1. Polycomb Protein Ezh1 Promotes RNA Polymerase II Elongation
Kambiz Mousavi, Hossein Zare, A. Hongjun Wang, Vittorio Sartorelli 
Molecular Cell 
Volume 45, Issue 2, Pages (January 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 45, 255-262DOI: (10.1016/j.molcel.2011.11.019)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Ezh1 and Ezh2 Are Bound to Distinct Genomic Regions in Proliferating Myoblasts
(A) Relative Ezh1 and Ezh2 transcript levels (normalized to Gapdh) during C2C12 myogenic differentiation (error bars represent SEM, n = 3).
(B) Protein levels (western blots) in 50% confluent myoblasts (MB) and 2 day differentiated myotubes (MT).
(C) Heat maps depicting the binding patterns of genomic landmarks (H3K4me3 and H3K27me3), Ezh1, and Ezh2 within −5/+5 kbp of transcription start site (TSS) in C2C12 MB; genes are ranked based on H3K4me3+ read density. In these heat maps, correlation between Ezh2 and H3K27me3 is 0.51 and between Ezh1 and H3K27me3, −0.14. Correlation between Ezh1 and H3K4me3 is 0.66.
(D) Ezh1 occupancy profile on H3K4me3+ transcriptionally active HOXA cluster and of Ezh2 on H3K27me3+ repressed HOXD gene cluster.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Ezh1 Is Recruited to Active Loci, and Its Genome-wide Occupancy Overlaps with Transcriptional Machinery
(A) Representative DNA agarose gels displaying association of endogenous Ezh1 (ChIP) with promoters of genes in C2C12 MB and MT (left panel) (n = 1); qPCR graph shows relative levels of Ezh1-bound promoter DNA in either MB or MT (right panel) (n = 1).
(B) Heat maps of tag densities representing binding distribution of H3K4me3, H3K27me3, Pol II (8WG16 ChIP), endogenous Ezh1, and bfEzh1 within −5/+5 kbp of transcription start site (TSS) in C2C12 MT cells (24–48 hr in DM). In these heat maps, correlation between Ezh1 and H3K4me3 is 0.58 and between Ezh1 and Pol II (8WG16 ChIP) Promoters are ranked based on H3K4me3+ read density.
(C) Examples of occupancy profiles on transcribed genes in both MB and MT (DES and MYOD1) and on genes that are only transcribed in MT (MYOG, MYLPF, and CKM).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Ezh1 Is a Positive Regulator of the Transcriptome and Required for Transcriptional Activation of Myogenic Genes
(A) Ezh1 transcript and protein levels are reduced after Ezh1i (i) as compared to control (C) C2C12 cells (n = 3; ∗p < 0.01) (error bars represent SEM).
(B) Average (global) reduction of Ezh1 occupancy profile after Ezh1i (p = 5e − 15) as measured by Ezh1+ read density within −5/+5 kbp of all transcription start sites (TSS).
(C) Ezh1 occupancy profile within −5/+5 kbp of TSS plotted against transcriptional output (measured as reads/kilobase/million, RPKM).
(D) Scatter plot showing changes in the transcriptome in proliferating MB versus early (100% cell confluency)-differentiating C2C12 cells (x axis) as well as after Ezh1i (y axis). Each dot represents relative levels of reads (RPKM) for each gene. Note that the upregulated (as determined by 2-fold cut-off) genes (red dots: 243 genes in total) during differentiation (e.g., Myog, Mylpf, Tnni1) are those affected by Ezh1i (y axis). Green dots represent 87 genes, which are naturally downregulated (as determined by 2-fold cut-off) during myogenic differentiation and stay elevated in Ezh1i cells. Right panel shows gene ontology of genes (1.5-fold cutoff) affected after Ezh1i. Count refers to the number of genes in each category.
(E) Reduction of Myog and Myh protein levels (immunoblot) at early (24 hr in DM) and late time points (48 hr in DM) after Ezh1i in C2C12 cells.
(F) Micrographs of C2C12 cells in late myogenic differentiation stage (48 hr in DM) stained for Myh (red) and DAPI (inset, blue).
(G) Reduction of Myog protein levels (immunoblot) in human skeletal muscle cells after Ezh1i. In this panel, early and late differentiation time points were set at 3 hr and 24 hr in DM, respectively.
(H) Early expression of Myog and Myh proteins (immunoblot) in C2C12 cells after overexpression of full-length mycEzh1 and not with SET domain-deleted (mycEzh1ΔSET) isoform. In this panel, early and late differentiation time points represent 100% cell confluency and 24 hr in DM, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Ezh1 Interacts with RNA Polymerase II and Promotes Transcription Elongation In Vivo
(A) Coimmunoprecipitation of endogenous Pol II (8WG16 and S2P) with Flag or Flag-biotin tandem-purified bfEzh1.
(B–D) Genome-wide Pol II (8WG16) (B), Pol II (S5P) (C), and Pol II (S2P) (D) occupancy profiles in control C2C12 cells and after Ezh1i, on genes upregulated during C2C12 differentiation (TSS, transcription start site; TES, transcription end site).
(E) Schematic of traveling ratio (TR) calculation. “A” represents Pol II tag density at promoter regions, whereas “B” symbolizes Pol II tag density within gene bodies.
(F) Illustrates right-handed shift of TR distribution, indicating reduced Pol II occupancy in gene bodies, at all genes after Ezh1i in early differentiating C2C12 cells (p = 5.8e − 116; Kolmogorov-Smirnov test).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions