Fig 1.
Clustering analysis of H3K4me2 profiles in undifferentiated C2C12 cells.
(A) H3K4me2 profiles identified by k-means clustering. The clustering is based on TSS and the corresponding number of genes is given for each cluster. Genes with multiple TSS can be present in more than one cluster. (B) Discriminant analysis shows a clear distinction of the six clusters identified by k-means clustering. (C) The box plot (25% to 75% quartile) shows the levels of gene expression from the different H3K4me2 clusters in Undiff C2C12 cells. The expression of cluster 1 and cluster 2 genes was compared using the Mann-Whitney U test. (D) GO enrichment analysis of cluster 1 genes using the DAVID functional annotation tool. Top ten biological process terms with an adjusted (Benjamini-Hochberg) P value ≤ 0.01 are indicated. GO terms related to muscle development are highlighted in red.
Fig 2.
Clustering analysis of H3K4me3 profile in undifferentiated C2C12 cells and comparison to H3K4me2 profiles.
(A) H3K4me3 profiles identified by k-means clustering. The clustering is based on TSS and the corresponding number of genes is given for each cluster. Genes with multiple TSS can be present in more than one cluster. (B) The box plot (25% to 75% quartile) shows the levels of gene expression from the different H3K4me3 clusters in Undiff C2C12 cells. The expression of cluster 1 and cluster 2 genes was compared using the Mann-Whitney U test. (C) Overlap of H3K4me2 and H3K4me3 cluster 1 genes in Undiff C2C12 cells. The P value is based on a hypergeometric test. (D) GO enrichment analysis of common cluster 1 genes using the DAVID functional annotation tool. Top ten biological process terms with an adjusted (Benjamini-Hochberg) P value ≤ 0.01 are indicated. GO terms related to muscle development are highlighted in red. (E) H3K4me2 and H3K4me3 enrichment profiles of selected muscle-relevant cluster 1 genes. The TSS is marked by an arrow. The y-axis indicates the ChIP-seq signal.
Fig 3.
Cluster 1 genes bound by MyoD.
(A) Overlap of common H3K4me2/3 cluster 1 genes in Undiff and Diff C2C12 cells. The number of genes, which gain, loose or have constitutive MyoD binding are indicated, including their respective number of differentially expressed genes (fold change (FC) ≥ 2). (B) Percentage of genes in the common H3K4me2/3 clusters bound by MyoD in Undiff and Diff C2C12 cells. Highest enrichment in Undiff and Diff (each in cluster 1) is indicated by the two red lines. The P values are based on two-sided Fisher's exact test. (C) Heatmap of differentially expressed genes in H3K4me2/3 cluster1, which gain MyoD during differentiation (22 genes with fold change ≥ 2 out of 95 genes). The numbers in the heatmap represent the FPKM (fragments per kilo bases of exons for per million mapped) values. Gene names in blue indicate the genes with the E-box motif (CANNTG) within a 30 bp region centered on the peak summit. Gene names underlined in blue are genes with the MyoD preferred E-box motif (CAGCTG) within a 30 bp region centered on the peak summit.
Fig 4.
Patz1 expression and MyoD binding during myogenic differentiation.
(A) RNA expression profile of Patz1, H3K4me2 enrichment profile, H3K4me3 enrichment profile and MyoD binding profile at the Patz1 promoter in Undiff and Diff C2C12 cells. All profiles are based on raw mapped reads. Position of the MyoD preferred E-box motif (CAGCTG) in the peak region is indicated. (B) ChIP analysis of MyoD occupancy levels and H3K4me2/3 enrichment at the Patz1 promoter. Error bars indicate the standard deviation from at least three independent experiments. The statistical significance of enrichment versus the IgG control was calculated using Student's t-test. (C) Expression levels (mRNA) of Patz1 in Undiff and Diff C2C12 cells were measured by real-time PCR in at least three independent experiments. The statistical significance of the difference in expression between Undiff and Diff C2C12 was calculated using Student's t-test.
Fig 5.
PATZ1 down-regulation by MyoD.
(A) RNA expression profile of PATZ1 and MYOD binding profile at the PATZ1 promoter in IMR-90 cells cultured with growth medium (GM) and differentiation medium (DM). All profiles are based on raw mapped reads. Position of the MyoD E-box motif (CANNTG) in the peak region is indicated. (B) Expression levels (mRNA) of PATZ1 in IMR-90 fibroblasts. P value was calculated using Student's t-test based on at least three independent experiments. (C) Luciferase reporter analysis in HEK293 cells transiently transfected with a Patz1 reporter-construct and MyoD expression plasmids or empty vector control. Measurements were performed in triplicates.