Identifying the FOXD3 transcriptome in melanoma. To know the transcriptional impact of FOXD3 in melanoma cells, we uti lized a microarray approach. We collected RNA from 3 unrelated mutant BRAF melanoma cell lines that have been engineered to inducibly express FOXD3 or the control gene galactosidase soon after five days of transgene induction. This time point was chosen according to maximal phe notypic adjustments previously observed. Comparison of gene signatures among the 3 cell lines produced around two,600 frequent genes differentially regulated by FOXD3 expressing cells compared together with the LacZ controls. Since a big quantity of altered genes may represent secondary targets of FOXD3, we sought to narrow the scope of FOXD3 regulated genes to direct transcriptional targets. We performed ChIP seq on V5 tagged FOXD3 IP from WM115TR FOXD3.
The results showed spe cific, reproducible enrichment foci across the genome selelck kinase inhibitor with a preference for promoter regions and bidirectional promoters. Analysis of genes situated proximal to FOXD3 enrichment web sites and displaying regulation by FOXD3 indicated a preference for genes involved in focal adhesions, ECM receptor interactions, MAPK and mTOR signaling, and also other processes involved in cancer, suggesting that FOXD3 is able to act as a major orches trator of transcription in melanoma. ERBB3 is actually a direct transcriptional target of FOXD3. Depending on our pre vious data displaying that FOXD3 promotes resistance to BRAF inhibition, we focused on genes that had been druggable, offered the translational nature from the study. We identified ERBB3 as a target upregulated by FOXD3 in the expression arrays and robust ly enriched by FOXD3 inside the ChIP seq evaluation. ERBB3 expression is increased in response to targeted therapies such as lapatinib in breast cancer and gefi tinib in lung cancer and is also important for melanoma survival and proliferation.
ChIP seq analysis showed that the initial intron of ERBB3 was enriched by FOXD3. This region is well conserved among species and functions as an enhancer region for ERBB3. Quantitative PCR showed dramatic enrichment of intron 1 over regular IgG only follow ing FOXD3 expression. Importantly, the V5 anti body did not enrich the promoter of an irrelevant Asaraldehyde gene, actin, inside a doxycycline dependent manner, verifying the specificity of FOXD3 enrichment. Enhanced expres sion on our microarrays coupled with binding of FOXD3 to the enhancer area suggests that FOXD3 straight upregulates the transcription of ERBB3. In support of this, IP of RNA polymerase II phosphoserine 2, a marker for transcrip tional elongation, considerably enriched ERBB3 intron 1 in cells expressing FOXD3. Additionally we found that FOXD3 elevated the expression of ERBB3 at each the mRNA and protein levels in WM115TR FOXD3 cells.