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Localization of the Rb tumor suppressor in MEFs during reprogramming to iPS and the consequence of Rb loss on the transcriptional profile and histone landscape in these cells

ABSTRACT: The reprogramming of differentiated cells to an embryonic stem cell-like state provides a powerful system to explore fundamental mechanisms of development, including how mammalian cells establish and maintain pluripotency and long-term self-renewal capability. Based on the similarities between embryonic stem cells and cancer cells, we investigated the potential role of the retinoblastoma tumor suppressor and cell cycle regulator RB in the reprogramming of fibroblasts into induced pluripotent stem cells (iPS cells). Herein we demonstrate that loss of RB function leads to both an acceleration of the reprogramming process and the generation of more iPS clones from fibroblasts. This effect is largely due to a restrictive role for RB at the early stages of reprogramming. Surprisingly, however, RB inactivation does not enhance the formation of iPS clones by accelerating the proliferation of cells undergoing reprogramming. Rather, a genome-wide investigation of RB targets indicates that RB binds to regulatory regions of pluripotency genes such as Sox2 and Oct4 and contributes to their full repression in differentiated cells. This effect correlates with the maintenance of a repressive chromatin structure at these loci. Accordingly, Rb-deficient fibroblasts can be reprogrammed into iPS cells even in the absence of exogenous Sox2, which is normally required to initiate reprogramming from fibroblasts. These experiments identify a novel barrier in the reprogramming process, mainly the repression of certain pluripotency genes such as Sox2 by RB, which provides a new link between tumor suppressor mechanisms and cellular reprogramming.

ORGANISM(S): Mus musculus

PROVIDER: GSE40594 | GEO | 2014/11/13

SECONDARY ACCESSION(S): PRJNA174435

REPOSITORIES: GEO

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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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Localization of the Rb tumor suppressor in MEFs during reprogramming to iPS and the consequence of Rb loss on the transcriptional profile and histone landscape in these cells

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