Chemogenetic CRISPR-Cas9 Screen to Study Cell Cycle Progression

(A) : Schematic of chemogenetic CRISPR-Cas9 screen.

(B) : Volcano plots of camptothecin chemogenetic screen results. The “Differential Gene Effect” was plotted against the -log10(p-value) for this effect for each gene targeted in the screen, as calculated by the Orobas pipeline. Red dotted line indicates the established cut-off. Highlighted dots are genes with known roles in response to each treatment, with blue or yellow dots indicate genes that when inactivated resulted in sensitivity or resistance, respectively, to camptothecin.

(C) : Representative STRING analysis networks for protein complexes with known roles in pathways that we identified as sensitive in the camptothecin chemogenetic screen. Blue dots in the STRING network indicate genes that when inactivated resulted in sensitivity to camptothecin.

(D) : Same as in (B) but for colchicine chemogenetic screen results.

(E) : Same as in (C) but for colchicine screen results.

(F) : Same as in (B) but for palbociclib chemogenetic screen results. (G): Same as in (C) but for palbociclib screen results.

Analysis of Camptothecin and Colchicine Chemogenetic Screen Reveals Novel Players in Cell Cycle Regulation

(A) : Dot plot comparison of the effect of gene mutation across three different screen conditions. Circle color indicates the strength of the positive or negative differential gene effect, circle size indicates the -log10(p-value) of the sgRNA enrichment.

(B) : Volcano plot of genes identified in the camptothecin chemogenetic screen, plotted as in Figure 1B with highlighted dots representing novel genes identified in the camptothecin screen.

(C) : Dot plot of Metascape analysis of significant genes that sensitized or de-sensitized cells to camptothecin. The-log10(p-value) of each term was plotted the enrichment was indicated by color of circle and the percentage of the input of genes associated with a given term is indicated by the size of the circle.

(D) : STRING analysis of genes identified from the analysis of the camptothecin screen. (E), (F) and (G) Same as in (B), (C) and (D) except for the colchicine screen.

Mutation of Mitochondria Genes Attenuates the Sensitivity to Palbociclib

(A) : Dot plot of the -log10(p-value) Metascape analysis of significant genes in the palbociclib chemogenetic screen. The enrichment of a given term is indicated by color of circle and the percentage of the input is indicated by the size of the circle.

(B) : Volcano plot of genes identified from our analysis of the palbociclib screen, plotted as in Figure 1D, with highlighted dots representing novel genes.

(C) : STRING networks of novel protein complexes identified in palbociclib screen. Dots in the STRING network indicate genes that when inactivated resulted in sensitivity (blue) or resistance (yellow) to palbociclib. (D): Dose-response curve of palbociclib-induced proliferation rescue in combination with oxidative phosphorylation inhibitors by PrestoBlue assay. Cells were grown in palbociclib with or without increasing concentrations of rotenone, TTFA or oligomycin. Data represents mean of three technical replicates, normalized to the initial dose of each inhibitor in indicated concentration of palbociclib, ±StdDev. *: p-value<0.05, **: p-value<0.005, ***: p-value<0.0005, n.s.: not significant, two tailed unpaired Student’s t-test.

Loss of Polycomb Repressive Complex Components Display Specific Resistance to Palbociclib

(A) : Volcano plot as in Figure 3B except with members of PR-DUB, PRC1 and PRC2 highlighted.

(B) : STRING analysis network of PRC components. Yellow dots indicate that inactivation of these genes conferred resistance to palbociclib.

(C) Dot plot of comparison of the effect of PRC2 complex member gene mutation across three different screen conditions, as in Figure 2B.

(D) : Dose-response curve of palbociclib-induced proliferation inhibition rescue with GSK126 by Crystal Violet assay. Data was normalized to untreated cells and represents the mean of three technical replicates, ±StdDev.

(E) : Results of competitive proliferation assay for each indicated time point, normalized to the initial GFP+/GFP- ratio of the pool. The performance of each sgRNA in 1.5µM palbociclib vs Mock is shown, after normalizing to control sgRNAs, ±SEM of the GFP+/GFP- ratios of three independent sgRNAs.

(F) : Dose-response curve of palbociclib-induced proliferation inhibition in MTF2Δ and JARID2Δ cells by Crystal Violet assay. Data represents mean staining of three monoclonal knockout cell lines, ±StdDev. (G): BrdU incorporation assay for wild-type, SUZ12Δ, MTF2Δ and JARID2Δ cell lines. Left – Representative BrdU incorporation vs propidium iodide flow cytometry traces. Right – Quantification of BrdU incorporation assay, mean of S-phase cells in three knockout lines ±StDev. *: p-value<0.05, n.s.: not significant, two-tailed unpaired Student’s t-test.

Polycomb 2.1 and PRC2.2 are Differentially Recruited to Promoters with CpG Island in HAP1

(A): Left - Western blots of wild-type, SUZ12Δ, MTF2Δ and JARID2Δ cell extracts probed with the indicated antibodies. Right - Quantification of H3K27me3 signal intensity, normalized to H3, ±StDev. *: p-value<0.05, n.s.: not significant, two-tailed unpaired Student’s t-test.

(B) : Venn diagrams of MTF2Δ or JARID2Δ compared to wild-type cells of left - promoters with decreased H3K27me3 signal in CUT&RUN experiment or right - increased transcript levels in RNA-Seq

(C) : Dot-plot of selected Metascape terms of protein coding genes displaying significantly increased or decreased levels of H3K27me3 or transcripts. Color of the circle indicates the -log10(p-value) of the term and the size of circle indicates the percentage of the genes from the input list were represented in that term.

(D) : Genome browser traces of promoters with decreased H3K27me3 and increased mRNAs that were dependent on MTF2 (left), JARID2 (center) or on the presence either MTF2 or JARID2 (right). Tracks represent combined BED files from two clonal biological replicates.

(E) : Representative heat map of H3K27me3 signal for 1,877 peaks overlapping with CGI. Genomic regions are ordered by the H3K27me3 read density intensity in wild-type cells then plotted for the same loci in MTF2Δ and JARID2Δ cells. Plots are of one of two biological replicate.

(F) : H3K27me3 signal averaged for all CGI-containing promoters for wild-type, MTF2Δ, and JARID2Δ cells.

CCND1 and CCND2 Expression is Increased in MTF2Δ Mutants

(A) : Scatterplot of genes whose log2 fold-changes for MTF2Δ/wild-type ratio of mRNA expression (x-axis) versus promoter H3K27me3 signal (y-axis) had an adjusted p-value of <0.05 and an adjusted p-value <0.1 where plotted.

(B) : Genome browser traces of H3K27me3 signal, transcript abundance and CGIs in the CCND1 and CCND2 promoters. Annotated CGIs indicated by green bar.

(C) : Top – Western blots of Cas9-expressing pools of cells transduced three independent sgRNAs targeting the indicated genes, probed with the indicated antibodies.

(D) : Western blots of whole-cell lysates from a panel of cell lines treated with 10µM GSK126 for the indicated time points, with listed antibodies.

(E) : Left – Western blots of whole-cell lysates from MDA-MB-231 and COS7 cells transduced with shRNA constructs shRNAs targeting SUZ12, MTF2, JARID2 or a scrambled control. Probed with indicated antibodies. Right – Quantification of western blots, CCND1 signal normalized to Vinculin. Each bar is the mean for two different shRNA expressing pools, error bars ±range. *: p-value<0.05, **: p-value<0.005, n.s.: not significant, two tailed unpaired Student’s t-test.

(F) : Left – Representative western blot of total RB1 and P-S807/8111-RB1 with increasing [palbociclib] in WT, MTF2Δ and JARID2Δ cells, probed with indicated antibodies. Right – Quantification of the ratio of P-S807/8111-RB1 to total RB1 signal plotted against [palbociclib], two biological replicates, error bars ±range.

