Subtype-specific Mechanism Of Gene Transcript Regulation By FOXF2 In Breast Cancer

BackgroundForkhead box F2(FOXF2)is involved in differentiation and development through transactivates or transrepresses diverse genes.Previous study demonstrated that FOXF2 is specifically expressed in basal-like breast cells and functions as an epithelial-mesenchymal transition(EMT)suppressor.But it exhibits opposing biological activities in luminal breast cancer by upregulating the transcription of EMT transcription factors.Transcriptional coregulators play a central role in regulating the expresssion of genes,and they are recruited by transcription factors(TFs)assemble into multisubunit complexes.NCOR1 as a common corepressor are core subunit and recruit HDAC3 assemble into a protein complex.SRC family are recruited by transcription factors and as a scaffolding protein joint the second coregulators into the protein complex.Thus,we investigated whether FOXF2 recruit coregulators in a subtype-specific way and then assembled into activation complexes or repression complexes to transactivates or transrepresses target genes.PurposesTo explore the subtype-specific mechanism of gene transcript regulation by FOXF2 in breast cancer.To investigate epigenetic modification mechanism involved in the function of FOXF2 transcriptional regulation.Methods1.IP assays were performed in MDA-MB-231 and MCF-7 cells to investigate whether FOXF2 is associated with multiple transcription corepressor such as NCOR1?HDAC3 and HDAC1 or coactivators such as p300 in vivo.2.ChIP and Re-Ch IP assays were performed in MDA-MB-231 cells to investigate whether FOXF2 recruit NCOR1 and HDAC3 binding to the promoter of target genes.Whether FOXF2 and p300 binding to the promoter of target genes together.Ch IP-qPCR assays were performed to investigate whether FOXF2 influence NCOR1 and HDAC3 binding activities or the chromatin structure.3.Dual-luciferase reporter assays were used to investigate the promoter activity in MDA-MB-231 and BT549 cells with NCOR1 knockdown or FOXF2 overexpression or FOXF2 overexpression plus NCOR1 knockdown,as well as the control cells.RT-qPCR and immunoblot assays were used to investigate whether NCOR1 affected the transrepress function of FOXF2 on target genes mRNA and protein expression in basal-like breast cancer.Results1.IP assays indicates that in basal-like breast cancer cells FOXF2 is associated with corepressor NCOR1 and HDAC3 assemble into a protein complex,while there is no interact between FOXF2 and HDAC1 or coactivator such as NCOA1.In luminal breast cancer cells FOXF2 is associated with coactivator p300 assemble into a protein complex which function as a transactivate complex.But there is no interaction between FOXF2 and corepressors NCOR1 or HDAC3,failed to assemble into a transrepress complex in luminal breast cancer cells.2.ChIP and Re-ChIP assays indicates that FOXF2 is associated with corepressor NCOR1 and HDAC3 assemble into a transrepress complex,binding to the promoter of target genes together in basal-like breast cancer cells.NCOA1 could bind to the FOXF2 promoter when activative NF-?B signaling pathway in luminal breast cancer cells.CHIP-qPCR assays were performed to indicates that FOXF2 enhance NCOR1 and HDAC3 binding activities to its target genes,and modify the chromatin structure such as histone deacetylase.3.Dual-luciferase reporter assays were used to indicate that NCOR1 mediates the FOXF2-regulated suppression of target genes promoter activity in basal-like breast cancer.In BLBC cells MDA-MB-231 and BT549,NCOR1 knockdown could partially influence target genes promoter activity.Forced expression of FOXF2 significantly decreased the promoter activity of target genes,while knockdown of NCOR1 could,at least partially,offset the decreased promoter activity of FOXF2-overexpressing cells.4.RT-qPCR and immunoblot assays revealed that NCOR1 mediates the FOXF2 transrepress target genes expression in basal-like breast cancer.In BLBC cells MDA-MB-231 and BT549,NCOR1 knockdown could partially influence target genes mRNA and protein expression.Forced expression of FOXF2 significantly decreased the mRNA and protein expression of target genes,while knockdown of NCOR1 could,at least partially,neutralize the decreased target genes expression of FOXF2-overexpressing cells.Conclusions1.In basal-like breast cancer cells,FOXF2 transrepresses target genes through the recruitment of NCOR1 and HDAC3,binding to promoter of target genes as a form of complexes.The multisubunit complexes with chromatin-modifying activities such as histone deacetylase provide a suppressed chromatin structure and promoter to suppress the gene expression.2.In luminal breast cancer cells,FOXF2 assembled into transactivate complexes through the recruitment of p300 to transactivate target genes.3.In luminal breast cancer cells,NCOA1 played a role in NF-?B transactivate FOXF2 expression...