1. Volume 165, Issue 2, Pages 331-342 (April 2016)
Suppression of Enhancer Overactivation by a RACK7-Histone Demethylase Complex 
Hongjie Shen, Wenqi Xu, Rui Guo, Bowen Rong, Lei Gu, Zhentian Wang, Chenxi He, Lijuan Zheng, Xin Hu, Zhen Hu, Zhi-Ming Shao, Pengyuan Yang, Feizhen Wu, Yujiang Geno Shi, Yang Shi, Fei Lan 
Cell 
Volume 165, Issue 2, Pages (April 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Identification of RACK7 and KDM5C Interaction and Their Binding Events at Active Enhancers and Super-Enhancers
(A) Schematic representation of the domain architecture of RACK7 protein.
(B) Venn diagram analysis of ChIP-seq peaks of RACK7, H3K4me1, and H3K27Ac. p value by Pearson’s chi-square test is shown.
(C) Total numbers of super-enhancers and RACK7 bound super-enhancers in ZR-75-30, MCF-7, and mESCs. The mESC H3K4me1 and H3K27Ac raw ChIP-seq data were downloaded from GEO (GSM and GSM594578; Creyghton et al., 2010).
(D) Tandem affinity-purified FLAG-HA-RACK7 (F.H.RACK7) protein complex was resolved and visualized by silver staining.
(E) Reciprocal immunoprecipitation between endogenous RACK7 and KDM5C.
(F) In vitro pull-down between recombinant RACK7 and KDM5C proteins.
(G) Venn diagram analysis shows the overlap between RACK7 and KDM5C co-bound regions and active enhancers defined by H3K4me1 and H3K27Ac; p value by Pearson’s chi-square test.
(H) Genome Browser tracks showing RACK7 and KDM5C ChIP-seq signals in parental ZR and KDM5C ChIP-seq in RACK7 KO1 cells at a select genomic location.
(I) KDM5C recruitment to chromatin in the parental and the RACK7 KO1 cells examined by the genome-wide analyses of ChIP-seq signals.
(J) KDM5C recruitment to chromatin in parental ZR and the RACK7 KO cells confirmed by ChIP-qPCR. q-PCR data are represented as mean ± SD from three biological replicates; ∗p < 0.05; ∗∗p < 0.01; t test.
See also Figure S1 and Tables S1 and S2.
Cell  , DOI: ( /j.cell )
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4. Figure 2
RACK7 and KDM5C Suppress H3K4me3 at Active Enhancers and Super-Enhancers
(A) Heatmap analyses of ChIP-seq signals of RACK7, KDM5C, and select histone modifications in the parental and RACK7 KO1 ZR cells, ranked by RACK7 ChIP-seq signals in parental ZR cells. All ChIP-seq signals are displayed from −10 kb to +10 kb surrounding the center of each annotated RACK7 peak.
(B) Normalized levels of H3K4me3 and H3K4me1 at the RACK7-bound super-enhancers in the parental and the RACK7 KO1 ZR cells. p values by ANOVA test are shown.
(C and D) H3K4 methylation states at three representative enhancers in parental ZR and RACK7 KO cell lines, shown by ChIP-seq snapshots (C) and confirmed by ChIP-qPCR (D).
(E and F) RACK7 binding (E) and H3K4me3 (F) level at three select enhancers examined by ChIP-qPCR in the parental, RACK7 KO1, and RACK7 KO1 containing a rescuing, wild-type RACK7 transgene.
(G) H3K4me3 levels at three selected enhancers in parental ZR and KDM5C KO cells.
In all panels, qPCR data are represented as mean ± SD from three biological replicates. ∗p < 0.05; ∗∗p < 0.01; t test. See also Figure S2 and Tables S1 and S2.
Cell  , DOI: ( /j.cell )
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5. Figure 3 RACK7 or KDM5C Loss Leads to an Increase of eRNA Production
(A) Heatmap analyses of H3K4me3 ChIP-seq and nascent RNA-seq data from the parental and RACK7 KO1 ZR cells ranked by RACK7 ChIP-seq signals at all enhancers in the parental ZR cells. Nascent RNA-seq signals are displayed from −2 kb to +2 kb, and ChIP-seq signals are displayed from −10 kb to +10 kb surrounding the centers of the annotated RACK7 peaks. Sense and antisense stands are indicated by “+” and “−”.
(B) Comparison of eRNA levels from RACK7-bound super-enhancers between the parental and RACK7 KO1 ZR cells, as assessed by nascent RNA-seq. p value by ANOVA test is shown.
(C) Snapshots showing eRNA increases at all three select RACK7-bound enhancers in RACK7 KO1 cells.
(D and E) Nascent RNA qRT-PCR confirmation of eRNA increase in the RACK7 KO (D) and KDM5C KO (E) cells from the same three select enhancers shown in (C). All qPCR data are represented as mean ± SD from three biological replicates. ∗p < 0.05; ∗∗p < 0.01; t test.
See also Figure S2 and Tables S1 and S2.
Cell  , DOI: ( /j.cell )
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6. Figure 4 Loss of RACK7 or KDM5C Leads to Hyper-activated Enhancers
(A) Expression comparison (using mRNA-seq data) of the adjacent genes to the RACK7-bound and unbound enhancers (left: top 2,000, and right: bottom 2,000) between the parental and RACK7 KO1 ZR cells. p values by ANOVA test are shown.
(B) Expression comparison (using mRNA-seq data) of the adjacent genes to the RACK7-bound super-enhancers between the parental and RACK7 KO1 ZR cell lines. p value by ANOVA test is shown.
(C) Snapshots showing mRNA increases from the nearest genes of the three select RACK7-bound enhancers in RACK7 KO1 cells.
(D and E) qRT-PCR confirmation of the expression changes of the three select target genes in the parental, RACK7 KO (D), and KDM5C KO (E) cells. Total mRNA samples were used. All qPCR data are represented as mean ± SD from three biological replicates. ∗p < 0.05; ∗∗p < 0.01; t test.
See also Figures S2 and S3 and Tables S1 and S2.
Cell  , DOI: ( /j.cell )
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7. Figure 5
Loss of RACK7 or KDM5C Promotes Tumorigenic Potential of ZR Cells
(A–C) In vitro soft agar (A), invasion (B), and migration (C) assays examining the anchorage-independent growth, invasion, and migration abilities of the parental, RACK7 KO1, and the RACK7 KO1 cells with a rescuing, wild-type RACK7 transgene.
(D) Xenograft growth (24 days; n = 10) analysis of the parental, RACK7 KO1, and the RACK7 KO1 cells with a rescuing, wild-type RACK7 transgene. Quantifications of tumor volume (upper) and representative images of the tumors (lower) are shown. Error bars represent SEM of the mean; ∗∗p < 0.01; t test. The animal protocols were approved by the Animal Welfare Committee of Shanghai Medical College, Fudan University.
(E and F) In vitro invasion (E) and migration (F) assays examining the invasion and migration abilities of the parental and KDM5C KO ZR cells.
(G) RACK7 expression is significantly lower in IDC than the paired DCIS tumors from six breast cancer patients (GDS2046; Schuetz et al., 2006).
In (A)–(C), (E), and (F), all data are represented as mean ± SD from three biological replicates; ∗∗p < 0.01; t test. See also Figure S4.
Cell  , DOI: ( /j.cell )
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8. Figure 6
RACK7 Suppresses Tumorigenesis in Part through Repressing the S100A Family of Oncogenes
(A) ChIP-seq profiles of RACK7, KDM5C, H3K4me1, and H3K27Ac in ZR and nascent RNA-seq and mRNA-seq profiles in the parental and RACK7 KO1 cells at S100A oncogene cluster. S100A4 and S100A16 are selected as examples to show increased eRNA and mRNA expression in the RACK7 KO1 cells (shadow).
(B) S100A4 protein levels in the parental and the RACK7 KO1 cells examined by western blotting.
(C) qRT-PCR showing the expression of multiple S100A oncogenes in the RACK7 KO1 cells compared to the parental ZR cells.
(D) S100A4 expression is significantly higher in IDC than the paired DCIS tumors from the same dataset shown in Figure 5G (GDS2046).
(E) S100A4 protein levels were examined by western blotting in the RACK7 KO1 cells with indicated treatment.
(F) The tumorigenic abilities of the RACK7 KO1 cells with the indicated treatments were examined and showed by the numbers of the invaded and migrated cells.
In (C), all data are represented as mean ± SD from three biological replicates; ∗p < 0.05 and ∗∗p < 0.01; t test.
Cell  , DOI: ( /j.cell )
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9. Figure S1
RACK7/KDM5C Protein Complex Binds Active Enhancers, Related to Figure 1
(A) Venn diagram analysis showing significant overlaps among ChIP-seq peaks of RACK7, p300 and active enhancers (defined as H3K4me1/H3K27Ac overlapped peaks excluding TSS ± 1k). p value by Pearson’s Chi-square test.