Dosing to Determine Inhibitor Concentration for Chemogenetic Screen

(S1A): Drug dosing experiments were performed to determine screening concentrations. Cells were counted during passage in increasing doses of camptothecin (left), palbociclib (center) and colchicine (right).

(S1B): Representative images of flow cytometry traces from untreated cells or cells treated with 0.7µM palbociclib, 9.2nM colchicine or 1nM camptothecin treated cells for three days, then stained propidium iodide. Plots represent the number of stained cells with a given propidium iodide intensity.

(S1C): Venn diagrams showing overlap for significant genes that sensitized (left) or de-sensitized cells (right) to each condition tested. Genes that were determined as significant in all three screens were omitted in further analyses.

(S1D): Dose-response curve of palbociclib-induced proliferation rescue in combination with oxidative phosphorylation inhibitors by PrestoBlue assay. Data represents mean of three technical replicates, normalized to the initial dose of each inhibitor in indicated concentration of rotenone, ±StdDev. (S1E): Same as in (S1D) but for TTFA.

(S1F): Same as in (S1D) but for Oligomycin.

Assays to Determine Resistance of PRC2 Component Mutants to CDK4/6 Inhibitors

(S2A): Schematic of internally controlled competitive proliferation assay used to validate chemogenetic results or knockout cell line proliferation when treated with palbociclib. In experiments where we generated pooled knockouts, GFP+ cells expressing Cas9 were mixed with GFP- cells without Cas9 (as in Figure 4E). For competitive proliferation experiments with monoclonal knockout cell lines, GFP+, Cas9 expressing cells were mixed with GFP- monoclonal knockout lines (as in Supplemental Figure 2C).

(S2B): Western blots demonstrating the efficacy of indicated sgRNA used in the competitive proliferation assay.

(S2C): Competitive proliferation assay for or monoclonal knockout cell lines. wild-type, MTF2Δ and JARID2Δ cell lines (GFP-) were mixed with wild-type cells expressing Cas9 and GFP (GFP+) and treated with either DMSO (mock) or 1.5µM palbociclib (left), 3.5µM ribociclib (center) or 0.4µM abemaciclib (right). Cells were split every three days and the GFP-/GFP+ ratio was assessed every six days by flow cytometry. (S2D): Western blot of protein extracts from cells treated with DMSO (mock) or 1.5µM palbociclib for 48 hours, probed with indicated antibody. PARP cleavage and BIM from protein extracts from RPE1 cells over-expressing a doxycycline-inducible HA-tagged BIM to induce apoptosis as a control.

Analysis of Changes in H3K27me3 Distribution in CUT&RUN and Differentially Expressed Genes in RNA-Seq Experiments

(S3A): Venn diagrams of the Gencode Annotations of promoters that had significantly up regulated (top row) and down regulated H3K27me3 (bottom row) for MTF2Δ (left) and JARID2Δ cells (right). Significant promoters were determined as having a log2 fold change ±1 and an adjusted p-value of <0.1.

(S3B): Same as in (S3B) only for our RNA-Seq experiments and significant promoters were determined as having a log2 fold change ±1 and an adjusted p-value < 0.05.

(S3C): Top - PCA plot of H3K27me3 peaks called by macs2 from CUT&RUN experiment done in biological duplicate. Bottom – PCA plot of RNA-seq reads for experiment in biological triplicate.

(S3D): Average H3K27me3 distribution over a 10kb window for 1,877 peaks overlapping with CGIs. Genomic regions are ordered by the H3K27me3 read density intensity in wild-type cells then plotted for the same loci in MTF2Δ and JARID2Δ cells. Plots are of one of two biological replicate.

(S3E): Bar plot of log10(p-value) of Reactome (teal bars) and MSigDB (red bars) terms associated with promoters of protein coding genes that contain at least one CGI.

