1. Volume 14, Issue 5, Pages 1156-1168 (February 2016)
The Histone Variant MacroH2A1.2 Is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1 
Stefania Dell’Orso, A. Hongjun Wang, Han-Yu Shih, Kayoko Saso, Libera Berghella, Gustavo Gutierrez-Cruz, Andreas G. Ladurner, John J. O’Shea, Vittorio Sartorelli, Hossein Zare 
Cell Reports 
Volume 14, Issue 5, Pages (February 2016)
DOI: /j.celrep
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2. Cell Reports 2016 14, 1156-1168DOI: (10.1016/j.celrep.2015.12.103)
Copyright © 2016 The Authors Terms and Conditions

3. Figure 1
Genome-wide Distribution of the Histone Variant MacroH2A1.2 and Associated Epigenetic Marks at Regulatory Regions of Skeletal Muscle Cells
(A) Genome-wide distribution of mH2A1.2 in C2C12 MBs and MTs.
(B) Co-localization of mH2A1.2 and epigenetic marks H3K4me3, H3K4me1, H3K27ac, and H3K27me3 in C2C12 MBs and MTs.
(C) ChIP-seq profiles of mH2A1.2 and H3K27ac at Myod1 and Desmin loci.
(D) ChIP-seq profiles of mH2A1.2 and H3K27me3 at Neurogenin2 and Wnt1 loci. Both H3K27ac and mH2A1.2 signals were corrected for input DNA.
(E) GSEA of genes assigned to MT-active enhancers bound by mH2A1.2 in MBs. Genes are ranked from left to right according to their Signal2Noise metric in MTs. The ES profile indicates that the gene set is enriched for upregulated genes in MT (p < 2.0e-4, false discovery rate [FDR] ∼0).
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4. Figure 2
Reducing MacroH2A1.2 Impairs Skeletal Muscle Cell Differentiation
(A and B) Myogenin protein and mRNA evaluated after siRNA against mH2A1.2 in C2C12 cells. GAPDH and histone H2A were used as loading controls. CTRi, control.
(C and D) Myogenin (C) and MHC (D) immunofluorescence staining of control and mH2A1.2i C2C12 cells prompted to differentiate for 2 days. DAPI identifies nuclei.
(E) mH2A1.2 and Myogenin mRNA expression in C2C12 cells transfected with FLAG-empty (CTR) or FLAG-mH2A1.2 (f-mH2A1.2) expression vector (0.8 μg mH2A1.2 plasmid/1 × 105 cells).
(F) Immunoblot for FLAG, Myogenin, and GAPDH in C2C12 cells transfected with FLAG-empty (CTR) or FLAG-mH2A1.2 vector.
Data are represented as mean ± SD.
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5. Figure 3
MacroH2A1.2 Regulates the Transcriptome of Differentiating Skeletal Muscle Cells
(A) Scatterplot shows the inhibitory effect of mH2A1.2 knockdown on transcriptome during differentiation. Each dot represents a gene, the x axis shows expression changes during differentiation in FLAG-empty vector (control; CTR), and the y axis shows the expression changes in mH2A1.2i versus CTR in MTs. Genes marked red and green are upregulated and downregulated during differentiation, respectively.
(B) Venn diagram illustrating number of genes upregulated in control C2C12 MT and downregulated in counterpart mH2A1.2i cells.
(C) GO for genes downregulated in differentiating mH2A1.2i C2C12 cells.
(D) GO for genes whose transcription remains elevated in differentiating mH2A1.2i C2C12 cells.
(E) RNA-seq profiles of downregulated genes Myog, Ckm, and Tnnt2 in differentiating control interference (CTRi) and mH2A1.2i C2C12 cells.
(F) RNA-seq profiles of upregulated genes Id3, Ccnd1, and Mcm5 in differentiating CTLi and mH2A1.2i C2C12 cells.
(G) Myogenin, Actc1, Myh3, Ckm, and Ccnd1 mRNAs were evaluated after siRNA against mH2A1.2 in C2C12 cells.
Data are represented as mean ± SD. ∗p < 0.01.
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6. Figure 4 MacroH2A1.2 Influences H3K27 Acetylation at Enhancer Regions
(A) Venn diagram representing ATAC-seq positive regions in C2C12 MBs and MTs.
