The ACF chromatin-remodeling complex is essential for Polycomb repression

  1. Elizabeth T Wiles  Is a corresponding author
  2. Colleen C Mumford
  3. Kevin J McNaught
  4. Hideki Tanizawa
  5. Eric U Selker  Is a corresponding author
  1. Institute of Molecular Biology, University of Oregon, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Forward genetics identifies ISWI complex members required for repression of H3K27-methylated genes.

(A) Selection scheme with reporter genes inserted at H3K27 methylation-marked loci to select for genes required for silencing. (B) Schematic of protein domains in ISWI and ACF1 with the changes identified in our selection (L430P and D161fs, respectively; marked with asterisks). The conserved nature of the changed residue in ISWI is highlighted for the designated species. (C) Serial dilution spot-test silencing assay for the indicated strains, which all contain PNCU07152::nat-1 on media with or without nourseothricin (NTC). (D), Serial dilution spot-test silencing assay for the indicated strains, which contain PNCU07152::nat-1 and PNCU5173::hph, on media with or without nourseothricin (NTC) or hygromycin (HYG). For complementation tests, wild-type copies of each gene were inserted at the his-3 locus (indicated at left as +iswiWTor +acf1WT). All spot tests were imaged after 48 hr at 32°C and performed at least twice. The number of cells spotted is indicated beneath the images.

Figure 1—figure supplement 1
Genetic mapping and growth rate analysis of mutants identified in the selection.

(A) Whole-genome sequencing of pooled mutant genomic DNA identified a region near the middle of LG VI (indicated by asterisk) that is enriched for Oak Ridge single-nucleotide polymorphisms (SNPs) and contained a point mutation in iswi. Each point represents a running average (window size is 10 SNPs; step size is 1 SNP). (B) Whole-genome sequencing of pooled mutant genomic DNA identified a region near the middle of LG III (indicated by asterisk) that is enriched for Oak Ridge SNPs and contained a two base pair insertion in acf1. Each point represents a running average (window size is 10 SNPs; step size is 1 SNP). (C) Linear growth rates as measured using race tubes for strains with the indicated genotypes. Points represent technical replicates.

Figure 2 with 2 supplements
ISWI and ACF1 interact in Neurospora crassa and are required for repression of a subset of SET-7-repressed genes.

(A) Schematic representation of ISWI-interactions found by immunoprecipitation followed by mass spectrometry. Proteins (ISWI/NCU03875, ACF1/NCU00164, IAF-3/NCU02684, IAF-1/NCU00412, IAF-2/NCU09388, HFP-1/NCU03073, and HFP-2/NCU06623) are depicted to scale. Arrows are drawn from the protein used as the ‘bait’ to the protein partner identified, and unique peptide counts are indicated. Dotted arrows indicate the peptide count was below 0.4% of the total spectrum threshold. Proteins in gray (HFP-1 and HFP-2) were identified as interacting partners but were not used as ‘bait.’ (B) Serial dilution spot-test silencing assay for the indicated strains on media with or without nourseothricin (NTC). All strains have PNCU07152::nat-1. The number of cells spotted is indicated beneath the images, which were generated after incubation for 48 hr at 32°C. Spot test assays were repeated at least twice. (C–G) Expression level (FPKM) for each gene in the indicated mutant strain plotted against the expression level in wild type. Two biological replicates were used for each mutant. Two biological replicates were perfomed twice for wild type. Differentially expressed (DE) genes were defined using a significance cutoff of log2fold change>2 for upregulated genes and log2fold change<–2 for downregulated genes with a p value <0.05. Gray dots indicate genes that are not considered DE. Upper left corner shows the total number of significantly up- and downregulated genes with the number of H3K27-methylated genes in parentheses. Significance for enrichment of H3K27-methylated genes in each DE gene set was calculated by Fisher’s exact test (FPKM - fragments per kilobase per million reads). (H) Venn diagram showing overlap between H3K27-methylated genes that are upregulated (log2fold change>2; p value <0.05) in ∆iswi and ∆acf1 strains. Significant overlap (p<8.036E−54) determined by hypergeometric probability test.

Figure 2—figure supplement 1
Summary of unique peptide counts from immunoprecipitation followed by mass spectrometry.

(A). Protein and affinity tag used as bait is listed at the top of the table and relevant interacting proteins (ISWI/NCU03875, ACF1/NCU00164, IAF-3/NCU02684, IAF-1/NCU00412, IAF-2/NCU09388, HFP-1/NCU03073, and HFP-2/NCU06623) are listed on the left side. Numbers indicate the unique peptide counts.

