Abstract
The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.
Highlights
Interphase eukaryotic nuclei contain two forms of chromatin [1]: densely DNA-stained regions termed heterochromatin and more diffusely stained regions called euchromatin
Both these transgenic lines carry an human CD2 (hCD2) transgene with a truncated locus control region (LCR - which is known to be necessary for chromosomal position-independent expression of the transgene) and have been shown to exhibit variegated expression of hCD2 protein on the surface of T cells, irrespective of the orientation of the truncated LCR [18]
The following analyses were performed on sorted hCD2 expressing and/or hCD2 non-expressing T cells isolated from mesenteric lymph nodes and spleens of CD2 1.3B and CD2 1.3A14 transgenic mice (Figure 1A). hCD2+ T cells from non-variegating CD2-LCR (called here, minigene 4 (MG4)) transgenic mice [31] were used as controls for some analyses
Summary
Interphase eukaryotic nuclei contain two forms of chromatin [1]: densely DNA-stained regions termed heterochromatin and more diffusely stained regions called euchromatin. Constitutive heterochromatin is enriched with specific chromatin modifications including histone H3 lysine 9 (H3K9) trimethylation (me3) [3,4,5], H4K20me3 [6,7,8] and DNA methylation [9,10,11,12], all of which have been impli-. Epigenetics & Chromatin 2009, 2:14 http://www.epigeneticsandchromatin.com/content/2/1/14 cated in gene silencing. These modifications may occur in a coordinated manner. Mice deficient in Suv39h, a H3K9 histone methyltransferase (HMTase), have reduced DNA methylation at their pericentric repeats [11], indicating the interdependence between these modifications. The relationship between H3K9me and DNA methylation has been implicated in the regulation of genes involved in early development and across species [13,14]
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