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. 2010 Jun;30(11):2584-93.
doi: 10.1128/MCB.01451-09. Epub 2010 Mar 29.

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene

Liangjun Wang  1 , Neal Jahren, Ellen L Miller, Carrie S Ketel, Daniel R Mallin, Jeffrey A Simon
Affiliations

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene

Liangjun Wang et al. Mol Cell Biol. 2010 Jun.

Erratum in

Abstract

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets.

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Figures

FIG. 1.
FIG. 1.
Domain organizations of SCM and two related PcG proteins. PH is a subunit of PRC1 (21, 75), and SFMBT is a subunit of PHO-RC (34). All three proteins share a C-terminal SPM domain (solid black) and copies of a Cys2-Cys2 zinc finger (gray). SCM and SFMBT also share multiple mbt repeats (hatched). “Q” represents a glutamine-rich domain. N-terminal portions of PH and SFMBT lacking homology domains are not shown. The mutant lesions of five newly characterized Scm alleles are displayed. Two are nonsense mutations (sz25 and sz29), and three are missense mutations (sz3, sz20, and sz36). The sz3 allele (boxed) is a pupal-lethal hypomorphic mutation used for wing disc ChIPs in this study. The sz20 missense change is identical to the independently isolated ScmSu(z)302 allele (2, 97).
FIG. 2.
FIG. 2.
SCM associates with a Polycomb response element (PRE) upstream of Ubx. (A) The map depicts the Ubx transcription start region and a PRE located ∼25 kb upstream within the bxd regulatory region. Numbered fragments above the map have been described previously (13, 93, 94) and were used in ChIP assays with signals obtained by endpoint PCR. Fragments 1 and 2, below the map, were used in ChIP assays employing Q-PCR signal detection. (B) ChIPs to detect distributions of PHO (left panel) and SCM (right panel) on the Ubx PRE in Drosophila S2 cells. Antibodies used for immunoprecipitations are indicated at the top, and amplified fragments are indicated at the left. “Mock” indicates control immunoprecipitation with protein A-agarose beads alone, and “Rp” indicates a control fragment from the RpII140 locus.
FIG. 3.
FIG. 3.
Consequences of SCM depletion and other PcG protein depletions in S2 cells. (A) Western blots to detect levels of individual PcG proteins after depletion by RNA interference. Labels at the left denote antibodies to detect the indicated PcG proteins or α-tubulin, which was used as a loading control. Labels at the top of each panel identify samples from mock-treated (GFP) or PcG-depleted S2 cells containing the indicated amounts of protein in μg. (B) RT-PCR analysis of Ubx and RpII140 expression after mock treatment (GFP) or treatment with the indicated PcG double-stranded RNAs. “+RT” indicates reverse transcriptase added, and “−RT” indicates a control with reverse transcriptase omitted from the reaction mixture. (C) ChIP analyses to detect associations of the indicated PcG proteins with Ubx PRE fragments b4 (left panel) and b5 (right panel). Labels at the left indicate antibodies (Ab) used in each chromatin immunoprecipitation, and labels at the top identify samples with individual PcG proteins depleted by RNAi. Lanes labeled “GFP” identify mock-depleted samples. PHO is a subunit of PHO-RC, SU(Z)12 is a subunit of PRC2, and PC is a subunit of PRC1.
FIG. 4.
FIG. 4.
Ubx desilencing in Scmsz3 mutant larvae. (A) Patterns of UBX accumulation revealed by immunostaining of a wild-type (WT) haltere disc (left), a wild-type wing disc (middle), and an Scmsz3/ScmH1 wing disc (right). (B) RT-PCR analysis of Ubx and RpII140 mRNA levels in wing discs isolated from wild-type or Scmsz3/ScmH1 mutant larvae. (C) Western blots to detect SCM or α-tubulin (loading control) in extracts from wild-type or Scmsz3/ScmH1 mutant larvae.
FIG. 5.
FIG. 5.
