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. 2015 Feb;35(4):662-74.
doi: 10.1128/MCB.01102-14. Epub 2014 Dec 8.

Panspecies Small-Molecule Disruptors of Heterochromatin-Mediated Transcriptional Gene Silencing

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Free PMC article

Panspecies Small-Molecule Disruptors of Heterochromatin-Mediated Transcriptional Gene Silencing

Emilie Castonguay et al. Mol Cell Biol. .
Free PMC article

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.

Figures

FIG 1
FIG 1
HMS-I1 and HMS-I2 disrupt centromeric heterochromatin integrity in fission yeast. (A) Schematic diagram showing the position of the cen1 imr1R(NcoI):kanMX (cen1-kanMX for short) reporter gene in centromere 1 relative to the dg and dh outer repeat (otr) elements, the innermost repeats (imr) and the central core (cnt). The position of heterochromatin and CENP-ACnp1 chromatin domains is indicated. Locations of primers used in panel E below are indicated by the gray and black bars. cen(dg) primers amplify a sequence present in all centromeres. (B) Plating assay of the cen1-kanMX strain on YES plates with 10 μg/ml G418 or without G418 (nonselective). (C) Growth curves of cen1-kanMX cells in YES media with 5 μM HMS-I1, 5 μM HMS-I2, and 10 mM nicotinamide (Nic), with 10 μg/ml G418 or without G418 (nonselective). (D) Chemical structures of HMS-I1 and HMS-I2. (E) qChIP analysis of H3K9me2 levels associated with the cen1-kanMX insertion or cen(dg) repeats, relative to act1+. (F) qChIP analysis of H3K9ac and H3K14ac levels associated with the cen1-kanMX insertion, relative to act1+. In panels E and F, cells were grown in media containing 5 μM HMS-I1 and 5 μM HMS-I2, with 10 μg/ml G418. Error bars indicate standard deviations for three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
FIG 2
FIG 2
HMS-I1 and HMS-I2 phenocopy loss of Clr3 or Sir2. (A) qChIP analysis of H3K9me2 levels associated with the silent mat2-mat3 region of the mating-type locus (mat), relative to act1+. Error bars indicate standard deviations from three biological replicates. (B) Quantification of mating-type gene transcripts (Pc, Pi, and Mc), relative to act1+ transcript levels and normalized to relative levels in untreated mat1Δ cells. Error bars indicate standard deviations from two biological replicates. (C) Northern blot analysis of enriched centromeric siRNAs. snoRNA58 (snR58) was used as a loading control. (D) Quantification of cen(dg) relative to act1+ transcript levels and normalized to relative levels in wild-type cells. Error bars indicate standard deviations for three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. HMS-I1 and HMS-I2 were used at a concentration of 5 μM in panels A to D. The media contained 10 μg/ml G418 in panels A, C, and D.
FIG 3
FIG 3
Clr3 is a putative target of HMS-I1 and HMS-I2. qChIP analysis of H3K9me2 levels associated with centromeric repeats [cen(dg)], relative to act1+. Cells were grown in the presence of 5 μM HMS-I1 or HMS-I2 and without G418 selection. Error bars indicate standard deviations for three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
FIG 4
FIG 4
HMS-I1 and HMS-I2 mimic the effect of SHREC mutations on gene expression. (A) Overlap between gene sets upregulated in cells grown in the presence of HMS-I1 and HMS-I2. (B) Expression profiles of cells grown in the presence of HMS-I1 or HMS-I2 compared to the indicated SHREC mutants. The P value (hypergeometric probabilities) of the overlap between gene lists is indicated in cases where a statistical significance was observed. Genes upregulated in cells that lack RNAi (ago1Δ and dcr1Δ) or the Sir2 HDAC (sir2Δ) were also compared but showed no significant overlap with those upregulated by HMS-I1 and HMS-I2 treatment. *, data from Hansen et al. (69); **, data from Wiren et al. (18); ***, data from the present study (see Data set S1 in the supplemental material). Cells were grown in YES medium with 5 μM HMS-I1 or HMS-I2 and 10 μg/ml G418 in panels A and B. Comparisons in panels A and B are restricted to annotated genes present in the Ekwall laboratory in-house database.
FIG 5
FIG 5
HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity in vitro without affecting recruitment of SHREC at heterochromatin. (A and B) qChIP analysis of Clr3 (Clr3-3XFLAG) (A) and Mit1 (Mit1-13xmyc) (B) recruitment at centromeric repeats [cen(dg)] and at the silent mat2-mat3 region of the mating-type locus (mat), relative to act1+. Cells were grown in the presence of 5 μM HMS-I1 or HMS-I2. Error bars indicate the standard deviations of three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. (C and D) Fluorescence-based deacetylation assays were used to detect inhibition of recombinant S. pombe Clr3 HDAC activity in vitro at the indicated concentrations of HMS-I1 (C) and HMS-I2 (D). Error bars indicate standard deviations for three replicates.
FIG 6
FIG 6
HMS-I1 and HMS-I2 alleviate transcriptional silencing of a GUS transgene in Arabidopsis L5 seedlings. (A) Histochemical staining of A. thaliana L5 transgenic seedlings for β-glucuronidase (GUS) activity after incubation with 10 μM HMS-I1, 10 μM HMS-I2, or solvent control. (B) Magnified view of root tissue. (C) Magnified view of leaf tissue.
FIG 7
FIG 7
HMS-I1 disrupts heterochromatin mediated transcriptional silencing in murine erythroleukemia (MEL) cells. (A) Schematic diagram of the β-globin/eGFP reporter inserted with (MEL RL5/pYB-αSat) or without (MEL RL5/pYB) adjacent α-satellite arrays at the RL5 locus in MEL cells (71). (B) FACS analysis to monitor eGFP expression by fluorescence of RL5/pYB or RL5/pYB-αSat MEL cells. Fluorescence was normalized relative to untreated DMSO controls, after consecutive days of growth in medium containing 10 μM HMS-I1 or 10 μM HMS-I2. Error bars indicate the standard deviations from three biological replicates. (C) qChIP analysis of H3K9me2 and H3K9me3 levels associated with the RL5/pYB-αSat β-globin–eGFP reporter gene and the endogenous mouse amylase gene after 3 days incubation with or without 10 μM HMS-I1. Error bars indicate standard deviations for three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
FIG 8
FIG 8
HMS-I1 reduces the activity of mammalian HDAC6 and HDAC10 in vitro. (A) Fluorescence-based deacetylation assays were used to detect inhibition of recombinant human HDAC6 and HDAC10 activity in vitro at the indicated concentrations of HMS-I1. Error bars indicate the standard deviations from two replicates. (B) Quantification of eGFP transcripts relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript levels after siRNA knockdown of HDAC6 and HDAC10 in MEL cells with a β-globin/eGFP reporter inserted with (RL5/pYB-αSat) or without (RL5/pYB) adjacent α-satellite arrays at the RL5 locus. Error bars indicate the standard deviations from three biological replicates. (C) Quantification of HDAC6 and HDAC10 transcripts relative to GAPDH transcript levels after siRNA knockdown of HDAC6 and HDAC10 in RL5/pYB and RL5/pYB-αSat MEL cells. Error bars indicate standard deviations for three biological replicates. *, P < 0.05.

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