Abstract
Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.
Highlights
Heterochromatin underpins gene repression, genome integrity, and chromosome segregation
Fission yeast is amenable to high throughput cell-based screens [36,37,38] and the integrity of its heterochromatin and associated gene silencing have been shown to be sensitive to the histone deacetylases (HDACs) inhibitor trichostatin A (TSA) [39, 40]
2-(2,3-dihydro-1,4-benzo[b][1,4] dioxin-6-yl)-6-methylimidazo[1,2-a]pyridine and N-(1-benzo[b]thiophen-2-yl)-4-(2-chloro-6-fluorobenzyl)piperazine-1-carboxamide, here referred to as HMS-I1 and HMS-I2, respectively, were identified as reproducible hits that increased the resistance of wild-type cen1-kanMX cells to G418 to a similar extent as nicotinamide (Fig. 1C and D)
Summary
Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. Fission yeast is amenable to high throughput cell-based screens [36,37,38] and the integrity of its heterochromatin and associated gene silencing have been shown to be sensitive to the HDAC inhibitor trichostatin A (TSA) [39, 40]. Unbiased small-molecule screens may identify novel compounds that inhibit the function of components of the RNAi-directed chromatin modification system in fission yeast, such as Dicer, Argonaute, Clr H3 lysine 9 methyltransferase and the various HDACs. Because small molecules identified from yeast screens may inhibit conserved orthologs [41,42,43,44], inhibitors of fission yeast heterochromatin integrity may yield insights into related processes in higher eukaryotes, including humans. The HDAC inhibitors vorinostat and romidepsin, as well as the histone lysine methyltransferase inhibitor chaetocin, have antitumorigenic activity [45, 46]
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