Telephone: 613-739-6737; Fax: 613-737-8803
Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 22 MAY 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 6, pages 1107–1120, June 2013
How to Cite
Campbell, P. A. and Rudnicki, M. A. (2013), Oct4 Interaction with Hmgb2 Regulates Akt Signaling and Pluripotency. STEM CELLS, 31: 1107–1120. doi: 10.1002/stem.1365
Author contributions: P.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; M.R.: financial support and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
first published online in STEM CELLS EXPRESS February 4, 2013.
- Issue online: 22 MAY 2013
- Version of Record online: 22 MAY 2013
- Accepted manuscript online: 14 MAR 2013 12:00AM EST
- Manuscript Accepted: 31 JAN 2013
- Manuscript Revised: 26 JAN 2013
- Manuscript Received: 15 OCT 2012
- Ontario Genomics Institute, the Stem Cell Network
- Canadian Institutes of Health Research
Additional Supporting Information may be found in the online version of this article.
|sc-12-0966_sm_SupplFigure1.tif||1095K||Supplemental Figure S1. Akt-dependence of Oct4-Hmgb2 interaction. (A) Interaction between Hmgb2 and WT (wild-type) Oct4 following transient transfection of Oct4 WT, T228, and T228D in 10T1/2 fibroblasts. EV represents the empty vector transfected negative control. (B) Hmgb2 interacts with WT Oct4 in 10T1/2 fibroblasts only when Akt signaling is active. Note heightened Akt activation in WT Oct4 transfected cells. EV represents the empty vector transfected negative control. WT is Oct4 wild-type.|
|sc-12-0966_sm_SupplFigure2.tif||1059K||Supplemental Figure S2. Alkaline Phosphatase staining in Oct4 over-expressing mESC sublines. (A) Representative alkaline phosphatase staining of mESC colonies. Size of each colony is indicated. (B) Quantitation of the alkaline phosphatase stained cells. Numbers represent the Mean ± SEM, n=4. Statistical significance: * p≤0.05 by two tailed Student's t-test.|
|sc-12-0966_sm_SupplFigure3.tif||1043K||Supplemental Figure S3. Western Analysis of luciferase lysates. Western analysis of mESCs lysates from luciferase assay described in Figure 6B.|
|sc-12-0966_sm_SupplFigure4.pdf||1294K||Supplemental Figure S4. Transcriptional synergy between Oct4 and Hmgb2 in 10T1/2 fibroblasts. (A) Luciferase assays as described in Figure 6B, except performed in 10T1/2 fibroblasts. Data depicted are Mean ± SEM, n=9. Statistical significance: * p≤0.05; ** p≤0.005, by Student's t-test. * Compared to Oct4 WT only (without Hmgb2) transfection. ** Compared to WT Oct4 co-transfected with Hmgb2. *** p≤0.05 as compared to Oct4 K118R only. (B), Schematic of Oct4 and Hmgb2 constructs employed in luciferase assays. NTAP denotes Nterminal 6X His-TEV-3X-FLAG epitope tagged Oct4 construct (Oct4 NTAP). DNA binding POU-specific domain (POUS) and POU-homeobox domain (POUH) are indicated in the red and green boxes, respectively. Hmgb2 construct contains an N-terminal MYC epitope tag (N-MYC). The two DNA binding HMG boxes of Hmgb2 are shown in blue. (C) Western blots of lysates from luciferase assays. Note doublet band upon co-transfection of Hmgb2 and the Oct4 K118R mutant and 60 kDa band detected by the anti-MYC epitope tag antibody directed against Hmgb2.|
|sc-12-0966_sm_SupplFigure5.tif||389K||Supplemental Figure S5. De-repression of Oct4 targets in Sumo mutant expressing mESCs. Luciferase assays were performed in J1 mESCs as previously described. The indicated Oct4 WT or mutant construct was co-transfected along with reporters for Cdx2, Ash2l, or Phc3. Schematic below each result indicates promoter/enhancer region within the genomic context of each transcript as well as the location of Oct, Sox, and G(A) motifs. Figure legend provides key to graphs.|