(B) Heatmap analyses of ChIP-seq signals of RACK7, H3K4me1, H3K27Ac and p300 in the parental ZR cells, ranked by RACK7 ChIP-seq signals. All ChIP-seq signals are displayed from −10 kb to +10 kb surrounding the center of each annotated RACK7 peak.
(C) Introduction of FLAG-HA-RACK7 (F.H.RACK7) expression in HeLa-S cells.
(D) The exogenous FLAG-HA-RACK7 (F.H.RACK7) expression shown by immunofluorescence.
(E) Polypeptides of RACK7 complex identified by tandem mass spectrometry.
(F) Gel filtration analysis of RACK7, KDM5C and LSD1 in RACK7 complex.
(G) Genome-wide distribution of KDM5C ChIP-seq peaks (8,487) in ZR cells.
(H) Distribution of RACK7 (Red) and KDM5C (Blue) ChIP-seq signals on an aligned unit of active enhancer (left) and super-enhancer (right), p values by ANOVA test.
(I) Protein levels of RACK7 and KDM5C in the parental and the two RACK7 KO ZR cell lines examined by western blotting.
Cell  , DOI: ( /j.cell )
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10. Figure S2
RACK7/KDM5C Regulates Enhancer Activity and Gene Expression, Related to Figures 2, 3, and 4
(A) H3K4me3 increase and H3K4me1 decrease at active enhancers grouped by the intensities of RACK7 ChIP-seq signals in the parental ZR p values by ANOVA test.
(B) Protein levels of RACK7 in the parental and RACK7 KO MCF-7 cell lines examined by western blotting.
(C) Heatmap analyses of ChIP-seq signals of RACK7 and select histone modifications in the parental and RACK7 KO MCF-7 cells, ranked by RACK7 ChIP-seq signals in the parental MCF-7 cells. All ChIP-seq signals are displayed from −10 kb to +10 kb surrounding the center of each annotated RACK7 peak on active enhancers.
(D) Heatmap analyses of ChIP-seq signals of RACK7 and select histone modifications at intergenic regions only in the parental and RACK7 KO1 ZR cells, ranked by RACK7 ChIP-seq signals in the parental ZR cells. Nascent RNA-seq signals are displayed from −2 kb to +2 kb and ChIP-seq signals are displayed from −10 kb to +10 kb surrounding the centers of the annotated RACK7 bound intergenic active enhancers.
(E) Global H3K4 methylation levels were not altered in the two RACK7 KO cell lines.
(F) Reintroduction of FLAG-RACK7 expression in RACK7 KO1 cells. The expression of exogenous FLAG-RACK7 is comparable to the endogenous one.
(G) The exogenous FLAG-RACK7 expression shown by immunofluorescence.
(H) Protein levels of KDM5C and RACK7 in the parental and KDM5C KO cell lines examined by western blotting.
(I) RACK7 bindings to three select enhancers in KDM5C KO were examined by ChIP-qPCR.
(J) Expression comparison (using mRNA-seq data) of the top 2,000 target genes whose promoters (TSS ± 500bp) are bound by RACK7 between the parental and RACK7 KO1 cells. p values by ANOVA test. All q-PCR data are represented as mean ± SD from three biological replicates. ∗p < 0.05; ∗∗p < 0.01, t test.
Cell  , DOI: ( /j.cell )
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11. Figure S3
RACK7/KDM5C-Bound DNA Sequences Possess Enhancer Activities, Related to Figure 4
Enhancer activities of RACK7/KDM5C-bound regions demonstrated by luciferase reporter assays.
(A) Genome coordinates of the 10 select RACK7/KDM5C binding sites used for the luciferase reporter assays.
(B and C) Enhancer activities, reflected by normalized firefly luciferase activities, of the 10 RACK7/KDM5C bound regions were tested in the parental, RACK7 KO1 and RACK7 KO1 containing a rescuing, wild-type RACK7 transgene (B) and KDM5C KO cell lines (C). All data are represented as mean ± SD from three biological replicates, ∗p < 0.05; ∗∗p < 0.01, t test.
Cell  , DOI: ( /j.cell )
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12. Figure S4
RACK7/KDM5C-Regulated Genes Are Enriched in Cell Adhesion-Related Genes, Related to Figure 5
(A) Comparison of the RACK7 ChIP-seq signal densities in the parental ZR cells at the enhancers next to the differentially upregulated genes and differentially downregulated genes upon RACK7 loss. p values by ANOVA test.
(B) GO analysis of the 270 differentially upregulated genes upon RACK7 loss.
(C and D) The indicated cell lines proliferated at similar rates in regular 2D cell culture.
Cell  , DOI: ( /j.cell )
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