Figure 2—figure supplement 2
iswi and acf1 are required for regulation of non-H3K27-methylated genes.

(A) Venn diagram showing the overlap of non-H3K27-methylated genes upregulated (log2FC >2; p value <0.05) for the indicated genotypes. (B) Venn diagram showing the overlap of non-H3K27-methylated genes downregulated (log2FC<–2; p value <0.05) for the indicated genotypes. Venn diagrams are not scaled relative to each other. (C) Venn diagram showing the overlap of H3K27-methylated genes upregulated (log2FC >2; p value <0.05) for the indicated genotypes. FC, fold change.

Figure 3 with 2 supplements
iswi and acf1 are required for wild-type H3K27me2/3 and H3K36me3 but loss of these methyl marks is not required for transcriptional upregulation.

(A, B) Scatter plots show the correlation of H3K27me2/3 at genes in wild type and ∆iswi or ∆acf1 based on biological replicates of ChIP-seq data. Green points (n=260 in ∆iswi and n=0 in ∆acf1) represent genes with increased H3K27me2/3 levels (at least twofold over wild type) and red points (n=341 in ∆iswi and n=193 in ∆acf1) represent genes with decreased H3K27me2/3 levels (at least twofold relative to wild type) in the indicated mutant. (C, D) Scatter plots show the correlation of H3K36me3 at genes in wild type and ∆iswi or ∆acf1 based on biological replicates of ChIP-seq data. Green points (n=1 in ∆iswi and n=0 in ∆acf1) represent genes with increased H3K36me3 levels (at least twofold over wild type) and red points (n=444 in ∆iswi and n=317 in ∆acf1) represent genes with decreased H3K36me3 levels (at least twofold relative to wild type) in the indicated mutant. (E, F) Scatter plots show the correlation between H3K27me2/3 and gene expression at H3K27-methylated genes (n=836) in the indicated mutants. Pearson correlation coefficient is reported. Red box indicates genes (n=92 in ∆iswi and n=66 in ∆acf1) that are significantly upregulated (log2 fold change>2) but show no significant loss of H3K27me2/3 (log2 fold change>–1). (G, H) Scatter plots show the correlation between H3K36me3 and gene expression at H3K27-methylated genes (n=836) in the indicated mutants. Pearson correlation coefficient is reported. (I) ChIP-seq tracks showing average level of H3K27me2/3 or H3K36me3 merged from two biological replicates for the indicated strains on LG III. Y-axis is 0–1000 RPKM for H3K27me2/3 tracks and 0–100 average read counts for H3K36me3 tracks. (J) Same as in (I), but for LG IV. (K) Enlarged ChIP-seq tracks showing the underlined region on LG IV from (J). Gene expression changes in ∆iswi are shown. (L) ChIP-qPCR data for H3K27me2/3 at the two genes used for the initial mutant selection (NCU05173 and NCU07152) in the indicated strains. Filled bars represent the mean of technical triplicates and error bars show standard deviation (** for p<0.01, * for p<0.05, and ns for not significant; all relative to wild type by unpaired t-test). Data are from one representative experiment that was performed three times.

Figure 3—source data 1

H3K27me2/3 ChIP-seq comparisons (∆iswi and ∆acf1).

https://cdn.elifesciences.org/articles/77595/elife-77595-fig3-data1-v4.xlsx
Figure 3—source data 2

H3K36me3 ChIP-seq comparisons.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig3-data2-v4.xlsx
Figure 3—source data 3

Comparison of H3K27me2/3 ChIP-seq data and RNA-seq data in ∆iswi and ∆acf1.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig3-data3-v4.xlsx
Figure 3—figure supplement 1
iswi and acf1 are not required for H3K36me2.

(A, B) Scatter plots show the correlation of H3K36me2 at genes in wild type and ∆iswi or ∆acf1 based on biological replicates of ChIP-seq data. Green points (n=9 in ∆iswi and n=1 in ∆acf1) represent genes with increased H3K36me2 levels (at least twofold over wild type) and red points (n=25 in ∆iswi and n=7 in ∆acf1) represent genes with decreased H3K36me2 levels (at least twofold relative to wild type) in the indicated mutant.

Figure 3—figure supplement 2
Loss of iaf-3, iaf-1, and iaf-2 results in minor changes in H3K27me2/3.

