Chromatin-modifying enzymes as modulators of reprogramming

Nature. 2012 Mar 4;483(7391):598-602. doi: 10.1038/nature10953.

Abstract

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Publication types

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

MeSH terms

  • Cellular Reprogramming* / genetics
  • Chromatin / genetics
  • Chromatin / metabolism*
  • DNA Methylation / genetics
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / metabolism
  • Enhancer of Zeste Homolog 2 Protein
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Histone-Lysine N-Methyltransferase
  • Histones / metabolism
  • Homeodomain Proteins / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism*
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / metabolism
  • Methylation
  • Methyltransferases / antagonists & inhibitors
  • Methyltransferases / biosynthesis
  • Methyltransferases / genetics
  • Methyltransferases / metabolism
  • Nanog Homeobox Protein
  • Polycomb Repressive Complex 2
  • Polycomb-Group Proteins
  • Proto-Oncogene Proteins c-myc / metabolism
  • RNA, Small Interfering
  • RNA-Binding Proteins / metabolism
  • Repressor Proteins / antagonists & inhibitors
  • Repressor Proteins / metabolism
  • Transcription Factors / antagonists & inhibitors
  • Transcription Factors / metabolism
  • YY1 Transcription Factor / antagonists & inhibitors
  • YY1 Transcription Factor / metabolism

Substances

  • Chromatin
  • DNA-Binding Proteins
  • Histones
  • Homeodomain Proteins
  • KLF4 protein, human
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Lin28A protein, human
  • MYC protein, human
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Polycomb-Group Proteins
  • Proto-Oncogene Proteins c-myc
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Repressor Proteins
  • Transcription Factors
  • YY1 Transcription Factor
  • YY1 protein, human
  • SUV39H1 protein, human
  • DOT1L protein, human
  • Methyltransferases
  • EZH2 protein, human
  • Enhancer of Zeste Homolog 2 Protein
  • Histone-Lysine N-Methyltransferase
  • Polycomb Repressive Complex 2

Associated data

  • GEO/GSE29253
  • GEO/GSE35791