Chromatin modifying enzymes as modulators of reprogramming

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.

[1]  J. Rinn,et al.  Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells , 2010, Nature Genetics.

[2]  Kakajan Komurov,et al.  Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes , 2010, Proceedings of the National Academy of Sciences.

[3]  G. Daley,et al.  Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells , 2009, Nature Biotechnology.

[4]  R. Stewart,et al.  Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences , 2009, Science.

[5]  Ji Luo,et al.  Cancer Proliferation Gene Discovery Through Functional Genomics , 2008, Science.

[6]  Rudolf Jaenisch,et al.  Single-gene transgenic mouse strains for reprogramming adult somatic cells , 2010, Nature Methods.

[7]  Yang Shi,et al.  Transcriptional repression by YY1, a human GLI-Krüippel-related protein, and relief of repression by adenovirus E1A protein , 1991, Cell.

[8]  T. Mikkelsen,et al.  Dissecting direct reprogramming through integrative genomic analysis , 2008, Nature.

[9]  E. Lander,et al.  Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. , 2008, Cancer research.

[10]  Yonghong Xiao,et al.  Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. , 2011, Cancer cell.

[11]  Megan F. Cole,et al.  Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells , 2005, Cell.

[12]  Lee E. Edsall,et al.  Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. , 2010, Cell stem cell.

[13]  J. Wrana,et al.  Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. , 2010, Cell stem cell.

[14]  Yi Zhang,et al.  hDOT1L Links Histone Methylation to Leukemogenesis , 2005, Cell.

[15]  J. S. Lee,et al.  RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs , 2005, Nucleic Acids Res..

[16]  G. Daley,et al.  High‐Efficiency RNA Interference in Human Embryonic Stem Cells , 2005, Stem cells.

[17]  Lars Bullinger,et al.  MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. , 2011, Cancer cell.

[18]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[19]  D. Reinberg,et al.  The Polycomb complex PRC2 and its mark in life , 2011, Nature.

[20]  Jill P. Mesirov,et al.  GSEA-P: a desktop application for Gene Set Enrichment Analysis , 2007, Bioinform..

[21]  David Landeira,et al.  ESCs require PRC2 to direct the successful reprogramming of differentiated cells toward pluripotency. , 2010, Cell stem cell.

[22]  E. Li,et al.  The Histone H3K79 Methyltransferase Dot1L Is Essential for Mammalian Development and Heterochromatin Structure , 2008, PLoS genetics.

[23]  Paul H Lerou,et al.  Generation of human-induced pluripotent stem cells , 2008, Nature Protocols.

[24]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[25]  G. Schotta,et al.  SU(VAR)3-9 is a Conserved Key Function in Heterochromatic Gene Silencing , 2003, Genetica.

[26]  T. Mikkelsen,et al.  Genome-wide maps of chromatin state in pluripotent and lineage-committed cells , 2007, Nature.

[27]  Shulan Tian,et al.  Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells , 2007, Science.

[28]  F. Bertucci,et al.  Gene expression profiling of breast cell lines identifies potential new basal markers , 2006, Oncogene.

[29]  George Q. Daley,et al.  Reprogramming of human somatic cells to pluripotency with defined factors , 2008, Nature.

[30]  T. Lenstra,et al.  Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms , 2011, Epigenetics & Chromatin.

[31]  S. Cole,et al.  Sequences Human Induced Pluripotent Stem Cells Free of Vector and Transgene , 2012 .