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Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
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dCas9-targeted locus-specific protein isolation method identifies histone gene regulators.

Chiahao Tsui1,2, Carla Inouye1,2, Michaella Levy3

  • 1Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine Center of Excellence, University of California, Berkeley, CA 94720.

Proceedings of the National Academy of Sciences of the United States of America
|March 7, 2018
PubMed
Summary

Researchers developed CLASP, a Cas9-targeted proteome purification method, to discover novel gene-regulatory factors. This approach identified Vig and Vig2 proteins regulating histone mRNA in Drosophila.

Keywords:
CRISPR/Cas9gene expressionhistone regulationreverse ChIP

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Eukaryotic gene regulation involves numerous proteins, with many gene-specific regulators yet to be identified.
  • Previous studies identified basal machinery and ancillary factors, but a comprehensive survey of locus-specific regulators is lacking.

Purpose of the Study:

  • To develop and apply a novel method for unbiased identification and functional testing of gene-regulatory factors associated with specific genomic loci.
  • To discover previously unknown proteins involved in the regulation of eukaryotic gene expression.

Main Methods:

  • Developed CLASP (Cas9 locus-associated proteome), an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy.
  • Utilized mass spectrometry and functional screens for unbiased isolation and characterization of associated regulatory factors.
  • Applied CLASP to the Drosophila melanogaster histone cluster in S2 cells.

Main Results:

  • Successfully identified several novel gene-regulatory factors associated with the Drosophila histone cluster.
  • Discovered Vig and Vig2 proteins that bind and regulate core histone H2A and H3 mRNA.
  • Demonstrated that Vig and Vig2 interact with the 3' UTRs of histone mRNAs.

Conclusions:

  • The CLASP method provides an efficient and unbiased approach to comprehensively survey the proteome directing gene expression at specific genomic loci.
  • Identified Vig and Vig2 as novel regulators of histone mRNA, highlighting their role in gene regulation.
  • CLASP is adaptable for various genomic loci and cell types, offering a versatile tool for discovering gene-regulatory factors.