Abstract
X-chromosome inactivation is a dosage compensation mechanism that equalizes X-linked gene expression between male and female mammals through the transcriptional silencing of most genes on one of the two X-chromosomes in females. With a few key exceptions, once the X-chromosome is inactivated replicated copies of that X-chromosome are maintained as inactive in all descendant cells. X-inactivation is therefore a paradigm of epigenetic inheritance. Imprinted X-inactivation is a specialized form of X-inactivation that results in the silencing of the paternally derived X-chromosome. Due to its parent-of-origin-specific pattern of inactivation, imprinted X-inactivation is a model of mitotic as well as meiotic, i.e., transgenerational, epigenetic inheritance. All cells of the early mouse embryo undergo imprinted X-inactivation, a pattern that is subsequently maintained in extraembryonic cell types in vivo and in vitro. Here, we describe both high- and low-throughput approaches to interrogate imprinted X-inactivation in the mouse embryo as well in cultured extraembryonic stem cells.
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References
Huynh KD, Lee JT (2003) Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature 426(6968):857–862
Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303(5658):644–649. https://doi.org/10.1126/science.1092727
Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S, Otte AP, Brockdorff N (2004) Reactivation of the paternal X chromosome in early mouse embryos. Science 303(5658):666–669
Takagi N, Sasaki M (1975) Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 256(5519):640–642
West JD, Frels WI, Chapman VM, Papaioannou VE (1977) Preferential expression of the maternally derived X chromosome in the mouse yolk sac. Cell 12(4):873–882
Harper MI, Fosten M, Monk M (1982) Preferential paternal X inactivation in extraembryonic tissues of early mouse embryos. J Embryol Exp Morphol 67:127–135
Lyon MF (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190:372–373
Maclary E, Buttigieg E, Hinten M, Gayen S, Harris C, Sarkar MK, Purushothaman S, Kalantry S (2014) Differentiation-dependent requirement of Tsix long non-coding RNA in imprinted X-chromosome inactivation. Nat Commun 5:4209. https://doi.org/10.1038/ncomms5209
Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J (1998) Promotion of trophoblast stem cell proliferation by FGF4. Science 282(5396):2072–2075
Kunath T, Arnaud D, Uy GD, Okamoto I, Chureau C, Yamanaka Y, Heard E, Gardner RL, Avner P, Rossant J (2005) Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132(7):1649–1661. https://doi.org/10.1242/dev.01715
Gayen S, Maclary E, Buttigieg E, Hinten M, Kalantry S (2015) A primary role for the Tsix lncRNA in maintaining random X-chromosome inactivation. Cell Rep 11(8):1251–1265. https://doi.org/10.1016/j.celrep.2015.04.039
Gayen S, Maclary E, Hinten M, Kalantry S (2016) Sex-specific silencing of X-linked genes by the Xist RNA. Proc Natl Acad Sci U S A 113(3):E309–E318
Sarkar MK, Gayen S, Kumar S, Maclary E, Buttigieg E, Hinten M, Kumari A, Harris C, Sado T, Kalantry S (2015) An Xist-activating antisense RNA required for X-chromosome inactivation. Nat Commun 6:8564. https://doi.org/10.1038/ncomms9564
Maclary E, Hinten M, Harris C, Sethuraman S, Gayen S, Kalantry S (2017) PRC2 represses transcribed genes on the imprinted inactive X chromosome in mice. Genome Biol 18(1):82. https://doi.org/10.1186/s13059-017-1211-5
Calabrese JM, Sun W, Song L, Mugford JW, Williams L, Yee D, Starmer J, Mieczkowski P, Crawford GE, Magnuson T (2012) Site-specific silencing of regulatory elements as a mechanism of X inactivation. Cell 151(5):951–963. https://doi.org/10.1016/j.cell.2012.10.037
Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, Handsaker RE, Lunter G, Marth GT, Sherry ST, McVean G, Durbin R, Genomes Project Analysis G (2011) The variant call format and VCFtools. Bioinformatics 27(15):2156–2158. https://doi.org/10.1093/bioinformatics/btr330
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. https://doi.org/10.1093/bioinformatics/bts635
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6):841–842. https://doi.org/10.1093/bioinformatics/btq033
Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169. https://doi.org/10.1093/bioinformatics/btu638
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. https://doi.org/10.1186/s13059-014-0550-8
Breese MR, Liu Y (2013) NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets. Bioinformatics 29(4):494–496. https://doi.org/10.1093/bioinformatics/bts731
Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A (1998) Non-invasive sexing of preimplantation stage mammalian embryos. Nat Genet 19(3):220–222
Kalantry S, Magnuson T (2006) The polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation. PLoS Genet 2(5):e66. https://doi.org/10.1371/journal.pgen.0020066
Kalantry S, Purushothaman S, Bowen RB, Starmer J, Magnuson T (2009) Evidence of Xist RNA-independent initiation of mouse imprinted X-chromosome inactivation. Nature 460:647–651. https://doi.org/10.1038/nature08161
Rossant J (2007) Stem cells and lineage development in the mammalian blastocyst. Reprod Fertil Dev 19(1):111–118
Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R, Willard HF (1991) A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349(6304):38–44
Hinten M, Maclary E, Gayen S, Harris C, Kalantry S (2016) Visualizing long noncoding RNAs on chromatin. Methods Mol Biol 1402:147–164. https://doi.org/10.1007/978-1-4939-3378-5_12
Nagy A, Gertsenstein M, Vintersten K, Behringer RR (2003) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Acknowledgments
This work was funded by an NIH training grant: Michigan Predoctoral Training in Genetics (T32GM007544) to M.C.; NIH National Research Service Award #5-T32-GM07544 from the National Institute of General Medicine Sciences to E.M.; an NIH Director’s New Innovator Award (DP2-OD-008646-01) to S.K.; a March of Dimes Basil O’Connor Starter Scholar Research Award (5-FY12-119); and the University of Michigan Endowment for Basic Sciences.
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Cloutier, M., Harris, C., Gayen, S., Maclary, E., Kalantry, S. (2018). Experimental Analysis of Imprinted Mouse X-Chromosome Inactivation. In: Sado, T. (eds) X-Chromosome Inactivation. Methods in Molecular Biology, vol 1861. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8766-5_14
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DOI: https://doi.org/10.1007/978-1-4939-8766-5_14
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