piggyBac insertional mutagenesis screen identifies a role for nuclear RHOA in human ES cell differentiation

Stem Cell Reports. 2015 May 12;4(5):926-38. doi: 10.1016/j.stemcr.2015.03.001. Epub 2015 Apr 9.

Abstract

The mechanisms regulating human embryonic stem (ES) cell self-renewal and differentiation are not well defined in part due to the lack of tools for forward genetic analysis. We present a piggyBac transposon gain of function screen in human ES cells that identifies DENND2C, which genetically cooperates with NANOG to maintain self-renewal in the presence of retinoic acid. We show that DENND2C negatively regulates RHOA activity, which cooperates with NANOG to block differentiation. It has been recently shown that RHOA exists in the nucleus and is activated by DNA damage; however, its nuclear function remains unknown. We discovered that RHOA associates with DNA and that DENND2C affects nuclear RHOA localization, activity, and DNA association. Our study illustrates the power of piggyBac as a cost-effective, efficient, and easy to use tool for forward genetic screens in human ES cells and provides insight into the role of RHOA in the nucleus.

Publication types

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

MeSH terms

  • Cell Differentiation / drug effects
  • Cell Nucleus / metabolism
  • Chromosome Mapping
  • DNA Transposable Elements / genetics*
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism*
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Human Embryonic Stem Cells / cytology
  • Human Embryonic Stem Cells / metabolism*
  • Humans
  • Mutagenesis, Insertional
  • Nanog Homeobox Protein
  • Tretinoin / pharmacology
  • rac1 GTP-Binding Protein / metabolism
  • rhoA GTP-Binding Protein / metabolism*

Substances

  • DNA Transposable Elements
  • Guanine Nucleotide Exchange Factors
  • Homeodomain Proteins
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Tretinoin
  • rac1 GTP-Binding Protein
  • rhoA GTP-Binding Protein