(A) Scatter plot showing the correlation of H3K27me2/3 at all genes in wild type and ∆iaf-3 based on biological replicates of ChIP-seq data. Green points represent genes with increased H3K27me2/3 (at least twofold over wild type) and red points represent genes with decreased H3K27me2/3 levels (at least twofold relative to wild type) in ∆iaf-3 strains. (B) Same as in (A), but for ∆iaf-1 strains. (C) Same as in (A), but for ∆iaf-2 strains. (D) ChIP-seq tracks showing average levels of H3K27me2/3 merged from two biological replicates for the indicated strains on the indicated chromosomes (linkage groups). Y-axis is 0–1000 RPKM for all tracks.

Figure 3—figure supplement 2—source data 1

H3K27me2/3 ChIP-seq comparisons (∆iswi, ∆acf1,iaf-3, ∆iaf-1, and ∆iaf-2).

https://cdn.elifesciences.org/articles/77595/elife-77595-fig3-figsupp2-data1-v4.xlsx
Figure 4 with 1 supplement
ACF1 localizes to H3K27me2/3-marked regions of the genome.

(A) Top track shows wild-type H3K27me2/3 levels based on ChIP-seq averaged from two biological replicates for one chromosome (LG III). Y-axis is 0–500 RPKM. Middle two tracks show DamID-seq average reads merged from two biological replicates for the indicated genotypes. Y-axis is 0–500 RPKM. Bottom track compares the DamID-seq reads from ∆set-7 strains to wild-type strains (shown above) displayed as the fold change between the two genotypes. Y-axis is –3–3. (B) Same as in (A), but showing an enlarged view of the right arm of LG III. Region shown is underlined in black in (A). (C) Average enrichment based on DamID-seq for each non-H3K27-methylated gene, scaled to 1 kb, ±500 base pairs, is plotted for the indicated strains. All lines represent average reads from two biological replicates except for Free-Dam which is from only one. TSS, transcription start site; TTS, transcription termination site. (D) Same as in (C), but for H3K27-methylated genes.

Figure 4—figure supplement 1
ACF1 localizes to H3K27me2/3-marked regions of the genome.

(A) DamID Southern blot with genomic DNA from the indicated strains digested with DpnI (I), DpnII (II), or left undigested (–). DpnII, which digests GATC sites without methylated adenines, shows the pattern of complete digestion in wild type. DpnI, which only digests GATC sites bearing adenine methylation, reveals the extent of methylation by Dam at probed regions (NCU05173 and Tel VIIL, H3K27-methylated; his-3, euchromatin). Ethidium bromide (Et-Br) shows total DNA. Biological replicates are shown for ACF1-Dam strains. EPR-1 is a presumptive H3K27 methyl-binding protein and was used as a positive control. EED and SET-7 are both members of the PRC2 complex and are required for H3K27 methylation. See source data for raw, uncropped images. (B) Top track shows wild-type H3K27me2/3 levels averaged from two biological replicates of ChIP on the indicated linkage group. Y-axis is 0–500 RPKM. Middle two tracks show DamID-seq average reads from two biological replicates for the indicated genotypes. Y-axis is 0–500 RPKM. Bottom track compares the DamID-seq reads from ∆set-7 to wild type (above) to show the fold change between the two genotypes. Y-axis is –3–3.

Figure 4—figure supplement 1—source data 1

Raw image for Et-Br gel.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data1-v4.zip
Figure 4—figure supplement 1—source data 2

Raw image for Southern blot probed with NCU05173.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data2-v4.pdf
Figure 4—figure supplement 1—source data 3

Raw image for Southern blot probed with Tel VIIL.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data3-v4.pdf
Figure 4—figure supplement 1—source data 4

Raw image for Southern blot probed with his-3.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data4-v4.pdf
Figure 4—figure supplement 1—source data 5

Raw, uncropped image for Et-Br gel with labels.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data5-v4.pdf
Figure 4—figure supplement 1—source data 6

Raw, uncropped image for Southern blot probed with NCU05173 with labels.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data6-v4.pdf
Figure 4—figure supplement 1—source data 7

Raw, uncropped image for Southern blot probed with Tel VIIL with labels.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data7-v4.pdf
Figure 4—figure supplement 1—source data 8

Raw, uncropped image for Southern blot probed with his-3 with labels.

https://cdn.elifesciences.org/articles/77595/elife-77595-fig4-figsupp1-data8-v4.pdf
Figure 5 with 2 supplements
ISWI and ACF1 position the +1 nucleosome at H3K27-methylated, upregulated genes.

