An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation

Proc Natl Acad Sci U S A. 2020 Mar 24;117(12):6571-6579. doi: 10.1073/pnas.1921786117. Epub 2020 Mar 10.

Abstract

MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.

Keywords: CRISPRi; MYC; lncRNAs; oncogenesis; transcriptional regulation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / chemistry
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / genetics
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / metabolism*
  • CRISPR-Associated Protein 9 / genetics
  • Capsid Proteins / metabolism
  • Cell Line, Tumor
  • Cell Proliferation / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Humans
  • Promoter Regions, Genetic
  • Protein Domains
  • Proto-Oncogene Proteins c-myc / metabolism*
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Long Noncoding / genetics*
  • RNA, Long Noncoding / metabolism
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Transcription, Genetic

Substances

  • Aptamers, Nucleotide
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Capsid Proteins
  • MXD1 protein, human
  • MYC protein, human
  • Proto-Oncogene Proteins c-myc
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Long Noncoding
  • Repressor Proteins
  • CRISPR-Associated Protein 9