Il ruolo della metilazione dell'istone H3 durante la formazione dell'eterocromatina facoltativa
Author(s)
Pasqualini, Barbara
Date Issued
February 20, 2009
Type
Doctoral Thesis
Abstract
The N-termini histone tails protrude from the nucleosome and are subject to posttranslational modifications, including acetylation, methylation, phosphorylation, ubiquitination, and ADPribosylation. During recent years, many histone modifying enzymes and severaI chromatinassociated proteins that specifically bind to these modifications have been identified. Particular histone modification patterns have been associated with distinct chromatin states and are proposed to represent an indexing mechanism that could extend the information potential of the genetic code.
In eukaryotes, histone H3 lysine 9 tri-methylation (K9H3m3) controls gene silencing and plays a central role for the establishment of constitutive heterochromatin. Histone H3 is selectively methylated at lysine 9 by a specific histone methyltransferase (HMTase) codified by Su(var)3-9 gene. The heterochromatin protein HPl binds by its chromodomain to methylated Iysine 9 of histone H3 to propagate the heterochromatic state. H4 trimethylation at Iysine 20 (K20H4m3) is also enriched at pericentric heterochromatin. K9H3 and K20H4 trimethylation seems to be important components of a repressive pathway that can index pericentric heterochromatin.
The possible role of the K9H3 trimethylation by SU(VAR)3-9 in the establishment of facultative heterochromatin has been poorly studied.
The chromosome system of mealybugs provides a dramatic example of facultative heterochromatinization, thus rcpresenting an ideaI model system to investigate such a phenomenon. In male mealybug embryos, the entire paternally-derived, haploid chromosome set becomes heterochromatic at mid-c1eavage, while in female embryos both the paternal plus the maternal complements remain euchromatic throughout ontogeny. In the mealybug Planococcus cifri, PCHET2, an HPl-like protein which is prcferentially associated with the male-specific facultative heterochromatin was previously identified.
In this work explored the role of a Su(var)3-9 ortholog in mealybugs. To this alm, I identified and cloned the Su(var)3-9-like HMTase by PCR amplification from genomic DNA. The nucleotide sequcnce obtained has an high degree of identity (98%) with Drosophila melanogaster's homolog sequence.
I made RNAi experiments with Su(var)3-9-like dsRNA and I showed that the quenching of K9H3-specific HMTase exprcssion in embryos leads to loss K9H3m3 staining concomitant with a loss of staining for PCHET2 (HPI-like), for K9H3m2, for K20H4m3, and the generation of abnormal cytological morphologies of chromatin and chromosomes which are very similar to those observed extinguishing pchet.? expression in embryos by RNAi. Then these data suggest us that the K9H3m3IHPlIK20H4m3 pathway is involved in facultative heterochromatinisation in mealybugs.
I aIso approached to this functional characterization of Su(var)3-9 using the chaetocin, a fungai metabolite discovered as the first inhibitor of a Iysine-specific histone methyltransferase. Chaetocin is spccific for thc methyItransferase SU(VAR)3-9 both in vitro and in vivo and may therefore be uscd to study hcterochromatin-mediated gene repression. Treated embryos showed a Ioss of K9H3m3 staining concomitant with a Ioss of staining for PCHET2, for K9H3m2, for K20H4m3, and the generation of abnormai cytoiogicai morphoIogies of heterochromatin and chromosomes, as I have aIreacly observed extinguishing Su(var)3-9-like expression in embryos by RNAi. The implications of these results wiIl be discussed.
Additional information
Dottorato di ricerca in genetica e biologia cellulare
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