Function of cell fate determinants during acquisition and loss of pluripotency

• Applicant: Professor Dr. Jens Christian Schwamborn
• Subject Area: Cell Biology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2011 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 195082371

• Final Report Year: 2014

Final Report Abstract

The emergence of the possibility to reprogram a terminally differentiated cell into a pluripotent stem cells (iPSCs) has provided great potentials for regenerative medicine. This was achieved by the enforced overexpression of four transcription factors, namely Oct4, Sox2, Klf4 and cMyc, in short OSKM, in mouse embryonic fibroblasts. Later on, the same was shown for adult human fibroblasts and further on for miscellaneous other adult somatic cells. Interestingly, variable efficiencies underlie the generation of iPSCs from the different sources using the different approaches, which implies the presence of a pool of cellular modulators that could have the ability to further fine-tune the generation of iPSCs. This does not only apply to the reprogramming process into pluripotent stem cells but also for the efficient differentiation of these cells into the required somatic cell type. Thus, deeper understanding of the underlying molecular mechanisms is still required. TRIM32 is an E3 ubiquitin ligase that belongs to the tripartite motif (TRIM) family. Previously, we have been shown that TRIM32 is regulating neuronal differentiation of neural stem cells. As a differentiation inducing factor, it is tempting to speculate that TRIM32 would play a counteracting role to previously described TRIM-NHL proteins and would rather constitute a potential hurdle for cellular reprogramming of somatic cells into iPSCs. In this project we demonstrated that TRIM32 is expressed in pluripotent stem cells whereas its expression increases with differentiation of these cells. Moreover our data support the concept that TRIM32 constitutes a barrier for cellular reprogramming since TRIM32 deficient mouse fibroblasts showed enhanced iPSC colony generation upon OSKM induction. Carefully analyzing the resulting TRIM32-ko iPSCs indicated slower differentiation kinetics with slower degradation of pluripotency associated proteins. These findings could be explained by the observation that TRIM32 is able to ubiquitinate Oct4, targeting it for proteasomal degradation and thereby regulating its degradation during cellular differentiation. All in all our data show that TRIM32 is a reprogramming roadblock that facilitates cellular transition to a more differentiated state by modulating the protein levels of Oct4 and c-Myc via ubiquitination.

Publications

• (2012) Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors. Cell Stem Cell 10(4), 465-72
  Han D.W., Tapia N., Hermann A., Hemmer K., Höing S., Araúzo-Bravo M.J., Zaehres H., Frank S., Moritz S., Greber B., Yang J.H., Lee H.T., Schwamborn J.C., Storch A., Schöler H.R.
  
• (2012). Discovery of Inhibitors of Microglial Neurotoxicity Acting Through Multiple Mechanisms Using a Stem Cell–Based Phenotypic Assay. Cell Stem Cell 11(5), 620-32
  Höing S., Rudhard Y., Reinhardt P., Glatza M., Stehling M., Wu G., Peiker C., Böcker A., Bunk E.C., Schwamborn J.C., Slack M., Sterneckert J., Schöler H.R.
  
• (2012). Inhibition of neuroinflammation activates adult neural stem cells and induces functional regeneration in a mouse model for Parkinson’s disease. Stem Cell Research & Therapy 3(4)
  Worlitzer M.M.A., Bunk E.C., Hemmer K., Schwamborn J.C.
  
• (2013). Regulatory feedback loop between TP73 and TRIM32. Cell Death Dis 4, e704
  Gonzalez-Cano L., Hillje A.L., Fuertes-Alvarez S., Marques M.M., Blanch A., Ian R.W., Irwin M.S., Schwamborn J.C., Marín M.C.
  
• (2013). Systemic analysis of neural stem cell maintenance mechanisms reveals the critical function of an E2F1-microRNA regulatory circuitry. Nucleic Acids Research 41(6), 3699-712
  Palm T., Hemmer K., Winter J., Fricke, I.B., Tarbashevich K., Shakib F., Rudolph I.M., Hillje A.L., De Luca P., Bahnassawy L., Madel R., Viel T., De Siervi A., Jacobs A.H., Diederichs S., Schwamborn J.C.
  
• (2013). The majority of newly generated cells in the adult mouse substantia nigra express low levels of Doublecortin, but their proliferation is unaffected by 6-OHDA-induced nigral lesion or Minocycline-mediated inhibition of neuroinflammation. Eur J Neurosci 38(5), 2684-92
  Worlitzer M.M., Viel T., Jacobs A.H., Schwamborn J.C.
  
• (2013). The Parkinson’s disease associated LRRK2 mutation R1441G inhibits neuronal differentiation of neural stem cells. Stem Cells Dev 22(18), 2487-96
  Bahnassawy L., Palm T., Nicklas S., Quandel T., Birzele F., Gillardon F., Schwamborn J.C.
  
• (2013). TRIM32 dependent transcription in adult neural progenitors regulates neuronal differentiation and olfactory learning. Cell Death Dis 4, e976
  Hillje A.L., Pavlou M.A., Beckmann E., , Worlitzer M.M., Bahnassawy L., Lewejohann L., Palm T., Sachser N., Schwamborn J.C.
  
• (2014). The Notch co-repressor protein NKAP is highly expressed in adult mouse subventricular zone neural progenitor cells. Neuroscience 266, 138-49
  Worlitzer M.M., Schwamborn J.C.