Unknown,Transcriptomics,Genomics,Proteomics

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JARID2-associated protein network drives rapid and efficient reprogramming to pluripotency.

ABSTRACT: Somatic cell reprogramming can be achieved by cell fusion with embryonic stem cells (ESCs), nuclear transfer into oocytes, or forced expression of transcription factors essential for ESC identity. Reprogramming by transcription factors is a less efficient and slower process than that by other methods. Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here, we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by combined transduction of Jarid2, a gene highly expressed in both ESCs and oocytes and genes encoding its associated proteins. We demonstrate that forced expression of Jarid2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, Jarid2 accelerated retroviral transgene silencing and Nanog promoter demethylation, confirming its promoting activity. We further reveal that JARID2 physically interacts with ESRRB, SALL4A and PRDM14, and show that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a Jarid2-dependent manner. Our findings provide an insight into the important roles of Jarid2 during reprogramming, and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers. We used microarrays to detect the up- and down-regulated genes in MEFs transduced with Jarid2 compared to empty vector control. Total RNA was extracted from mouse embrionc fibroblasts (MEFs) transduced with empty vector (control) or the indicated genes at 3 days post-transduction, and two iPSC clones from MEFs transduced with OSKM plus Jarid2. Gene expression profile was analyzed by the Affymetrix GeneChip microarray system.

ORGANISM(S): Mus musculus

SUBMITTER: Yasushi Okazaki

PROVIDER: E-GEOD-52200 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).

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JARID2-associated protein network drives rapid and efficient reprogramming to pluripotency.

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