Alternative promoter usage. TPM values were determined for genomic regions identified in Nepal et al. (31) to correspond to canonical and alternative promoters, associated with the same genes (n = 1612). Genes with significant expression (TPM > 5 in either the canonical or alternative promoter region in both the G1 and S/G2/M populations) were taken for further analysis (n = 231). (A) Plot of correlation of alternative promoter utilization, normalized to canonical promoter expression, between G1 and S/G2/M populations. Dashed lines denote threshold of 2-fold change in normalized alternative promoter expression between the populations (n = 79). These differentially utilized genes were segregated by whether a change in the expression of the canonical or alternative promoter, in either population, was responsible for the 2-fold change in the expression of the alternative promoter, normalized to canonical promoter expression. (B) Diagrammatic summary of group selection criteria. In brief, relative to the G1 population; Canonical down: S/G2/M alternative promoter expression unchanged, but canonical expression depleted (n = 12); Alternative down: S/G2/M alternative promoter expression depleted, but canonical expression unchanged (n = 22); Canonical up: S/G2/M alternative promoter expression unchanged, but canonical expression enhanced (n = 10); Alternative up: S/G2/M alternative promoter expression enhanced, but canonical expression unchanged. Situations falling outside of these criteria were discarded (n = 9). Full selection criteria shown in materials and methods. (C) Heatmap visualizing the log2 odds ratio of selected promoter motif occurrence for each group. Occurrence is scored in each group vs. occurrence in the rest of the data set (n = 80) (*P< 0.05, **P< 0.01, ***P< 0.001, Fisher's exact test). (D) UCSC genome browser view of the inositol polyphosphate-5-phosphatase B (inpp5b) promoters with CAGE-seq tracks showing enhanced alternative promoter usage in the S/G2/M population versus G1, an example of a member of ‘Alternative up’ group. The position of a CCAAT motif relative to the alternative promoter is shown. RNA-seq tracks, imported from the ‘Promoterome CAGE and nucleosome positioning’ publicly available trackhub (URL: http://trackhub.genereg.net/promoterome/danRer7/index.html) (17,31) show that this alternative promoter drives a somitogenesis specific transcript.
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