Free and chromatin-associated mono-, di-, and trimethylation of histone H4-lysine 20 during development and cell cycle progression

Abstract

Methylation of specific amino acids in histone tails is responsible for packaging DNA into condensed, repressed chromatin, and into open chromatin that is accessible to the transcription machinery. Monomethylation and trimethylation of histone H4-lysine 20 (H4-K20) control the formation of repressed chromatin. Using antibodies that specifically recognize the three methyl marks of histone H4-K20, we characterized their regulation during the cell cycle and throughout development. We find free mono- and trimethylated histone H4-K20 in unfertilized Drosophila eggs and in S2 tissue culture cells. Soluble mono-. di-, and trimethylated H4-K20 are also found in HeLa cells. These soluble modified histones may represent a pool of free histones that can rapidly be incorporated into chromatin. The three methyl marks are each regulated differentially during development and their detection on western blots does not overlap with their detection on chromosomes. Monomethylated H4-K20 is detected on condensed chromosomes throughout development, while di- and trimethylated H4-K20 are detected on metaphase chromosomes at specific stages. Our results suggest that the detection of methylated H4-K20 on chromosomes may reveal chromatin packaging rather than the distribution of the methyl marks.

Functional Characterization of the Drosophila Hmt4-20/Suv4-20 Histone Methyltransferase

“…We previously studied the distribution of the three H4K20 methyl marks and showed that they are developmentally regulated and are detected cell-cycle-specifically on chromosomes (Karachentsev et al 2007). The requirement of the H4K20 monomethyl mark is fundamental in vertebrates and flies.…”

“…In vertebrates, Suv4-20-dependent histone H4-K20 trimethylation is strongly associated with constitutive heterochromatin at the chromocenter. The me3 mark appears to be organ-specifically distributed and to change during the life of an animal (Karachentsev et al 2007).…”

Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells

“…This and the observed loss of H4K20me3 mark in cancers (Fraga et al, 2005;Pogribny et al, 2006) seem to support the notion that it is a heterochromatin maintenance marker. Studies in Drosophila have reinforced this idea by showing that the H4K20me3 mark, which in this species is not detectable in nuclei until mid-gastrulation (Karachentsev et al, 2007), was sufficient for heritable maintenance of heterochromatin in the absence of other heterochromatin hallmarks (Phalke et al, 2009). In Xenopus, histone H4K20me3 was not detected until after gastrulation (Dunican et al, 2008).…”

DNA polymerase alpha interacts with PrSet7 and mediates H4K20 monomethylation in Drosophila

“…Primary antibodies were purchased from Abcam (V5 and PCNA mouse monoclonal antibodies); Sigma (mouse anti-tubulin monoclonal antibodies): Rockland (rabbit anti-histone H2AvD); and Upstate (antimonomethyl-H4K20) (Karachentsev et al, 2007). Rabbit polyclonal antibodies against DNA polymerase a and the mouse monoclonal antibody against the DNA polymerase a 180-kDa subunit were as reported previously (Melov et al, 1992;Yamaguchi et al, 1992).…”