Molecular mechanisms of gene silencing mediated by DNA methylation.
DNA methylation and chromatin modification operate along a common pathway to repress transcription; accordingly, several experiments demonstrate that the effects of DNA methylation can spread in cis and do not require promoter modification. In order to investigate the molecular details of the inhibitory effect of methylation, we microinjected into Xenopus oocytes a series of constructs containing a human CpG-rich sequence which has been differentially methylated and cloned at different positions relative to a specific promoter. The parameters influencing the diffusion of gene silencing and the importance of histone deacetylation in the spreading effect were analyzed. We demonstrate that a few methylated cytosines can inhibit a flanking promoter but a threshold of modified sites is required to organize a stable, diffusible chromatin structure. Histone deacetylation is the main cause of gene repression only when methylation does not reach levels sufficient to establish this particular structure. Moreover, contrary to the common thought, promoter modification does not lead to the greater repressive effect; the existence of a competition between transactivators and methyl-binding proteins for the establishment of an open conformation justifies the results obtained.
Curradi
Michela
M
Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi dell'Insubria, 21052 Busto Arsizio (Va), Italy.
Izzo
Annalisa
A
Badaracco
Gianfranco
G
Landsberger
Nicoletta
N
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D009942:Organometallic Compounds; D010455:Peptides; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics*
D002843:Chromatin; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D036341:Intercellular Signaling Peptides and Proteins; D009942:Organometallic Compounds; C074094:PC 1038; D010455:Peptides; C012589:trichostatin A; D004247:DNA; D006655:Histone Deacetylases