Abstract
Imprinted X chromosome inactivation (iXCI) balances the expression of X-linked genes in preimplantation embryos and extraembryonic tissues in rodents. Long noncoding Xist RNA drives iXCI, silencing genes and recruiting Xist-dependent chromatin repressors. Some domains on the inactive X chromosome include repressive modifications specific to constitutive heterochromatin, which show no direct link to Xist RNA. We explored the relationship between Xist RNA and chromatin silencing during iXCI in vole Microtus levis. We performed locus-specific activation of Xist transcription on the only active X chromosome using the dCas9-SAM system in XO vole trophoblast stem cells (TSCs), which allow modeling iXCI events to some extent. The artificially activated endogenous vole Xist transcript is truncated and restricted ~ 6.6 kb of the exon 1. Ectopic Xist RNA accumulates on the X chromosome and recruits Xist-dependent modifications during TSC differentiation, yet is incapable by itself repressing X-linked genes. Transcriptional silencing occurs upon ectopic Xist upregulation only when repressive marks spread from the massive telomeric constitutive heterochromatin to the X chromosome region containing genes. We hypothesize that the Xist RNA-induced propagation of repressive marks from the constitutive heterochromatin could be a mechanism involved in X chromosome inactivation.
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Abbreviations
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- FISH:
-
Fluorescence in situ hybridization
- H3K27me3:
-
Histone H3 trimethylated at lysine K27
- H3K4me2:
-
Histone H3 dimethylated at lysine K4
- H3K9me3:
-
Histone H3 trimethylated at lysine K9
- H4K20me3:
-
Histone H4 trimethylated at lysine K20
- PCR:
-
Polymerase chain reaction
- SAM system:
-
Synergistic Activation Mediators system
- TSC:
-
Trophoblast stem cells
- XCI:
-
X chromosome inactivation
- rXCI:
-
Random X chromosome inactivation
- iXCI:
-
Imprinted X chromosome inactivation
- Xi:
-
Inactive X chromosome
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Acknowledgements
The study was carried out using the resources of the Common Facilities Center of Microscopic Analysis of Biological Objects, ICG SB RAS (https://ckp.icgen.ru/ckpmabo/). We thank S.I. Bayborodin for technical assistance.
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This work was supported by the State project of the Institute of Cytology and Genetics FWNR-2022–0015.
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AIS, SMZ, and ISZ conceived and designed the study. AIS, NAR, and ISZ conducted the experiments, analyzed data, and prepared figures. AIS and NAR wrote the manuscript. SMZ contributed to methods and models. All the authors read and approved the manuscript.
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Supplementary Fig. 1
Immunofluorescent staining of metaphase spreads and nuclei with antibodies to histone modifications in R1 TSC culture (53, X) of vole M. levis (a-d). H3K4me2 is an active chromatin modification. H3K9me3, H4K20me3, a histone methyltransferase SETDB1, HP1-gamma, and KAP1protein are constitutive heterochromatin marks. Chromosomes and nuclei are counterstained with DAPI (blue). X chromosomes are indicated by arrows. MS4 is a DNA repeat specific for the X chromosome telomeric heterochromatin block in vole M. levis. Scale bar is 10 μm.(PNG 2828 kb)
Supplementary Fig. 2
Representative image of metaphase spread from clone #19 showing a diploid autosomal set and two X-chromosomes with extended Н3K9me3 distribution (green) from their constitutive heterochromatin to gene-bearing part. X chromosomes are indicated by arrows. H3K4me2 mark (red) indicates active chromatin. MS4 (white) is a DNA repeat specific for the X chromosome telomeric heterochromatin block in vole M. levis. The table shows the number and portion of metaphases with one (1X), two (2X), and three (3X) X chromosomes per a diploid autosomal set s in TSC clones #19 and #32.(PNG 995 kb)
Supplementary Fig. 3
X-linked transcripts of the Hprt1, Pgk1, Rab9, and Sb1.8 genes and Xist RNA detected by RNA FISH in vole R1 (53, X) and R2 (54, XX) TSC lines. Nuclei are counterstained with DAPI (blue). Gene transcripts are detected in red; signals from Xist RNA are in green.(PNG 2943 kb)
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Shevchenko, A.I., Rifel, N.A., Zakian, S.M. et al. Constitutive heterochromatin propagation contributes to the X chromosome inactivation. Chromosome Res 30, 289–307 (2022). https://doi.org/10.1007/s10577-022-09706-4
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DOI: https://doi.org/10.1007/s10577-022-09706-4