PcG chromatin associations at the Ubx PRE in Scm mutant wing discs. ChIP assays were used to detect PRE binding of PHO, SU(Z)12, PC, and SCM in wild-type or Scmsz3/ScmH1 wing discs as revealed by endpoint PCR (A) and quantitative (real-time) PCR (B). (A) The left panel shows ChIPs using wild-type wing discs, and the right panel shows ChIPs of Scmsz3/ScmH1 wing discs. Antibodies used for immunoprecipitations are indicated at the top, and amplified fragments are indicated at the left. “Mock” indicates control immunoprecipitation with protein A-agarose beads alone. “Rp” indicates a control fragment from the RpII140 locus. (B) Bar graphs depict Q-PCR ChIP signals obtained using antibodies against the indicated PcG proteins and chromatin samples from wild-type (solid bars) or Scmsz3/ScmH1 (hatched bars) wing discs. Error bars show standard deviations from the mean determined using at least two independent ChIP samples for each PcG protein and at least six separate Q-PCRs. For the SU(Z)12 and PC ChIP data (bottom panels), Student's t test yields a P value of ≤0.02 for all comparisons of Scm mutant versus wild type. “PRE1” and “PRE2” correspond to fragments 1 and 2, respectively, in Fig. 2A.
FIG. 6.
FIG. 6.
Consequences of PC knockdown in wing discs. (A and B) Phenotypes of adult flies bearing the UAS-Pc shRNA construct without a GAL4 driver (A) or combined with the A9-GAL4 wing disc driver (B). (C) Western blots to detect PC or α-tubulin (loading control) in wing discs from wild-type (WT) or UAS-Pc shRNA; da-GAL4 larvae. (D) RT-PCR analysis of Ubx and RpII140 mRNA levels in wing discs isolated from wild-type or UAS-Pc shRNA; da-GAL4 larvae. (E) Bar graphs depict Q-PCR ChIP signals obtained using antibodies against the indicated four PcG proteins and wing disc chromatin samples from wild-type (solid bars) or UAS-Pc shRNA; da-GAL4 (hatched bars) larvae. Error bars show standard deviations from the mean determined using a single ChIP for each PcG protein and six independent Q-PCRs.
FIG. 7.
FIG. 7.
Model for chromatin association of PcG complexes. A PcG target gene with an upstream Polycomb response element (PRE). In the first step, PHO-RC, consisting of subunits PHO and SFMBT (34), binds to the PRE via DNA-binding activity of PHO. PRC2 is then recruited, possibly through contact with PHO (93), which methylates local nucleosomes on H3-K27. PRC1 associates with the target gene via interactions with methylated H3-K27 and/or with PHO (42, 43) or PRC2 (60). Red circles depict trimethyl-H3-K27 deposited by PRC2, and red squares depict ubiquitylated H2A on K119 created by PRC1 and/or the PRC1-related dRAF complex (12, 37, 92). The present ChIP data suggest that SCM is recruited independently of PRC2 and PRC1, possibly in parallel to PHO-RC. The protein labeled “X” (green) represents a hypothetical DNA-binding protein that could partner with SCM, akin to PHO partnership with SFMBT. Gray circles represent nucleosomes, and the curved open arrow depicts deposition of histone modifications. The arrow pointing rightward represents the transcription start site.
FIG. 8.
FIG. 8.
Alignment of Cys2-Cys2 zinc fingers in SCM, SFMBT, and PH. Amino acid residues are from the two zinc fingers in Drosophila SCM, the single zinc fingers in Drosophila SFMBT and PH, and the single zinc fingers in the three human PH homologs. This type of zinc finger, termed an FCS zinc finger (IPR012313), is also found in the l(3)mbt [lethal (3) malignant brain tumor] protein, which shares homology domains but is not known to function with PcG proteins. Two human SCM homologs and two human SFMBT homologs apparently lack these FCS zinc fingers. Presumed zinc-coordinating cysteines are shown in bold. The residues shown from each protein are indicated at the right. A consensus sequence is shown at the bottom.
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