BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration

Abstract

Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ~50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized. Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAF(V600E) protein. To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAF(V600E) expression. To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAF(V600E)-mutant human melanomas. BRAF(V600E) regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts. These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription. Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs. BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11. Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.

Figure 1.

Oncogenic BRAF^V600E remodels the transcriptome in melanoma. (A) Schematic of experimental workflow and RNA-seq data analysis. (B) Pie chart showing number and categories of transcripts regulated by BRAF^V600E. Numbers of transcripts changed in all three samples in the same direction ± twofold versus pooled normal melanocyte control. (C) Heatmap showing hierarchical clustering of protein-coding transcript expression in primary melanocytes overexpressing BRAF^V600E and in primary melanomas (± twofold, changed in all three samples). (D) Top 10 unique gene ontology (GO) terms associated with protein-coding transcripts up-regulated by BRAF^V600E.

Figure 2.

Multi-tissue expression profile of annotated lncRNAs and novel transcripts. (A) Publicly available RNA-seq data (ENCODE project) for multiple cell types were downloaded from the UCSC Genome Browser. The melanoma column expresses an aggregate from our RNA-seq data indicating if each transcript was concordantly up-regulated (yellow) or down-regulated (blue) by BRAF in melanoma. Red/white scaling indicates expression level in other cell types. (B) Melanocyte DNaseH (ENCODE) distribution over the 109 BRAF-regulated transcripts is shown. The following charts (C–H) depict signal distribution (ENCODE) over the 109 BRAF-regulated transcripts. (C) Distribution of H3K4me3 in H1ES cells. (D) Distribution of RNA pol II in H1ES cells. (E) Distribution of DNaseH in H1ES cells. (F) Distribution of H3K4me3 in K562 cells. (G) Distribution of RNA pol II in K562 cells. (H) Distribution of DNaseH in K562 cells. (TSS) Transcription start site; (TES) transcription end site.

Figure 3.

Oncogenic BRAF^V600E regulates expression of many novel transcripts. (A) Frequency distribution plot showing coding potential analysis (CPC scores) of novel transcripts discovered by de novo assembly. One thousand five hundred random annotated ncRNAs and 1500 random protein-coding genes were also analyzed for reference. (B) Analysis of BRAF up-regulated novel lncRNAs in a publicly available melanoma RNA-seq data set. We remapped and reanalyzed Berger et al. (2010) data and derived expression values for novel transcripts up-regulated in all three of our samples. Robustly up-regulated novel transcripts (with negative CPC scores) were clustered with Berger et al. (2010) data revealing recurrently highly expressed lncRNAs. (C) Histograms of raw RNA-seq data in control sample, in melanocytes overexpressing BRAF^V600E and in both primary melanomas (Mel. 1, Mel. 2). Scripture and Trinity assembly for BANCR is also shown. y-axis is number of RNA-seq reads normalized for mapping variation. (D) CPC score for BANCR. Examples of lncRNAs HOTAIR and XIST and protein-coding genes BRAF and TP53 are shown for reference. (E) BANCR expression in primary melanomas validated by qRT-PCR. (F) BANCR expression in melanocytes overexpressing BRAF^V600E or NRAS^G12V measured by qRT-PCR. Data are means from three experiments ± SD. (G) BANCR Northern blot using RNA from control melanocytes (MCs), melanocytes overexpressing BRAF^V600E, or sk-mel-5 melanoma cells. (H) Schematic of BANCR locus.

Figure 4.

BANCR loss impairs melanoma cell migration. (A) Quantification of BANCR knockdown by qPCR using two independent shRNAs. (B) Microarray analysis of Colo829 melanoma cells following shRNA knockdown of BANCR. (C) Significant gene ontology (GO) terms associated with genes repressed by BANCR loss. (D) Representative images showing amount of cell migration at day 0 and day 2. Melanoma cells were infected with lentivirus expressing either nontargeting control shRNA (control) or one of two duplicate independent shRNA sequences targeting BANCR (shRNA 1, shRNA 2). Green highlight, shown for clarity, is a mask applied by the analysis software for accurate, unbiased migration quantification. (E) Quantification of replicate images (n = 6 per timepoint) taken over migration assay timecourse. Values are means from two independent experiments ± SD. Statistical analysis performed by one-way ANOVA, **P ≤ 0.01, *P ≤ 0.05 versus control. (E) Quantification of replicate images (n = 3 per timepoint) taken over migration assay timecourse ± CXCL11. Cells were infected with shRNA control lentivirus and treated with vehicle (PBS), infected with BANCR shRNA, and treated with vehicle or infected with BANCR shRNA and treated with CXCL11 (10 ng/mL). (G) Representative images showing amount of cell migration at day 0 and day 2.