Long non-coding RNAs required for MYC-driven cell proliferation

Dear All,

Cell Signal Unit (Yamamoto Unit) would like to inform you of a seminar by Dr. Peter K. Vogt from the Scripps Research Institute.

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Date: Wednesday, February 12, 2020

Time: 13:00-14:00

Venue: D015, Level D, Lab 1

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Speaker:

Dr. Peter K. Vogt, The Scripps Research Institute

Title:
Long non-coding RNAs required for MYC-driven cell proliferation

Abstract:
MYC is a basic helix-loop-helix leucine zipper protein that dimerizes with a related smaller protein MAX to bind to DNA. The dimer functions as a transcription regulator. MYC is bound to all active promoters and may therefore be a universal activator of transcription. ItMYC regulates the expression of large numbers of coding and long non-coding RNAs (lncRNAs). Since gain of function in MYC plays an important role in the genesis and progression of numerous human cancers, some of the lncRNAs regulated by MYC probably act as significant factors in this process.

Our goal in this study was to identify MYC-regulated lncRNAs that are required for MYC-mediated cell proliferation. We applied CRISPR interference (CRISPRi) to the identification of these non-coding transcripts and used the human lymphoid cell lines P493-6 and RAMOS for our studies. RAMOS is a cell line derived from Burkitt lymphoma, and P493-6 is an engineered cell line which carries MYC as a doxycycline-regulatable transgene. We identified 320 non-coding loci that play a positive role in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cell. We validated these results by RT-qPCR and by CRISPRi competition assays with individual GFP-expressing single-guide RNA constructs. We also showed that MYC binds to the promoter of candidate genes identified in the CRISPRi screen.

In the course of building and testing our CRISPRi library, we discovered that the repressor domain SID, derived from the MYC antagonist MXD1 is highly effective in P493-6 and RAMOS cells and superior to the routinely used KRAB domain. We could also show that  SID remains effective as a fusion protein to the MS2 aptamer-binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system for the controlled repression of targeted genes. It will open the door to a functional analysis of growth-promoting lncRNAs.

Host:
Prof. Tadashi Yamamoto

We hope to see many of you at the seminar.

Best regards,

Yuki Nakagawa

Research Unit Administrator

Cell Signal Unit