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. 2022 Sep 19;14(18):4540.
doi: 10.3390/cancers14184540.

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines

Matea Kurtović  1 , Nikolina Piteša  1 , Nenad Bartoniček  2   3 , Petar Ozretić  1 , Vesna Musani  1 , Josipa Čonkaš  1 , Tina Petrić  1 , Cecile King  4 , Maja Sabol  1
Affiliations

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines

Matea Kurtović et al. Cancers (Basel). .

Abstract

Background: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma.

Methods: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR.

Results: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq.

Conclusions: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.

Keywords: GLI targets; HH-GLI pathway; MAPK pathway; melanoma; targeted therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
HH-GLI pathway activity in melanoma cell lines. (A) Western blot analysis of relative protein expression levels of GLI1, GLI2, GLI3 and PTCH1 in a panel of 14 melanoma cell lines. FL refers to the full-length protein, while R refers to the repressor form. (B) Average gene expression of GLI1, GLI2, GLI3 and PTCH1 relative to the housekeeping gene RPLP0 summarized according to the mutational background of the tested panel of melanoma cell lines. (C) Heatmap showing MTT proliferation assay on 14 melanoma cell lines. Cells were treated with three different HH-GLI pathway inhibitors in five doses, during 24, 48 and 72 h. Green color indicates high cell viability and red color indicates low viability (cell death). The uncropped blots are shown in File S1.
Figure 2
Figure 2
Expression profiles of CHL-1, A375, and MEL224 cell lines with transfected GLI1, GLI2 and GLI3. (A) MDS plot of A375 cell line. (B) Upset plot of differentially expressed genes across all cell lines and GLI proteins. (C) Heatmap of expression of top DEGs sorted by logFC across all cell lines. (D) Gene enrichment analysis of DEGs across cell lines, transfection with GLI1-3 vs. control. Upper half of the plot shows KEGG pathway analysis, while the lower part shows categories of diseases. On x-axis: normalized enrichment scores. Size of the circles denote ratio of DE genes in pathways. Color denotes significance, with gray and blue circles denoting non-significant enrichments and red denotes significant enrichment.
Figure 3
Figure 3
ChIP-seq analysis of GLI1-3 binding across cell lines. (A). Binding of peaks on the whole genome. (B) Binding of peaks that centered on the promoter regions (C) Overlap of GLI1-3 binding sites: left image shows overlapping of peaks on whole genome, and right image shows overlapping of peaks on the promotor regions only. FDR value vas set to 0.2. (D) Heatmap of binding of GLI1-3 TFs across promoter sites. (E) Peak distribution in PTCH1 and EBI3 loci.
Figure 4
Figure 4
Validation of 21 selected DEGs. (A) Schematic representation of choosing DEGs for qPCR validation. (B) Volcano plot represents previously identified targets of HH-GLI signaling in red and targets selected for validation in this study in blue for each GLI protein. (C) Heatmap showing qPCR validation of GLI target genes identified by both ChIP-seq and RNA-seq on seven melanoma cell lines (A375, SKMEL24, MEL224, SKMEL2, MEL505, CHL-1 and MEWO) with overexpressed GLI1, GLI2 or GLI3. The experiment was repeated two times in triplicates. Validation was performed for 21 DEGs and two known pathway targets PTCH1 and GLI1 as controls. The relative expression level of each gene was determined using the 2−ΔΔCt method with RPLP0 as the internal reference gene. Heatmap shows log2 fold change values, ND stating that expression levels could not be detected after 37th cycle.
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