(B) Heatmaps of tag densities representing distribution of ATAC-seq signals in C2C12 MBs (red), MTs (blue), and mH2A1.2 binding (green) in MBs.
(C) Average profile of H3K27ac signal in C2C12 MBs (black line), MTs (blue line), and MT-mH2A1.2i (red line) for constitutive enhancers. WT, wild-type.
(D) Average profile of H3K27ac signal in MBs (black line), MTs (blue line) and MT-mH2A1.2i (red line) for MT-specific enhancers.
(E) GSEA of genes assigned to constitutive enhancers. Genes are ranked from left to right according to their Signal2Noise metric in MB-control (MB-CTR) versus MB-mH2A1.2i. The ES profile indicates that the gene set is enriched for downregulated genes in mH2A1.2i-MB (p < 2.0e-4, FDR < 10%).
(F) GSEA of genes assigned to MT-specific enhancers. Genes are ranked from left to right according to their Signal2Noise metric in MT-control (MT-CTR) versus MT-mH2A1.2i. The ES profile indicates that the gene set is enriched for genes strongly downregulated in mH2A1.2i-MT (p < 2.0e-4, FDR < 10%).
(G) ChIP-qPCR for H3K27ac at the Myogenin and Myh3 loci in control (CTR), mH2A1.2i, and mH2A1.2i C2C12 cells transfected with mH2A1.2 expression vector.
Data are represented as mean ± SD. ∗p < 0.01.
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7. Figure 5
Genome-wide Analysis of Pbx1 and MyoD Binding in Skeletal Muscle Cells
(A and B) Genome-wide distribution of Pbx1 binding in C2C12 MBs and MTs.
(C and D) Venn diagrams representing Pbx1 and MyoD peaks in C2C12 MBs and MTs.
(E) MyoD and Pbx1 distribution relative to MT-specific ATAC-seq regions.
(F) ChIP-seq tracks at the Tnnt1 and Myh3 loci. Bottom to top: mH2A1.2 in MBs (red track), H3K4me3 in MTs (green track), H3K27ac in MTs (yellow track), H3K4me1 in MTs (blue track), Pbx1 in MTs (blue tracks), and MyoD in MTs (orange track). The ChIP-seq signals were corrected for input DNA.
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8. Figure 6
MacroH2A1.2 Regulates Recruitment of Pbx1 at Muscle Enhancer Regions
(A) Average profile of Pbx1 signal in MT-WT (blue line) and differentiating mH2A1.2i C2C12 cells (red line) at constitutive enhancers.
(B) Average profiles of Pbx1 signal in MT-WT (blue line), in MT-mH2A1.2i (red line), and in MB-WT at MT-specific enhancers.
(C) ChIP-seq tracks at the Myogenin locus. Top to bottom: mH2A1.2 in MTs and MBs (red tracks); H3K4me1 in MBs and MTs (light blue tracks); H3K4me3 in MBs and MTs (green tracks); H3K27ac in MBs and MTs (yellow tracks); MyoD in MTs (orange track); Pbx1 in MTs (blue tracks); and ATACseq signal in MBs and sMT (purple tracks). Turquoise shading identifies an H3K27ac+/H3K4me1+/Pbx1+/MyoD+/H3K4me3− region.
(D) ChIP-seq tracks at the Myogenin locus. Top to bottom: H3K27ac in MBs, MTs, and MT_mH2A1.2i (yellow tracks); Pbx1 in MT-control (MT-CTR) and mH2A1.2i (blue tracks).
(E) Summary of Pbx1and MyoD occupancy in MBs and MTs, expression in MBs and MTs, expression in mH2A1.2i cells, and assignment to MT-specific enhancers of Pbx1-dependent genes reported in Berkes et al. (2004).
(F) Pbx1 ChIP-qPCR in CTR and mH2A1.2-overexpressing (2 μg mH2A1.2 plasmid/1 × 105 cells).
(G) ChIP-qPCR for Pbx1 at the Myogenin locus in control (CTR), mH2A1.2i, and mH2A1.2i C2C12 cells transfected with mH2A1.2 expression vector.
(H) ChIP-qPCR for MyoD at the Myogenin locus in control (CTR) and mH2A1.2i C2C12 cells.
Data are represented as mean ± SD. ∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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