(A) Histogram of the number of H3K27-methylated SD genes (spectral density score for nucleosome order>2; n=358) that have the +1 nucleosome shifted downstream >30 base pairs when compared to wild type in the indicated mutant strains. Each point represents biological replicate 1, biological replicate 2, or analysis of the merged replicates and filled bar is the average of all three values. P values were determined with an unpaired t-test. (B) Venn diagram showing overlap of H3K27-methylated SD genes with a +1 nucleosome shifted downstream >30 bp when iswi or acf1 is deleted. P value was determined by hypergeometric probability test. (C–F) Average nucleosome signal at all H3K27-methylated SD genes plotted from MNase-seq data for the indicated mutants and wild type. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates for the strains indicated in the key. Arrows in (C) and (D) indicate the shifted +1 nucleosome. (G) Average nucleosome signal at SD genes that are upregulated (FDR <0.05) and marked by H3K27 methylation in ∆acf1 strains. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates. The boxed, shaded region is enlarged in the lower panel. (H) Same as panel (G), but for H3K27-methylated SD genes that are not upregulated in ∆acf1 strains. (I) Average nucleosome signal at SD genes that are upregulated (FDR <0.05) and marked by H3K27 methylation in ∆iswi strains. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates. Arrow indicates the shifted +1 nucleosome. (J) Same as (I), but for H3K27-methylated SD genes that are not upregulated in ∆iswi strains. (K, L) Average nucleosome signal at SD genes that are upregulated (FDR <0.05) and not marked by H3K27 methylation in ∆iswi (K) and ∆acf1 (L) strains. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates. (M) Same as (I), but for H3K27-methylated SD genes that are upregulated in ∆set-7.

Figure 5—figure supplement 1
ISWI and its interacting partners have minor effects on nucleosome repeat length in Neurospora crassa.

(A) Autocorrelation function is plotted for all genes (n=9730) in the indicated strains. Biological replicates and the average of the two replicates are shown. The vertical dotted blue line indicates wild-type nucleosome position and the vertical dotted red line indicates mutant nucleosome position. Average repeat length for each strain is shown on the right. (B) Same as in (A), but for all H3K27-methylated genes (n=836).

Figure 5—figure supplement 2
Nucleosome shifts are specific to genes that are H3K27-methylated and upregulated in ∆iswi and ∆acf1.

(A) Histogram of the number of SD genes (spectral density score for nucleosome order>2; n=7753) that have the +1 nucleosome shifted downstream >30 base pairs when compared to wild type in the indicated mutant strains. Each point represents biological replicate 1, biological replicate 2, or analysis of the merged replicates and filled bar is the average of all three values. P values were determined with an unpaired t-test. (B) Plot of the average nucleosome signal at all SD genes. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates for the indicated strain. (C, D) Average nucleosome signal at all H3K27-methylated SD genes plotted from MNase-seq data for the indicated mutants and wild type. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates for the strains indicated in the key.

Figure 6 with 1 supplement
Multifaceted repression in facultative heterochromatin.

(A) Clustered heatmap made using mRNA-seq data for combined biological replicates of the indicated mutant strains. All H3K27-methylated genes that had reads in mRNA-seq data were included (n=821). Clusters (C1–C5) were determined by eye. (B) Model depicting our current framework of factors responsible for maintaining gene silencing in regions marked by H3K27 methylation. Loss of this methyl-mark itself is sufficient to activate a fraction of genes, in part because of loss of the H3K27 methyl-specific factor EPR-1. Repression of many other genes, in H3K27-methylated domains and elsewhere, depend on both H3K36 methylation by ASH1 and both components of the ACF complex (ISWI and ACF). Gray partial square represents an unknown H3K36 methyl binding protein. TF represents unknown transcription factor(s) that could recruit/direct the ACF complex.

Figure 6—figure supplement 1
Loss of ash1 function does not result in a downstream nucleosome shift.

(A) Average nucleosome signal in ash1Y888Fand wild-type strains at SD genes that are upregulated (FDR <0.05) and marked by H3K27 methylation in ∆iswi strains. The three colored lines represent biological replicate 1, biological replicate 2, and the average of the replicates.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Neurospora crassa)MauricevilleFGSC 2225N51mat A; Mauriceville
Strain, strain background (N. crassa)Wild typeFGSC 2489N3752mat A; Oak Ridge
Strain, strain background (N. crassa)Wild typeFGSC 4200N3753mat a; Oak Ridge
Strain, strain background (N. crassa)Sad-1; his-3Wiles et al., 2020N3756mat A; Sad-1; his-3
Strain, strain background (N. crassa)set-7FGSC#
11182
N4718mat a; ∆set-7::hph
Strain, strain background (N. crassa)set-7Jamieson et al., 2018N4730mat A; ∆set-7::bar
Strain, strain background (N. crassa)ash1Y888FBicocca et al., 2018N4878mat A; his-3; ash1Y888F::3xFLAG::hph
Strain, strain background (N. crassa)pNCU07152::nat-1;set-7Wiles et al., 2020N5807Mat A; pNCU07152::nat-1;set-7::bar
Strain, strain background (N. crassa)pNCU07152::nat-1Wiles et al., 2020N5808mat a; pNCU07152::nat-1
Strain, strain background (N. crassa)iswiFGSC 11780N6170mat A; ∆iswi::hph
Strain, strain background (N. crassa)iswiThis studyN6171mat a; ∆iswi::hph
Strain, strain background (N. crassa)Mutant hunt strainWiles et al., 2020N6279mat a; pNCU05173::hph; pNCU07152::nat-1; his-3
Strain, strain background (N. crassa)iswiL430P original mutantThis studyN6606mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; iswiL430P
Strain, strain background (N. crassa)pNCU07152::nat-1; ∆iswi::hphThis studyN6727mat a; pNCU07152::nat-1; ∆iswi::hph
Strain, strain background (N. crassa)ash1Y888FThis studyN6876mat a; ash1Y888F::3xFLAG::nat-1
Strain, strain background (N. crassa)ash1Y888FThis studyN6877mat a; ash1Y888F::3xFLAG::nat-1
Strain, strain background (N. crassa)EPR-1-DamWiles et al., 2020N7525mat A; epr-1::10xGly::Dam::nat-1
Strain, strain background (N. crassa)EPR-1-Dam; ∆eedWiles et al., 2020N7538mat a; epr-1::10xGly::Dam::nat-1;eed::hph
Strain, strain background (N. crassa)Free-Dam; ∆set-7This studyN7476mat A; ∆set-7::hph;his-3+::NLS(SV40)::Dam::3xFLAG::nat-1
Strain, strain background (N. crassa)Free-Dam; ∆set-7This studyN7477mat a; ∆set-7::hph;his-3+::NLS(SV40)::Dam::3xFLAG::nat-1
Strain, strain background (N. crassa)Free-DamThis studyN7802mat A; his-3+::NLS(SV40)::Dam::3xFLAG::nat-1
Strain, strain background (N. crassa)iswiL430P complement-ation strainThis studyN7810mat a; pNCU05173::hph; pNCU07152::nat-1; his-3+::Pccg-1::3xFLAG::iswiWT; iswiL430P
Strain, strain background (N. crassa)pNCU07152::nat-1;iaf-2This studyN7941mat a; pNCU07152::nat-1;iaf-2::hph
Strain, strain background (N. crassa)acf1D161fs original mutantThis studyN7953mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; acf1D161fs
Strain, strain background (N. crassa)pNCU07152::nat-1;acf1This studyN7956mat a; pNCU07152::nat-1;mus-52::baracf1::hph
Strain, strain background (N. crassa)pNCU07152::nat-1;iaf-3::hphThis studyN7960mat A; pNCU07152::nat-1;iaf-3::hph
Strain, strain background (N. crassa)pNCU07152::nat-1;iaf-1:hphThis studyN7961mat a; pNCU07152::nat-1;iaf-1:hph
Strain, strain background (N. crassa)iaf-3This studyN7966mat A; ∆iaf-3::hph
Strain, strain background (N. crassa)ACF1-HAThis studyN7971mat a; ∆mus-52::bar acf1::HA::hph
Strain, strain background (N. crassa)IAF-1-HAThis studyN7973mat A; ∆mus-52::bar; iaf-1::HA::hph
Strain, strain background (N. crassa)∆iaf-2This studyN7988mat a; ∆iaf-2::hph
