V体育平台登录 - Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2012 Dec 7;11(6):783-98.
doi: 10.1016/j.stem.2012.09.011. Epub 2012 Oct 25.

Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system

Shannon M Buckley  1 , Beatriz Aranda-Orgilles, Alexandros Strikoudis, Effie Apostolou, Evangelia Loizou, Kelly Moran-Crusio, Charles L Farnsworth, Antonius A Koller, Ramanuj Dasgupta, Jeffrey C Silva, Matthias Stadtfeld, Konrad Hochedlinger, Emily I Chen, Iannis Aifantis
Affiliations

VSports - Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system

Shannon M Buckley et al. Cell Stem Cell. .

"VSports手机版" Abstract

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates. VSports手机版.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Mapping of the mouse ES cell Ubiproteome
A) Scheme depicting the proteomic strategy followed using SILAC and MS/MS technology. B) Ingenuity Pathways-generated network with Nanog and Oct4 as nodes. Molecules in red show a higher ubiquitination rate in self-renewal conditions and in green in differentiated cells. Color intensities reflect ubiquitination detection values. White marks molecules that belong to the network and were not detected in our study. In orange are marked pluripotency factors only detected in the label-free experiments. Lines without arrows mean binding. Lines with arrows mean acting on. Dotted lines reflect an indirect interaction. C) Heat-map illustrating fold change of ubiquitinated peptides differentially detected in self-renewing ES cells and in differentiated cells. D) Venn diagram showing the overlap between the mouse phospho-proteome (Li et al, 2011) and the ubi-proteome obtained in this study. Common proteins are underlined in the pluripotency network in dark grey.
Figure 2
Figure 2. Half-life of key pluripotency factors is regulated by the UPS
A) Differential expression of ES nuclear proteins in the presence of MG132. Proteins with increased expression in the presence of MG132 are indicated by an arrow (> 2 fold increase). X-axis shows the ratios of MS/MS spectra identified in the MG132 treatment over the MS/MS spectra identified in the DMSO treatment for each protein in the natural logarithm (ln). Y-axis shows the number of proteins collectively in each binned ratio. B) Representative annotated MS/MS spectra of the Oct4 and c-Myc remnant–containing peptides obtained by immunoprecipitation in pluripotent ES cells. The sequence of the ubiquitinated peptide and the diglycine-modified lysine (K*) are indicated. C) In vivo ubiquitination experiments of pluripotency factors in self-renewing conditions. D) In vivo ubiquitination of Nanog, Oct4 and Sox2 in self-renewal and 24 hours of differentiation with RA using ubiquitin pull-down enrichment. E) Western blot analysis of Nanog, Oct4, Sox2 and Cullin1 half-life in self-renewal and 24 hours of differentiation using 50µg/mL of cycloheximide. G) Quantification of the half-life of proteins depicted in E. Plots represent normalized intensity relative to actin.
Figure 3
Figure 3. UPS-targeted siRNA screen identifies genes required to maintain ES cell self-renewal and pluripotency
Nanog-GFP ES cells were transfected with pools of siRNAs under conditions of self-renewal and analyzed by FACS 48h later. A) Dot plot representing Z-score for all siRNAs. B) siRNA pool validation by individual siRNAs. C) FACS plots depicting loss of Nanog-GFP following siRNA transfection. D) Knockdown efficiency by qRT-PCR after siRNA transfection. E) Relative expression of pluripotency genes (Oct4, Nanog, Zfp42); endoderm genes (Gata6, Sox17); mesoderm (Meox1 and Mixtl1) and ectoderm genes (Nk2.2 and Fgf5) by qRT-PCR. Data represented as +SEM; N=3. *p-value <0.05, **p-value<0.01. F) Heat-map illustrating gene expression (log2). Selected pluripotency and differentiation genes are shown. G) Immunofluorescence and bright field images 48 hrs following siRNA knockdown. Staining for Oct4 (Green), Phalloidin (Red), and DAPI (Blue).
Figure 4
Figure 4. UPS-targeted siRNA screen identifies genes required for optimal ES cell differentiation
Nanog-GFP ES cells were transfected with pools of siRNAs and differentiated for 48hrs followed by FACS analysis. A) Dot plot representing z-score of all siRNAs. B) Differential expression of Nanog-GFP compared to NonTarget with individual siRNAs. C) Histogram FACS plots showing retention of GFP following siRNA transfection. D) Relative knockdown after siRNA transfection by qRT-PCR. E) Relative expression of pluripotency genes (Oct4, Nanog, Zfp42); endoderm genes (Gata6, Sox17); mesoderm (Meox1 and Mixtl1) and ectoderm genes (Nk2.2 and Fgf5), by qRT-PCR. Data represented as +SEM; N=3. *p-value<0.05, **p-value<0.01. F) Immunofluorescence and bright field images 72 hrs following siRNA knockdown. Staining for Oct4 (Green), Phalloidin (Red), and DAPI (Blue). G) Heat-map illustrating changes in gene expression (log2). Selected pluripotency and differentiation genes are shown.
Figure 5
Figure 5. Psmd14, a component of the 19S proteasome lid, is required for optimal ES cell function
