Characterization of the interaction sites in the genome of the PVT1 lincRNA associated with the androgen receptor in Prostate Cancer lineages

• Grant number: 20/02976-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): July 01, 2020
• Effective date (End): April 30, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Sergio Verjovski Almeida
• Grantee: Maria Gabriela Berzoti Coelho
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 18/23693-5 - Mechanisms of action of long non-coding RNAs involved with gene activation programs in eukaryotes, AP.TEM

Abstract

The Androgen Receptor (AR) is a transcription factor that mediates the effect of the androgen hormone and modulates the gene transcription program in prostate cells. little is known about the interaction of non-coding intergenic long RNAS (lincRNAs) with transcription factors, especially in the regulation of complex events such as the gene regulation program induced by the transcription factor AR in LNCaP cells. In a paper published in 2018, we observed that among the more than 600 lincRNAs linked to AR in LNCaP Prostate Cancer cells was PVT1, an oncogenic lincRNA, which is known to be increased in several types of cancer, including Prostate Cancer. The PVT1 lincRNA is known to bind to EZH2, a subunit of Polycomb Repressor Complex 2 (PRC2) and this leads to inhibition of the transcription of some protein-coding genes in certain types of cancer. However, until now the effect of PVT1 lincRNA on large-scale gene regulation in the same tissue has not been studied. Recent data from our laboratory, not yet published, obtained by doctoral student Alexandre Videira, show that the Knock-Down (KD) of PVT1 in LNCaP cells in culture, in the presence of androgen, significantly affects (q-value <0.01) transcription of hundreds of target genes, and show that dozens of androgen-inhibited genes may have their expression partially restored, fully restored or even overactivated after silencing the PVT1 lincRNA. These data confirm the role of lincRNA PVT1 as a transcription modifier, functioning as an inhibitor of the transcription of target genes, possibly through the formation of a lincRNA PVT1-AR-PRC2 complex. In the current proposal, we will use ChIP-Seq to immunoprecipitate the histone mark H3K27me3 followed by large-scale sequencing of the co-immunoprecipitated genomic DNA, in control LNCaP cells or with PVT1 silencing. We expect to map the genomic sites of the target genes of AR and PVT1, where there should be a decrease in the deposition of the H3K27me3 mark when PVT1 is silenced, failing to recruit EZH2 to those sites. We will do ChIP-Seq with anti-EZH2, to confirm that there has been a decrease in the occupation of the sites by EZH2 when PVT1 is silenced. We will also map the occupation of the PVT1 lincRNA throughout the genome, and we hope to reveal all the sites of interaction between PVT1 and chromatin in LNCaP, using the "Chromatin Isolation by RNA purification" (ChIRP) method that was used for the first time to map the binding sites in the genome of the HOTAIR lincRNA and the Polycomb complex. In addition to sequencing the captured genomic DNA, we will do mass spectroscopy (MS) analysis of the proteins captured in the ChIRP assay, which are part of the lincRNA: chromatin: protein complex, and we hope to reveal the other protein partners of the PVT1 lincRNA, in addition to EZH2. (AU)