Strain, strain background (N. crassa)∆iaf-2This studyN7989mat a; ∆iaf-2::hph
Strain, strain background (N. crassa)∆iaf-1FGSC 12715N7990mat a; ∆iaf-1::hph
Strain, strain background (N. crassa)∆iaf-1This studyN7992mat a; ∆iaf-1::hph
Strain, strain background (N. crassa)acf1This studyN8016mat a; ∆acf1::hph
Strain, strain background (N. crassa)acf1This studyN8017mat a; ∆acf1::hph
Strain, strain background (N. crassa)∆iaf-3This studyN8018mat A; ∆iaf-3::hph
Strain, strain background (N. crassa)IAF-3-HAThis studyN8071mat A; pNCU07152::nat-1; iaf-3::HA::hph
Strain, strain background (N. crassa)IAF-2-HAThis studyN8075mat a; pNCU07152::nat-1; iaf-2::HA::hph
Strain, strain background (N. crassa)ACF1-Dam; ∆set-7This studyN8113mat A;set-7::hph; ∆mus-52::bar acf1::Dam::nat-1
Strain, strain background (N. crassa)ACF1-Dam; ∆set-7This studyN8114mat a;set-7::hph; ∆mus-52::bar acf1::Dam::nat-1
Strain, strain background (N. crassa)ACF1-DamThis studyN8115mat A; ∆mus-52:bar acf1::Dam::nat-1
Strain, strain background (N. crassa)acf1D161fscomplement-ation strainThis studyN8142mat a; pNCU05173::hph; pNCU07152::nat-1; his-3+::Pccg-1::acf1WT::mCherry; acf1D161fs
Strain, strain background (N. crassa)ACF1-DamThis studyN8146mat a; ∆mus-52::bar acf1::Dam::nat-1
Strain, strain background (N. crassa)pNCU07152::nat-1;hfp-1This studyN8197mat a; pNCU07152::nat-1;hfp-1::hph
Sequence-based reagenthH4_qPCR_FP
(4082)
Jamieson et al., 2013ChIP-qPCR primerCATCAAGGGGTCATTCAC
Sequence-based reagenthH4_qPCR_RP (4083)Jamieson et al., 2013ChIP-qPCR primerTTTGGAATCACCCTCCAG
Sequence-based reagentNCU07152_promoter_FP
(6565)
Wiles et al., 2020ChIP-qPCR primerCGGTTCCAAAACTGCCCCTGTG
Sequence-based reagentNCU07152_promoter_RP
(6645)
Wiles et al., 2020ChIP-qPCR primerCTCAGCGGGGTATATCAACGGC
Sequence-based reagentNCU05173_promoter_FP
(6567)
Wiles et al., 2020ChIP-qPCR primerGCATTACCCTCGACAGGGTCTG
Sequence-based reagentNCU05173_promoter_RP
(6646)
Wiles et al., 2020ChIP-qPCR primerGCTACCACCATGTGAAGCTCTGG
Sequence-based reagenthis-3_FP
(1665)
Klocko et al., 2019Southern probe primersGACGGGGTAGCTTGGCCCTAATTAACC
Sequence-based reagenthis-3_RP
(3128)
Klocko et al., 2019Southern probe primersCGATTTAGGTGACACTATAG
Sequence-based reagentTel_VIIL_FP
(5271)
Wiles et al., 2020Southern probe primersGGCATCCGTGGGTGTCCCAG
Sequence-based reagentTel_VIIL_RP
(5272)
Wiles et al., 2020Southern probe primersTTCCCGTCCCTACCAGGCAT
Sequence-based reagentNCU05173_FP
(6567)
Wiles et al., 2020Southern probe primersGCATTACCCTCGACAGGGTCTG
Sequence-based reagentNCU05173_RP
(6568)
Wiles et al., 2020Southern probe primersCCTGTTCGAGTTATCGGTGTTG
Antibodyα-H3K27me2/3 (mouse monoclonal)Active MotifCat. #39536Chromatin immunoprecipitation
(2 µl ChIP-seq; 3 µl ChIP-qPCR)
Antibodyα-H3K36me2
(rabbit polyclonal)
AbcamCat. #ab9049Chromatin immunoprecipitation
(2 µl)
Antibodyα-H3K36me3
(rabbit polyclonal)
AbcamCat. #ab9050Chromatin immunoprecipitation
(2 µl)
Antibodyα-HA
(mouse monoclonal)
MBLCat. #180-3Immunoprecipitation
(20 µl)
Antibodyα−FLAG M2 affinity gel (mouse monoclonal)Sigma-AldrichCat. #A2220Immunoprecipitation
(400 µl)
Peptide, recombinant proteinHA peptideThermo Fisher ScientificCat. #26184Elution
Peptide, recombinant protein3× Flag peptideAPExBIOCat. #A6001Elution

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Elizabeth T Wiles
  2. Colleen C Mumford
  3. Kevin J McNaught
  4. Hideki Tanizawa
  5. Eric U Selker
(2022)
The ACF chromatin-remodeling complex is essential for Polycomb repression
eLife 11:e77595.
https://doi.org/10.7554/eLife.77595