Study of regulatory mechanisms of gene expression that determine immunosuppressive potential for application to cancer immunotherapy

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K16818
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 50020:Tumor diagnostics and therapeutics-related
• Research Institution: Nagoya University
• Principal Investigator: Hiramatsu Hiroaki 名古屋大学, 医学系研究科, 研究員 (70827253)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 部位特異的ChIP法 / dCas9 / 共免疫沈降法 / 転写因子複合体

Outline of Final Research Achievements

Gene expression is regulated by transcription factor (TF).TFs-DNA complex organizes 3d structure of chromatin. Chromatin immunoprecipitation (ChIP) is useful for identifying binding sites of specific TFs, but it requires the identification of molecular targets in advance and can not examine single promoter regions. In this study, I evaluated a method to isolate site specific TFs by using the dead Cas9 (dcas9) system. I developed a viral vector-based method and a recombinant protein-based method and tried to immunoprecipitate TF complexes. Although genomic DNA could be immunoprecipitated, unfortunately, it was not possible to obtain sufficient quantities of the proteins in the TF complex to allow identification of new associated proteins by mass spectrometry. But I found that optimization of the choice of protein crosslinkers, setting of conditions of crosslinking and sample disruption procedures were crucial to achieve this objective.

Free Research Field

生化学

Academic Significance and Societal Importance of the Research Achievements

dCas9を用いた部位特異的ChIP法では、sgRNAを変更するだけで、動物種や既存の抗体の質に縛られず、あらゆる遺伝子の、特異的なゲノム領域の解析に有効である。リコンビナントdCas9を用いた方法では、遺伝子導入の難しい細胞種や初代細胞を用いた解析への応用が期待できる。DNAを用いた解析では、極少量のDNAであっても配列を同定することが可能であるが、質量分析によるタンパク質の同定には、fmol～amolの量が必要であるため、効率の良いタンパク質回収法の確立は挑戦的であるが 意義深いものである。