Giorgio Prantera

Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals

Abstract. We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.

Heterochromatin and tri-methylated lysine 20 of histone H4 in animals

Tri-methylated lysine 20 on histone H4 (Me(3)K20H4) is a marker of constitutive heterochromatin in murine interphase and metaphase cells. Heterochromatin marked by Me(3)K20H4 replicates late during S phase of the cell cycle. Serum starvation increases the number of cells that exhibit high levels of Me(3)K20H4 at constitutive heterochromatin. Me(3)K20H4 is also present at the centromeric heterochromatin of most meiotic chromosomes during spermatogenesis and at the pseudoautosomal region, as well as at some telomeres. It is not present on the XY-body. During murine embryogenesis the maternal pronucleus contains Me(3)K20H4; Me(3)K20H4 is absent from the paternal pronucleus. On Drosophila polytene chromosomes Me(3)K20H4 is present in a `punctate pattern at many chromosomal bands, including the chromocenter. In coccids it is present on the facultatively heterochromatinised paternal chromosome set. We also present evidence that Me(3)K20H4 is dependent upon H3-specific Suv(3)9 histone methyltransferase activity, suggesting that there may be `epigenetic cross-talk between histones H3 and H4.

DNA methylation changes during mouse spermatogenesis

Genomic imprinting in mammals is thought to be mediated by differences in the methylation level of cytosine residues in the genome. These differences in DNA methylation are thought to be generated during the development of the germ line. To characterize the profile of global methylation of the mouse genome during male gametogenesis, we have quantified the relative level of methylation in individual cells during meiosis and spermatogenesis. A decrease in the level of DNA methylation is observed from meiotic cells to elongated spermatids. The erasure of the somatic pattern of methylation during spermatogenesis suggests the existence of a subsequent mechanism generating the parental specific methylation patterns leading to genomic imprinting of specific alleles.

Inverted meiosis and meiotic drive in mealybugs

In the males of lecanoid coccids, or mealybugs, an entire, paternally derived, haploid chromosome set becomes heterochromatic after the seventh embryonic mitotic cycle. In females, both haploid sets are euchromatic throughout the life cycle. In mealybugs, as in all homopteran species, chromosomes are holocentric. Holocentric chromosomes are characterized by the lack of a localized centromere and consequently of a localized kinetic activity. In monocentric species, sister chromatid cohesion and monopolar attachment play a pivotal role in regulating chromosome behavior during the two meiotic divisions. Both these processes rely upon the presence of a single, localized centromere and as such cannot be properly executed by holocentric chromosomes. Here we furnish further evidence that meiosis is inverted in both sexes of mealybugs and we suggest how this might represent an adaptation to chromosome holocentrism. Moreover, we reveal that at the second meiotic division in males a monopolar spindle is formed, to which only euchromatic chromosomes become attached. By this mechanism the paternally derived, heterochromatic, haploid chromosome set strictly segregates from the euchromatic one, and it is then excluded from the genetic continuum as a result of meiotic drive.

Imprinted Facultative Heterochromatization in Mealybugs

In lecanoid Coccids, or mealybugs, the male development is accompanied by the facultative heterochromatization of the entire, paternally derived, haploid chromosome set. This epigenetic phenomenon occurs in all the cells of mid-cleavage male embryos. Consequently, the Coccid chromosome system offers a powerful tool for gaining insights into the structure of facultative heterochromatin, and into the epigenetic mechanisms of its imprinted, developmentally regulated formation. This paper will present new data and summarize recent studies on genomic imprinting and facultative heterochromatization in mealybugs. First, the existence and the possible role of DNA methylation as an epigenetic modification that fulfills the requisites of the imprinting process in mealybugs will be considered. The second part of this paper will focus on proteins involved in the facultative heterochromatization process. In particular, the involvement of an HP-1-like protein in the silencing of the paternally derived haploid chromosome set and its interaction with the lysine 9 methylated isoform of histone H3 will be discussed.

Effect of Hoechst 33258 on Chinese hamster chromosomes

Cells of the Chinese hamster strain C-125 were treated for different time intervals with H 33258, a bibenzimidazole derivative. The same compound was used to stain fixed cells of the same strain. — H 33258 induced in cells in culture specific areas of reduced spiralization on the metaphase chromosomes of some cells. These probably correspond to DNA segments rich in A-T bases interspersed along the chromosomes. Probably H 33258 acts during S period of cell cycle. — The banding obtained by staining with H 33258 is similar to that induced by quinacrine dihydrochloride but shows a better resolution.

Effects of DAPI on human leukocytes in vitro

DAPI (4-6-diamidino-2-phenylindole), a fluorochrome specific for AT-rich DNA, was supplied for 24 h at various concentrations to human leukocytes in culture. This treatment caused the appearance on the chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1,9, and 16, a short region on the long arm of chromosomes 1 and 2, and the distal heterochromatic part of the long arm of the Y chromosome were despiralized. The despiralization pattern of DAPI is compared with those previously obtained with Hoechst 33258 and Distamycin A.

A Study of the Heterochromatin of Asellus Aquaticus (Crust. Isop.)

SUMMARYThe constitutive heterochromatin of Asellus aquaticus can be differentially stained using the C-banding technique. It is present in the proximity of the NOs and, only rarely, in some of the other telomeres. Part of the A. aquaticus male population presents a heteromorphic chromosome pair due to the presence on one of the metacentric chromosomes of a heterochromatic area localized about half way along each arm. All the heterochromatin of the genome fluoresces brightly when stained with chromomycin A3 but remains dull after staining with quinacrine mustard. It seems therefore reasonable to argue that this is constituted by GC-rich DNA. An analysis of the late-labelling pattern reveals the presence of two chromosomes which replicate later than the constitutive heterochromatin.

Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3-HP1-Me(3)K20H4 pathway.

Using RNA interference (RNAi) we have conducted a functional analysis of the HP1-like chromobox gene pchet2 during embryogenesis of the mealybug Planococcus citri. Knocking down pchet2 expression results in decondensation of the male-specific chromocenter that normally arises from the developmentally-regulated facultative heterochromatinisation of the paternal chromosome complement. Together with the disappearance of the chromocenter the staining levels of two associated histone modifications, tri-methylated lysine 9 of histone H3 [Me(3)K9H3] and tri-methylated lysine 20 of histone H4 [Me(3)K20H4], are reduced to undetectable levels. Embryos treated with double-stranded RNA (dsRNA) targeting pchet2 also exhibit chromosome abnormalities, such as aberrant chromosome condensation, and also the presence of metaphases that contain `lagging chromosomes. We conclude that PCHET2 regulates chromosome behavior during metaphase and is a crucial component of a Me(3)K9H3-HP1-Me(3)K20H4 pathway involved in the facultative heterochromatinisation of the (imprinted) paternal chromosome set.

Silver staining analysis of nucleolar-organizer activity during spermatogenesis of Asellus aquaticus (Crustacea, Isopoda)

The Ag-staining technique was employed to investigate the activity of the nucleolar organizer during spermatogenesis of the isopod crustacean Asellus aquaticus. The most interesting results of this investigation were: (1) The NORs remain continuously Ag-stained for the whole of spermatogenesis until maturation of the sperm, contrary to the situation in the other species so far described; (2) In the gonial mitotic cells the NORs of a single pair, in the meiotic cells of both pairs of chromosomes that have the NORs, are Ag-stained and therefore active; (3) Some of the individuals examined exhibited additional NORs.