Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells - PubMed (original) (raw)

Shigeyuki Magi 2, Giuseppe Jurman 1, Masayoshi Itoh 3 4 5, Hideya Kawaji 3 4 5, Timo Lassmann 3 4 6, Erik Arner 3 4 7, Alistair R R Forrest 3 4, Piero Carninci 3 4, Yoshihide Hayashizaki 3 5, Carsten O Daub 3 4 8; FANTOM Consortium; Mariko Okada-Hatakeyama 2, Cesare Furlanello 1

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Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells

Marco Mina et al. Sci Rep. 2015.

Abstract

The analysis of CAGE (Cap Analysis of Gene Expression) time-course has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. To identify the most prominent transcriptional regulations induced by growth factors in human breast cancer, we apply here the Complexity Invariant Dynamic Time Warping motif EnRichment (CIDER) analysis approach to the CAGE time-course datasets of MCF-7 cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We identify a multi-level cascade of regulations rooted by the Serum Response Factor (SRF) transcription factor, connecting the MAPK-mediated transduction of the HRG stimulus to the negative regulation of the MAPK pathway by the members of the DUSP family phosphatases. The finding confirms the known primary role of FOS and FOSL1, members of AP-1 family, in shaping gene expression in response to HRG induction. Moreover, we identify a new potential regulation of DUSP5 and RARA (known to antagonize the transcriptional regulation induced by the estrogen receptors) by the activity of the AP-1 complex, specific to HRG response. The results indicate that a divergence in AP-1 regulation determines cellular changes of breast cancer cells stimulated by ErbB receptors.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

(a) The signal transduction pathway activated upon stimulation of MCF-7 cells by HRG or EGF. (b) Comparison of MAPK proteins abundance after the stimulation of the same ligands (10 nM) by HRG (black pattern) and EGF (red pattern), quantified by Western blot analysis. (c) Comparison of MAPK proteins phosphorylation levels after the stimulation of the same ligands (10 nM) by HRG (black pattern) and EGF (red pattern), quantified by Western blot analysis. Western blot experiments were performed twice. Each dot in panels a and b represents the quantified values in an independent experiment, and lines represent the average of these values.

Figure 2

Figure 2. Characterization of SRF, cFOS, cJUN, FOSL1, RARAlpha and DUSP5 genes at expression and protein levels.

(a) CAGE analysis captures the quantitative differences in transcriptional changes induced by EGF (red)- and HRG (black)-stimulated signaling pathways in MCF-7 cells. (b) Western blot analysis was performed to quantify protein levels after the stimulation of the same ligands (10 nM). (c) Western blot analysis was performed to quantify protein phosphorylation levels after the stimulation of the same ligands (10 nM). Western blot experiments were performed twice. Each dot in panels b and c represents the quantified values in an independent experiment, and lines represent the average of these values.

Figure 3

Figure 3

Figure 4

Figure 4. Intersection of the regulatory maps inferred by CIDER for the MCF-7 HRG- and EGF-induced time-courses.

This network of 800 predicted interactions associates 12 TFs (large blue/pink nodes) to 386 target promoters (gray, pale green and red nodes). The cluster of 36 pale green nodes includes 30 genes coding for Histone proteins (Supplementary Data 4). The cluster of red nodes includes the FOS TF.

Figure 5

Figure 5. The SRF-rooted regulatory sub-network of MCF-7 cells in response to HRG- and EGF-induction.

Solid gray edges indicate the transcriptional regulations inferred by CIDER for both HRG and EGF time-courses. Dashed gray edges indicate the transcriptional regulations inferred by CIDER only for the HRG time-course. The interplay between JUN, JUNB, FOS and FOSL1 in the AP-1 complex at protein level is represented by the undirected black lines. The plots on the left show the expression patterns of the four clusters 1 (red), 7 (orange), 8 (green) and 9 (blue).

Figure 6

Figure 6. The expression patterns of the HRG Cluster 1

(a) and the EGF Cluster 2 (b), enriched for the Jaspar MA0476.1 motif (SRF transcription factor). Left: representation of the average expression pattern (lines) and 10%–90% quantiles interval (colored regions). Right: selection of Gene Ontology terms most enriched (top tables) and list of genes included in the clusters (gene names and Entrez ids).

Figure 7

Figure 7

(a) FOSL1 regulation by FOS in HRG-induced MCF-7 cells. cFOS expression (red curve) and protein abundance (dark gray region) rapidly increase after HRG-induction. Consistently, FOSL1 expression (orange curve) increases in the 45–120 min time span, and decreases as FOS protein abundance decreases. FOSL1 protein level, instead, is sustained up to 8 hrs. (b) DUSP5/DUSP10 regulation by AP-1 complex. DUSP5/10 expression (green curves) increases in the 45–120 min time span, and decreases as FOS protein abundance decreases. (c) RARA regulation by AP-1 complex. RARA expression (blue curve) increases peaks in the 100–180 min time interval, and slowly decreases at late time-points.

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