Identification of additional/alternative factors required for cellular reprogramming

• Antragsteller: Professor Dr. Hans Robert Schöler, seit 8/2012
• Fachliche Zuordnung: Biochemie ; Zellbiologie
• Förderung: Förderung von 2008 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 66126906

• Erstellungsjahr: 2011

Zusammenfassung der Projektergebnisse

When it comes to inducing pluripotency, Oct4 is the only factor that cannot be replaced by other members of the same family. The Oct4 and Pou2 genes evolved from a POU class V gene ancestor, but it is unknown whether pluripotency induced by Oct4 gene activity is a feature specific to mammals or was already present in ancestral vertebrates. Different vertebrate Pou2 and Oct4 homologues can induce pluripotency in mouse and human fibroblasts. The inability of zebrafish Pou2 to establish pluripotency is not representative of all Pou2 genes, as medaka Pou2 and axolotl Pou2 are able to reprogram somatic cells into pluripotent ones. Therefore, induction of pluripotency is not a feature specific to mammals, but existed in the common Oct4/Pou2 ancestral vertebrate. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. The linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein's surface. Point mutations in this αhelix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein-protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. To further elucidate the role of Oct4 during reprogramming, the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types was studied. A significant upregulation of pluripotent markers could not be detected. Therefore, Oct4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Oct4 overexpression upregulated the gene Mgarp in all analyzed cell types. Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, Oct4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs. ESCs are characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the TFIID complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Those results show that TFIID is critical for transcription-factor-mediated reprogramming.

Projektbezogene Publikationen (Auswahl)

• Evaluating the Potential of Putative Pluripotent Cells Derived from Human Testis. Stem Cells 29:1304-9, 2011
  Ko K, Reinhardt P, Tapia N, Schneider-Kramann R, Araúzo-Bravo MJ, Han DW, Greber B, Kim J, Kliesch S, Zenke M, Schöler HR
  
• Autologous Pluripotent Stem Cells Generated from Adult Mouse Testicular Biopsy. Stem Cell Rev 8(2):435-44, 2012
  Ko K, Wu G, Araùzo-Bravo M, Kim J, Francine J, Greber B, Mühlisch J, Joo J, Sabour D, Frühwald, Tapia N, Schöler H
  
• Reprogramming to pluripotency is an ancient trait of vertebrate Oct4 and Pou2 proteins. Nat Commun 3:1279, 2012
  Tapia N, Reinhardt P, Duemmler A, Wu G, Araúzo-Bravo MJ, Esch D, Greber B, Cojocaru V, Rascon CA, Tazaki A, Kump K, Voss R, Tanaka EM, Schöler HR
  
• Zfp296 is a novel, pluripotent-specific reprogramming factor. PLoS One 7(4):e34645, 2012
  Fischedick G, Klein DC, Wu G, Esch D, Höing S, Han DW, Reinhardt P, Hergarten K, Tapia N, Schöler HR, Sterneckert JL
  
• A central role for TFIID in the pluripotent transcription circuitry. Nature 495(7442):516-9, 2013
  Pijnappel WW, Esch D, Baltissen MP, Wu G, Mischerikow N, Bergsma AJ, van der Wal E, Han DW, Bruch HV, Moritz S, Lijnzaad P, Altelaar AF, Sameith K, Zaehres H, Heck AJ, Holstege FC, Schöler HR, Timmers HT
  
• A unique Oct4 interface is crucial for reprogramming to pluripotency. Nat Cell Biol 15(3):295-301, 2013
  Esch D, Vahokoski J, Groves MR, Pogenberg V, Cojocaru V, Vom Bruch H, Han D, Drexler HC, Araúzo-Bravo MJ, Ng CK, Jauch R, Wilmanns M, Schöler HR
  
• Establishment of totipotency does not depend on Oct4A. Nat Cell Biol 15(9):1089-97, 2013
  Wu G, Han D, Gong Y, Sebastiano V, Gentile L, Singhal N, Adachi K, Fischedick G, Ortmeier C, Sinn M, Radstaak M, Tomilin A, Schöler HR
  
• BRG1 is required to maintain pluripotency of murine embryonic stem cells. Biores Open Access 3(1):1-8, 2014
  Singhal N, Esch D, Stehling M, Schöler HR
  
• Counteracting activities of OCT4 and KLF4 during reprogramming to pluripotency. Stem Cell Reports 2(3):351-65, 2014
  Tiemann U, Marthaler AG, Adachi K, Wu G, Fischedick GU, Araúzo-Bravo MJ, Schöler HR, Tapia N