A-B) Relative expression of Psmd14 mRNA and protein during ES cell differentiation with RA. C) Scheme depicting 26S proteasome components and Psmd14 interacting partners. The bait, Psmd14 (purple), and interacting partners encircled in blue. D) Western blots with antibodies against Psmd14, Psmd11, Psmd12, Psmb4, K48- and K63-linkage specific linked, as well as pan-ubiquitin following Psmd14 knockdown in ES cells. E) Immunofluorescence showing K48-Ubiquitin accumulation in response to Psmd14 RNAi. F) Relative expression of genes following induction of exogenous Psmd14 in ES cells. G) Western blot analysis after 4 days of differentiation in the presence or absence of Dox. H) Relative expression of pluripotency genes Oct4, Nanog and Zfp42 (Rex1); and genes representing endoderm (Gata6), mesoderm (T) and ectoderm (Nestin) by qRT-PCR after 4 days of differentiation in the presence or absence of Dox. I) Western blot showing StrepII/Flag-tagged Psmd14WT, Psmd14H113Q, or Psmd14C120S mutant protein expression in targeted KH2 cells in the presence or absence of Dox. J) Western blot analysis with antibodies against Flag (Psmd14), Psmd11 and Psmd12 antibodies following elutions of StrepII-purified Flag-tagged Psmd14 proteins. K) Bar graph representing mean fluorescent intensity (MFI) of SSEA1-expressing cells following shRNA transfection against Psmd14 in the presence or absence of Dox and normalized to non-target control. L) Western blot analysis of Oct4 and K48-linkage specific polyubiquitinated proteins in ES cells following knockdown of endogenous Psmd14 and expression of Psmd14WT, Psmd14H113Q or Psmd14C120 proteins. Data represented as +SEM; N=3. *p-value <0.05, **p-value<0.01.
Figure 6
Figure 6. Fbxw7 targets c-Myc for degradation during ES cell differentiation
A) Western blot of pluripotency factors following Fbxw7 silencing in Day 0 and 2 of differentiation. B) Co-transfection of Fbxw7 and pluripotency factors in 293T cells followed by immunoprecipitation. Lane 1 and 2; whole cell extract and Lane 3 and 4; immunoprecipitates. C) qRT-PCR of Fbxw7 and c-Myc during differentiation. D) c-Myc protein expression during differentiation with or without 10µM MG132 treatment. E) Immunoblot of known Fbxw7 substrates at Day 0 and Day 2 of differentiation. F and G) Intracellular FACS staining with anti-c-Myc following shRNA transduction and differentiation with RA. H) Histogram representing retention of GFP 72hrs following siRNA transfection and 48hrs addition of differentiation media. Bar graph on the right represents MFI of ES cells in one representative experiment (n=3). I-J) MycT58AER ES cells following depletion of Fbxw7 with shRNA were differentiated for 2 days (−LIF+5µM RA) without or in the presence of 10nM 4-OHT, and analyzed by I) immunoblot for c-Myc, J) SSEA1 expression Bar graph represents MFI of SSEA1 in one representative experiment (n=2), and K) morphology of colonies. Data represented as +SEM; N=3. *p-value <0.05, **p-value<0.01.
Figure 7
Figure 7. Psmd14 and Fbxw7 regulate cellular reprogramming
A) Images of ubiquitously expressed actin-RFP 6 days post siRNA transfection and Dox (OKSM) induction demonstrating morphology changes. B) AP+ ES-like colony count at Day 14 following siRNA knockdown of Fbxw7 and Psmd14 relative to +Dox control. C) Absolute number of AP+ ES-like colonies at day 14 following siRNA transfection and addition of Dox of MEFs. One representative experiment is shown (n=4). D) AP staining 14 days following siRNA transfection and addition of Dox. E) Reprogramming efficiency of OKSM MEFs expressing shRNAs against non-target, Psmd14, or Fbxw7 to generate AP+ ES-like colonies. Colonies were enumerated at day 14. F) Morphology of representative colonies following transduction of shRNAmir at day 14. Green; shRNAmir transduced cells. G) Efficiency of OSK MEFs expressing shRNAs against NonTarget, or Fbxw7 to generate AP+ ES-like colonies. Colonies were enumerated at day 14. H) Proteins identified and quantified in MEFs and iPS in previous studies, showing proteins highly expressed in iPS and the number of the ones that are ubiquitinated. I) Overlap between ubiquitinated proteins in ES and iPS and comparison with ubiquitinated proteins in MEFs. J) Self-Renewal network, as depicted in Figure 1D, demonstrating proteins that have been identified to be ubiquitinated in iPS and ES (grey). Proteins also ubiquitinated in MEFs are marked in yellow. Proteins in orange were found ubiquitinated in iPS and MEFs and proteins in purple were exclusively found in iPS. Data represented as +SEM; N=3. *p-value <0.05, **p-value<0.01.
See this image and copyright information in PMC

Comment in

  • UPS delivers pluripotency.
    Okita Y, Nakayama KI. Okita Y, et al. Cell Stem Cell. 2012 Dec 7;11(6):728-30. doi: 10.1016/j.stem.2012.11.009. Cell Stem Cell. 2012. PMID: 23217415

References

    1. Adams J, Kauffman M. Development of the proteasome inhibitor Velcade (Bortezomib) Cancer investigation. 2004;22:304–311. - V体育官网入口 - PubMed
    1. Aghajan M, Jonai N, Flick K, Fu F, Luo M, Cai X, Ouni I, Pierce N, Tang X, Lomenick B, et al. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat Biotechnol. 2010;28:738–742. - PMC - PubMed
    1. An J, Yang DY, Xu QZ, Zhang SM, Huo YY, Shang ZF, Wang Y, Wu DC, Zhou PK. DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein. Molecular cancer. 2008;7:32. - "V体育ios版" PMC - PubMed
    1. Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 2003;17:126–140. - PMC - PubMed
    1. Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis. 2006;44:23–28. - PubMed

Publication types

MeSH terms

VSports app下载 - Associated data