Pol-II ChIP-seq data quality. (A) Plot to show the distribution of ChIP-seq reads as normalized read counts per million around known transcription start site (TSS) of known genes in the five tissues. High Pol-II enrichment is observed around known TSS. (B) Real-time-PCR results show the enrichment of Polr2a promoter in Pol-II ChIP-enriched DNA from five mouse tissues (brain, lung, liver, spleen and kidney). The primers (sequences provided in the ‘Materials and Methods’ section) used in this ChIP-PCR cover both the promoter region of Polr2a and the downstream control region. The promoter region of Polr2a gene shows high recruitment of Pol-II while no significant enrichment is observed in downstream Polr2a region. (C) Enrichment of ChIP-seq reads around Polr2a sgene. The black box under the wiggle profile shows the position of the predicted promoters and the line below identifies the peaks that are statistically significant. The y-axis represents the normalized read counts per million mapped reads. All five tissues show huge enrichment of read around promoter region of Polr2a gene. The enrichment profile is low inside gene for all tissues.

Identification of Pol-II peaks related to promoters. (A) Cumulative distribution of Pol-II peaks around known transcription start site (TSS) of known genes in five tissues. The distribution shows that many Pol-II bound peaks are either upstream or downstream of known TSS. (B) Percentage of significant Pol-II peaks predicted as promoter and non-promoter peaks by our program in five tissues. The graph indicates that majority of the Pol-II peaks do not correspond to promoters (C) Percentage of CpG-rich promoter and non-promoter peaks in five tissues. (D) Distribution of annotated peak promoters relative to known TSS for both protein coding and non-coding genes.

Identification of novel promoters and experimental verification. (A–C) The wiggle profile shows the enrichment of Pol-II and prediction of novel promoters for mouse KIAA1632 (A) and GPM6 (B) gene in brain, kidney, liver, lung, and spleen tissues and (C) shows the Pol-II profile on a non-promoter region that lies within the transcripts (AK090117 and U58494). Y-axis shows the normalized read counts/million mapped reads and the black boxes below the Pol-II-binding profile represent the identified novel promoters by our program. The two novel promoters shown above have a corresponding conserved promoter in human genome. (D) Luciferase activity of the novel promoters in five distinct cell lines. The x-axis represents either the vector alone background (pGL3 basic) or the activity of novel promoters (NP1-10) and non-promoter region (ctrl1 and 2). On the y-axis, the normalized luciferase activity has been plotted in a logarithmic scale to the base of 2. Thus, the value of 1on the y-axis represents a 2-fold activity of the promoter. Only if a promoter shows an activity that is more than 2-fold it is considered as active in that cell line. NP7 does not show any luciferase activity in any of the five cell lines. Luciferase activity is expressed as log2 of the fold change over the vector alone (pGL3basic) after normalization with Renilla-luciferase for transfection efficiency. Dll1 promoter has been included as a positive control and Ctrl1, 2 represents the negative controls (non-promoter regions).

Alternative promoter usage in five mouse tissues (A) Wiggle profile showing examples of alternative promoter usage identified by our analysis in the five mouse tissues. For Adar1 and Hdgf our approach has identified the use of two and four known promoters among the five tissues, which are indicated by arrows on the transcripts at the bottom of the figure. Y-axis shows the normalized read count per million reads, the black box below the Pol-II enrichment shows the position of the identified promoters and the black line under it indicates the significantly enriched peaks of each track. It is evident that only few of the enriched peaks reside in the promoter region. (B and C) Pie chart shows distribution of alternative promoter usage for protein coding (B) and non-coding genes (C) in the five tissues analyzed from mouse.

Identification of tissue specific promoters and their relationship to CpG islands. (A) Box plot shows the normalized read counts of promoters that have been assigned to be brain specific in all five mouse tissues. The plot shows that the brain specific promoters indeed show specific and increased Pol-II binding in brain relative to other tissues. (B and C) Tissue specificity of promoters and its association with CpG richness. An inverse relationship is observed between tissue-specific promoters and CpG richness when we look at either the Shannon entropy (B) or normalized read count fold change (C) measures of tissue specificity across all five tissues. The left-hand side of y-axis represents the total promoter count. The right side of y-axis represents fraction of CpG-rich promoters. The x-axis in (B) represents Shannon entropy and in (C) it represents log2 (fold change) in read count value around annotated promoters.

Correlation of Pol-II enrichment on promoters with the expression of corresponding transcripts. (A and B) Based on the mRNA expression estimated by Cufflinks software the promoters were divided into four groups for brain and liver (high, medium, low and very low). The normalized read/tag count of Pol-II bound DNA for each group of promoters at base pair resolution from –1 kb to +1 kb relative to TSS was calculated and plotted as a function of distance from TSS. Total reads of gene promoter were counted based on our annotation for each group in each tissue. The x-axis represents distance of reads from annotated TSS and the y-axis represents total number of reads per million mapped reads.