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Brumbaugh, Justin

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Brumbaugh

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Justin

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Brumbaugh, Justin

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2014 - 20175

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Author's Publications: search.filters.isAuthorOfPublication.a605d94a-3765-4cc2-9890-5648c8ef9f93

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Now showing 1 - 5 of 5
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    A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2
    (Elsevier, 2016) Borkent, Marti; Bennett, Brian D.; Lackford, Brad; Bar-Nur, Ori; Brumbaugh, Justin; Wang, Li; Du, Ying; Fargo, David C.; Apostolou, Effie; Cheloufi, Sihem; Maherali, Nimet; Elledge, Stephen J.; Hu, Guang; Hochedlinger, Konrad
    Summary The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.
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    Small molecules facilitate rapid and synchronous iPSC generation
    (2014) Bar-Nur, Ori; Brumbaugh, Justin; Verheul, Cassandra; Apostolou, Effie; Pruteanu-Malinici, Iulian; Walsh, Ryan M.; Ramaswamy, Sridhar; Hochedlinger, Konrad
    The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2, and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is protracted and inefficient, resulting in heterogeneous cell populations. Here we show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-beta inhibitor (termed “AGi”) facilitates more synchronous and rapid iPSC formation from a variety of mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 hours of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.
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    The histone chaperone CAF-1 safeguards somatic cell identity
    (2016) Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Lucy; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee; Ang, Cheen Euong; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y.; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad
    Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
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    Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    (Springer Nature, 2017) Choi, Jiho; Huebner, Aaron; Clement, Kendell; Walsh, Ryan M.; Savol, Andrej J; Lin, Kaixuan; Gu, Hongcang; Di Stefano, Bruno; Brumbaugh, Justin; Kim, Sang-Yong; Sharif, Jafar; Rose, Christopher M.; Mohammad, Arman; Odajima, Junko; Charron, Jean; Shioda, Toshihiro; Gnirke, Andreas; Gygi, Steven; Koseki, Haruhiko; Sadreyev, Ruslan; Xiao, Andrew; Meissner, Alexander; Hochedlinger, Konrad
    Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo1. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.
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    Lineage conversion induced by pluripotency factors involves transient passage through an iPS cell stage
    (2016) Bar-Nur, Ori; Verheul, Cassandra; Sommer, Andreia Gianotti; Brumbaugh, Justin; Schwarz, Benjamin; Lipchina, Inna; Huebner, Aaron; Mostoslavsky, Gustavo; Hochedlinger, Konrad
    Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, was reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4+ cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.