2002b).
In the Drosophila gene disruption project, a single, homozygous lethal P element insertion into the 5'-untranslated region of the first exon of CG3307 [l(3)neo41] was isolated. This P element is deleted by Df(3R)red31 and is lethal over the deficiency. This mutant was crossed with a green fluorescent protein (GFP)-tagged TM3 balancer chromosome in order to identify homozygous mutants (pr-set7-/-). Although heterozygous mutants (pr-set7+/-) generated no obvious phenotype, homozygote mutants die as late second instar larvae (Nishioka, 2002b).
In order to determine if the homozygote mutant Drosophila larvae lack methylation on H4-K20, wild-type, homozygote mutant and heterozygote, GFP-positive sibling second instar larvae were collected. Western blot analysis was carried out on homozygote mutant and wild-type extract using anti-methyl H4-K20 antibodies. The results demonstrate that the methyl H4-K20 modification clearly exists in the wild-type (pr-set7+/+) larval extract as well as in their heterozygous siblings (pr-set7+/-). In contrast, the homozygous (pr-set7-/-) larval extract yielded no detectable methyl H4-K20. These observations demonstrate that Drosophila pr-set7 encodes the major H4-K20 HMT in Drosophila and that methylation at this residue is essential for development and viability (Nishioka, 2002b).
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date revised: 27 December 2005
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