|sc-12-0966_sm_SupplFigure6.tiff||491K||Supplemental Figure S6. Oct4 SUMOylation at K118 mediates transcriptional repression in mESC. Pooled stable mESC sublines expressing the indicated constructs were harvested for QRT-PCR and ChIP-QPCR analysis to ascertain the effect of Oct4 WT and mutant over-expression to the indicated transcriptional target in undifferentiated J1 mESCs. All analyses were performed as previously described.|
|sc-12-0966_sm_SupplFigure7.tif||479K||Supplemental Figure S7. Oct4 K118R mutant and Hmgb2 block RnapII at Phc1 locus (A) Luciferase assays performed in J1 mESCs as previously described using Phc1 promoter luciferase reporter. Lower schematic represents genomic context of region used in reporter and indicates location of Oct, Sox, and G(A) motifs. Legend for all panels can be found in Supplementary Figure S4. (B) RT-PCR analysis of Phc1 expression in stably expressing Oct4 WT and mutant clones in mESC using primers located in Exons 13 and 14. (C) RT-PCR analysis of Phc1 transcript level expression in J1 mESC clones stably expressing the indicated Oct4 NTap construct using primers tiling down Phc1 transcript. (D) Diagram of Phc1 exon structure with location of ChIP-qPCR primers indicated. POUS and G(A) motifs identified at +2337, located in intron 2. (E) Increased enrichment of Oct4 (Oct4 specific and FLAG epitope tag antibody) and Hmgb2 are observed at the Phc1 +2337 regulatory element. (F) Tiling ChIP-qPCR at Phc1 locus shows increased RnapII at promoter in the K118R mutants. (G) H3K36me3 extends into coding region. (H) Increased enrichment of H2A K119Ubn1 suggests initiation of the DNA damage response at this locus. ChIP-PCR shows increased Oct4 and Hmgb2 enrichment at the +2337 region of Phc1. Data suggests that loss of Oct4 sumoylation mediates RnapII block and repression of Phc1 through increased binding of Oct4 and Hmgb2 at Oct/G(A) motif at +2337. All analysis performed as previously described.|
|sc-12-0966_sm_SupplFigure8.tif||422K||Supplemental Figure S8. Altered enrichment of transcriptional regulators and histone modifications in Oct4 K118R mutant sublines. Extended analysis of chromatin occupancy at the Cdx2 promoter, Hoxb1 ARE, and Phc1 promoter by ChIP in J1 mESCs and pooled, stable clones expressing Oct4 WT or the indicated mutant constructs by ChIP. Analysis as previously described.|
|sc-12-0966_sm_SupplFigure9.tif||103K||Supplemental Figure S9. Oct4 K118R mutant impedes cell cycle progression. (A) Enumeration of mitotic figures in J1 and stable pooled clones of J1s expressing NTAP empty vector, WT, and K118R mutant Oct4. Statistical significance: * p≤0.05; ** p≤0.005; *** p≤0.001, n=4. (B) Representative DAPI staining of pooled stable clones of J1 mESC overexpressing the Oct4 K118R used for enumeration of mitotic figures. Blue arrow denotes representative cell blocked at prometaphase. Blue arrowhead denotes representative cell blocked at anaphase to telophase transition. DAPI staining was performed in 4 independent experiments. Images shown are at 20× magnification.|
|sc-12-0966_sm_SupplFigure10.tif||2301K||Supplemental Figure S10. Oct4 PTMs modulate APC/C Substrates. Western analysis of J1 and pooled stable J1 clones expressing the indicated WT and mutant NTAP constructs. Similar to the V118A mutant, the C214A and combined V118A/C214 mutants abrogate only the sumoylation consensus motifs and therefore retain the ability to undergo other potential modification at K118 or K215. Altered detection of cell cycle regulatory APC/C substrates Ccne1, Ccnb1, p55Cdc, and Cdkn1a is observed in the Oct4 mutant expressing clones.|
|sc-12-0966_sm_SupplFigure11.tif||539K||Supplemental Figure S11. Oct4 sumoylation is required for dimerization. (A) Lysates from the transient transfections described in Figure 6G are provided.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.