(S3F): Bar plot of -log10(p-value) for the enrichment of a given transcription factors from ENCODE and ChEA databases binding to the list of promoters with overlapping GGIs and H3K27me3 peaks.

Analysis of Differential H3K27me3 Distribution and Transcript Expression of D-type Cyclins in CUT&RUN and RNA-Seq Data Sets

(S4A): Volcano plot of DESeq2 calculated changes in log2 fold-change in H3K27me3 signal in promoters versus the log10(p-value) in enrichment in MTF2Δ cells determined by CUT&RUN. CCND1 and CCND2 location within the dataset are indicated by yellow dots.

(S4B): Same as in (S4A) but for transcript abundance determined by RNA-seq of MTF2Δ cells.

(S4C): Volcano plot of DESeq2 calculated changes in log2 fold-change in H3K27me3 signal in promoters versus the log10(p-value) in enrichment in JARID2Δ cells determined by CUT&RUN. CCND1 and CCND2 location within the dataset are indicated by yellow dots.

(S4D): Same as in (S4C) but for transcript abundance determined by RNA-seq of JARID2Δ cells.

(S4E): Scatter plot of log2 fold-change in transcript abundance vs H3K27me3 promoter signal for genes with an adjusted p-value <0.1 in our CUT&RUN and adjusted p-value <0.05 in our RNA-Seq from JARID2Δ cell lines.

(S4F): Genome browser traces of H3K27me3, transcript coverage and CGI location within the CCND3 promoter region.

Regulation of D-type Cyclin Expression by PRC2.1 and PRC2.2

(S5A): Quantification of protein signal from western blot in Fig. 6C for CCND1 (left), CCND2 (center), and CCND3 (right) normalized to Actin. Each bar is the mean for three biological replicates, error bars ±StDev.

*: p-value<0.05, **: p-value<0.005, ***: p-value<0.0005, n.s.: not significant, two tailed unpaired Student’s t-test.

(S5B): Western blots of whole-cell lysates of three-independently isolated monoclonal SUZ12Δ, MTF2Δ and JARID2Δ knockout cell lines probed with the indicated antibodies.

(S5C): qRT-PCR relative quantification of CCND1, CCND2 and CCND3 mRNA levels in wild-type, SUZ12Δ, MTF2Δ and JARID2Δ cells, three biological replicates, performed in technical triplicate, ±StDev.

*: p-value<0.05, **: p-value<0.005, ***: p-value<0.0005, n.s.: not significant, two tailed unpaired Student’s t-test.

(S5D): Dot plot of log2 fold-change for indicated mRNAs in MTF2Δ and JARID2Δ cells. Established cut-off for significant log2-fold change indicated by dashed grey line.

(S5E): Western blot for a panel of G1 regulators from lysates of wild-type, SUZ12Δ, MTF2Δ and JARID2Δ cell lines from three knockout cell lines probed with indicated antibodies.

(S5F): Competitive proliferation assay of CCND1 and CCND2 overexpression cell lines resistance to palbociclib. Wild-type, dox-inducible CCND1 and dox-inducible CCND2 polyclonal HAP1 cell lines (GFP-) were mixed with wild-type cells expressing GFP (GFP+) and treated with either DMSO (mock) or 1.5µM palbociclib, in the presence or absence of 500ng/mL doxycycline. Mock and palbociclib-containing media, with or without doxycycline was replaced daily. Cells were split and GFP-/GFP+ ratio was assessed by flow cytometry every three days. Fitness of each overexpression of each pool was determined by first normalizing the GFP-/GFP+ ratio to the minus doxycycline control and then the ratio of GFP-/GFP+ between the mock and palbociclib conditions.

(S5G): Co-immunoprecipitation of 2xFLAG-2xStrep-CDK6 expressed in wild-type, SUZ12Δ, MTF2Δ and JARID2Δ knockout cell lines, probed with the indicated antibodies.