Abstract
Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.
Highlights
The acquisition of pluripotency in the epiblast, a transient population of cells with unrestricted developmental potential during early embryogenesis, is controlled by a core set of transcription factors that include OCT4, SOX2 and NANOG (Nichols et al, 1998; Avilion et al, 2003; Chambers et al, 2003; Mitsui et al, 2003; Silva et al, 2009)
To directly screen in an unbiased manner for cofactor requirements that support OCT4 and SOX2 mediated activation, we developed an in vitro transcription assay that faithfully recapitulates OCT4/ SOX2 and coactivator-dependent gene activation observed in embryonic stem (ES) cells using purified components to reconstitute the human transcriptional apparatus (Fong et al, 2011)
We previously have shown an activity present in a partially purified protein fraction, Q0.3, that is required for the XPC coactivator complex to stimulate a full, synergistic activation of the human Nanog proximal promoter by OCT4 and SOX2 but is dispensable for basal or Sp1-activated transcription (Rodda et al, 2005; Fong et al, 2011)
Summary
The acquisition of pluripotency in the epiblast, a transient population of cells with unrestricted developmental potential during early embryogenesis, is controlled by a core set of transcription factors that include OCT4, SOX2 and NANOG (Nichols et al, 1998; Avilion et al, 2003; Chambers et al, 2003; Mitsui et al, 2003; Silva et al, 2009) This undifferentiated, pristine stem state can be captured as embryonic stem (ES) cells (Evans and Kaufman, 1981; Martin, 1981; Brook and Gardner, 1997), regenerated from somatic cells by cell fusion and nuclear transfer (Yamanaka and Blau, 2010), or by the ectopic expression of defined transcription factors (Takahashi and Yamanaka, 2006; Yu et al, 2007). Perhaps the involvement of this rather elaborate collection of cofactors arose from the need for ES cells to significantly expand their transcriptional repertoire in order to accommodate the wide range of gene expression responses governing self-renewal and the transition
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