The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting

Abstract

Pluripotency of embryonic stem (ES) cells is controlled by defined transcription factors1,2. During differentiation, mouse ES cells undergo global epigenetic reprogramming, as exemplified by X-chromosome inactivation (XCI) in which one female X chromosome is silenced to achieve gene dosage parity between the sexes3,4,5. Somatic XCI is regulated by homologous X-chromosome pairing6,7 and counting8,9,10, and by the random choice of future active and inactive X chromosomes. XCI and cell differentiation are tightly coupled11, as blocking one process compromises the other8,12 and dedifferentiation of somatic cells to induced pluripotent stem cells is accompanied by X chromosome reactivation2. Recent evidence suggests coupling of Xist expression to pluripotency factors occurs13, but how the two are interconnected remains unknown. Here we show that Oct4 (also known as Pou5f1)14 lies at the top of the XCI hierarchy, and regulates XCI by triggering X-chromosome pairing and counting. Oct4 directly binds Tsix and Xite, two regulatory noncoding RNA genes of the X-inactivation centre15,16, and also complexes with XCI trans-factors, Ctcf and Yy1 (ref. 17), through protein–protein interactions. Depletion of Oct4 blocks homologous X-chromosome pairing and results in the inactivation of both X chromosomes in female cells. Thus, we have identified the first trans-factor that regulates counting, and ascribed new functions to Oct4 during X-chromosome reprogramming.

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Figure 1: Oct4 and Sox2 bind Tsix and Xite.
Figure 2: Oct4–Ctcf and Sox2–Yy1 interactions, and Tsix transcriptional activation.
Figure 3: Oct4 knockdown disrupts X–X pairing and results in aberrant Xist expression in female ES cells.
Figure 4: Ectopic Xist expression from both X chromosomes in Oct4-deficient ES cells.

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Acknowledgements

We thank N. Stavropoulos and J. Lopilato for their contributions to the early phase of this work. We also thank J. Zhao, J. A. Erwin and J. Ahn for critical reading of the manuscript and all laboratory members for stimulating discussion. This work was funded by a National Institutes of Health grant to J.T.L. (GM58839). J.T.L. is an Investigator of the Howard Hughes Medical Institute.

Author Contributions M.E.D. designed, performed and analysed the Xite/Tsix bioinformatics screen, EMSA, tests of protein–protein interactions, luciferase assays, knockdowns and qRT–PCR; S.S.S. designed, performed and analysed the pairing and counting experiments; and S.F.P. designed, performed and analysed the Xist bioinformatics screen and the quantitative ChIP assays. N.X. participated in initial pairing analyses. J.T.L. formulated, analysed and directed the study and wrote the paper.

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Correspondence to Jeannie T. Lee.

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Donohoe, M., Silva, S., Pinter, S. et al. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting. Nature 460, 128–132 (2009). https://doi.org/10.1038/nature08098

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