Abstract
In vole Microtus levis, cells of preimplantation embryo and extraembryonic tissues undergo imprinted X chromosome inactivation (iXCI) which is triggered by a long non-coding nuclear RNA, Xist. At early stages of iXCI, chromatin of vole inactive X chromosome is enriched with the HP1 heterochromatin-specific protein, trimethylated H3K9 and H4K20 attributable to constitutive heterochromatin. In the study, using vole trophoblast stem (TS) cells as a model of iXCI, we further investigated chromatin of the inactive X chromosome of M. levis and tried to find out the role of Xist RNA. We demonstrated that chromatin of the inactive X chromosome in vole TS cells also contained the SETDB1 histone methyltransferase and KAP1 protein. In addition, we observed that Xist RNA did not contribute significantly to maintenance of X chromosome inactive state during iXCI in vole TS cells. Xist repression affected neither transcriptional silencing caused by iXCI nor maintenance of trimethylated H3K9 and H4K20 as well as HP1, KAP1, and SETDB1 on the inactive X chromosome. Moreover, the unique repertoire of chromatin modifications on the inactive X chromosome in vole TS cells could be disrupted by a chemical compound, DZNep, and then restored even in the absence of Xist RNA. However, Xist transcript was necessary for recruitment of an additional repressive histone modification, trimethylated H3K27, to the inactive X chromosome during vole TS cell differentiation.
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Acknowledgments
We thank S.I. Bayborodin for the technical assistance.
Funding
The work was funded by the Russian Academy of Sciences (program II.2P/VI.60-1 No 0324-2016-0032 and the Russian Foundation for Basic Research (grant No. 15-04-03947a).
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Fig. S1
A) SETDB1 distribution on metaphase spreads of vole XY TS cells. X and Y chromosomes are shown by arrows. B) HP1-gamma (green) and H4K20me3 (red) in nuclei of vole trophoblast cells after two weeks of differentiation. Metaphase chromosomes and nuclei were counterstained with DAPI (blue). MS4 is a DNA repeat specific for a giant telomeric block of constitutive heterochromatin on X chromosome of M. levis. (GIF 116 kb)
Fig. S2
Chromatin modifications on Xi in DZNep treated and untreated vole TS cells. MS4 repeat marks presence of two X chromosomes in nuclei of DZNep treated TS cells. (GIF 185 kb)
Fig. S3
DZNep treatment does not affect the level of Xist expression in vole TS cells. Relative level of Xist expression was examined by semi-quantitative RT-PCR. Blue bars represent untreated TS cell line, orange bars do the TS cell treated with DZNep. Xa – / Xi +, the original TS cell line, R2, and TS cell clones without the deletion; Xa Δ / Xi +, TS cell clones with the deletion in the Xist promoter and normal Xist expression; Xa – / Xi Δ, TS cell clones with the deletion in the Xist promoter and depleted Xist expression. R1, XO TS cell line. (GIF 71 kb)
Fig. S4
A) RNA FISH detecting monoallelic Hprt1 expression from Xa in XX TS cell nuclei with presence and absence of Xist RNA on Xi. Xi is enriched with H3K9me3. Xa – / Xi +, Xist is properly expressed; Xa – / Xi Δ, Xist is repressed. Scale bar is 10 μm. В) RNA FISH signals of monoallelically expressed Pgk1 (red) and Hprt1 (green) were revealed in a close proximity within nucleus. IF, immunofluorescent staining. (GIF 512 kb)
Table S1
Antibodies used in the study (DOCX 15 kb)
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Shevchenko, A.I., Grigor’eva, E.V., Medvedev, S.P. et al. Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis . Chromosoma 127, 129–139 (2018). https://doi.org/10.1007/s00412-017-0650-9
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DOI: https://doi.org/10.1007/s00412-017-0650-9