The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Purification of Stem Cell Coactivator-A (SCC-A) required for OCT4/SOX2-dependent activation of the Nanog gene.
(A) Chromatography scheme for purification of Q0.3 from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of …
Figure 1—figure supplement 1
The DKC1 and the XPC coactivator complexes are highly enriched in the transcriptionally active phosphocellulose 1 M KCl (P1M) and Ni-NTA flowthrough (Ni-FT) fractions.
Comparative western blot analysis of NT2 nuclear extract (NE), phosphocellulose 0.3 M, 0.5 M, 1 M KCl fractions (P0.3, P0.5, P1M, respectively), and Ni-NTA flowthrough (Ni-FT) using antibodies …
Figure 2 with 1 supplement
SCC-A is the dyskerin (DKC1) complex.
(A) Silver stained SDS-PAGE gel of Poros-HE peak activity fraction in Figure 1E with protein identities determined by mass spectrometry analysis. (B) Silver stained SDS-PAGE gel of recombinant DKC1 …
Figure 2—figure supplement 1
5’ end radiolabeling of RNAs co-purified from recombinant DKC1 complexes.
(A) Schematics of RNA labeling procedure. RNAs are treated with tobacco acid pyrophosphatase (TAP) to remove 5′ cap and dephosphorylated with APex heat-labile alkaline phosphatase before being …
Figure 3
DKC1-associated small RNAs modulate transcriptional coactivator activity.
(A) Purification scheme of endogenous DKC1 ribonucleoprotein complexes (RNPs). A partially purified fraction (Ni-FT) containing the bulk of the DKC1 RNPs in NT2 cells (See Figure 1—figure supplement …
Figure 4 with 3 supplements
Mechanism of Coactivation by the DKC1 Complex.
(A) Co-dependent activation of Nanog transcription by the XPC complex, the DKC1 complex, and SCC-B. Recombinant XPC and DKC1 complexes purified from Sf9 cells, and SCC-B purified from bacteria were …
Figure 4—figure supplement 1
Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact.
Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide …
Figure 4—figure supplement 2
DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion.
RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure …
Figure 4—figure supplement 3
MNase digestion moderately increases DKC1 coactivator activity.
Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC …
Figure 5 with 2 supplements
The DKC1 complex is recruited to regulatory regions of key pluripotency genes in mouse and human ES cells.
(A) Co-occupancy of DKC1, OCT4, and SOX2 on enhancers of Oct4, Nanog, Sox2, but not Fgf4, in mouse ES cell line D3. Chromatin immunoprecipitation (ChIP) analysis of DKC1 occupancy on control and …
Figure 5—figure supplement 1
Crosslinking DKC1 to chromatin requires protein–protein crosslinker ethylene glycol bis[succinimidylsuccinate] (EGS) in addition to formaldehyde (FA).
Mouse ES cell line D3 is crosslinked with FA for 5 min (left), 10 min (middle), or with EGS for 30 min followed by FA for 5 min (right). ChIP analysis of DKC1 occupancy on control (−251) and …
Figure 5—figure supplement 2
SOX2 is enriched on the regulatory regions of Oct4, Nanog, and Fgf4 in mouse ES cells.
Mouse ES cell line D3 is crosslinked with EGS and FA. Enrichment of SOX2 compared to control IgGs is analyzed as described in Figure 5—figure supplement 1.
Figure 6 with 1 supplement
The DKC1 complex is required for stem cell maintenance.
(A) Downregulation of the DKC1 complex upon retinoic acid (RA)-induced differentiation of mouse ES cell line D3. Western blot analyses of whole cell extracts prepared from D3 cells (mESC D3 WCE) …
Figure 6—figure supplement 1
shRNA-mediated knockdown of DKC1 in mouse ES cells does not compromise housekeeping gene expression.
Quantification of housekeeping gene Gapdh mRNA levels in control (NT), XPC, and DKC1 knockdown D3 cells are analyzed as in (D). Error bars represent standard deviation (n = 3).
Figure 7 with 1 supplement
The DKC1 complex is required for mesenchymal-to-epithelial transition (MET) during somatic cell reprogramming.
(A) Depletion of DKC1 blocks somatic cell reprogramming. CF-1 mouse embryonic fibroblasts (MEFs) are infected with lentiviruses expressing OCT4, KLF4, SOX2, and c-MYC (STEMCCA) and reverse …
Figure 7—figure supplement 1
Somatic cell reprogramming is blocked by DKC1 depletion.
Induced MEFs depleted of DKC1 by two independent shRNAs (shDKC1-1 and shDKC1-2) as described in Figure 7A are plated along with MEFs infected with control non-target lentiviruses onto 24-well plates …
Figure 8 with 3 supplements
Fast cycling somatic cell state conducive to iPS cell generation requires DKC1.
(A) MEFs depleted of DKC1 by two independent shRNAs (shDKC1-1 and shDKC1-2), along with MEFs infected with control non-target lentiviruses, were analyzed using the CellTrace CFSE Proliferation Assay …
Figure 8—figure supplement 1
Ultrafast cycling MEF population contains the bulk of reprogramming activity.
Sorted MEF populations (CFSE-Lo, Med-Lo, Med-Hi, and Hi) in Figure 7B are plated on feeders in 24-well plates at indicated cell numbers. AP-positive colonies are stained as described in Figure 7A.
Figure 8—figure supplement 2
Ultrafast cycling somatic cell state can be induced in secondary OKSM MEFs.
(A) Labeling and sorting of CFSE-labeled MEFs carrying an integrated dox-inducible transgene expressing OCT4, KLF4, SOX2, and c-MYC (OKSM MEFs) are performed and analyzed as described in Figure 8B. …
Figure 8—figure supplement 3
Ultrafast cycling OKSM MEFs have elevated Dkc1 levels.
Dkc1 expression is upregulated in the ultrafast cycling MEFs upon OKSM expression. mRNA levels of Dkc1 and Oct4 in sorted MEF populations described in (C) are compared to D3 ES cells and uninduced …
Additional files
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Supplementary file 1
Primer sequences.
- https://doi.org/10.7554/eLife.03573.023
