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. 2011 Apr 25;6(4):e19040.
doi: 10.1371/journal.pone.0019040.

Evolution from XIST-independent to XIST-controlled X-chromosome inactivation: epigenetic modifications in distantly related mammals

Julie Chaumeil  1 , Paul D Waters, Edda Koina, Clément Gilbert, Terence J Robinson, Jennifer A Marshall Graves
Affiliations

Evolution from XIST-independent to XIST-controlled X-chromosome inactivation: epigenetic modifications in distantly related mammals

Julie Chaumeil et al. PLoS One. .

Abstract

X chromosome inactivation (XCI) is the transcriptional silencing of one X in female mammals, balancing expression of X genes between females (XX) and males (XY). In placental mammals non-coding XIST RNA triggers silencing of one X (Xi) and recruits a characteristic suite of epigenetic modifications, including the histone mark H3K27me3. In marsupials, where XIST is missing, H3K27me3 association seems to have different degrees of stability, depending on cell-types and species. However, the complete suite of histone marks associated with the Xi and their stability throughout cell cycle remain a mystery, as does the evolution of an ancient mammal XCI system. Our extensive immunofluorescence analysis (using antibodies against specific histone modifications) in nuclei of mammals distantly related to human and mouse, revealed a general absence from the mammalian Xi territory of transcription machinery and histone modifications associated with active chromatin. Specific repressive modifications associated with XCI in human and mouse were also observed in elephant (a distantly related placental mammal), as was accumulation of XIST RNA. However, in two marsupial species the Xi either lacked these modifications (H4K20me1), or they were restricted to specific windows of the cell cycle (H3K27me3, H3K9me2). Surprisingly, the marsupial Xi was stably enriched for modifications associated with constitutive heterochromatin in all eukaryotes (H4K20me3, H3K9me3). We propose that marsupial XCI is comparable to a system that evolved in the common therian (marsupial and placental) ancestor. Silent chromatin of the early inactive X was exapted from neighbouring constitutive heterochromatin and, in early placental evolution, was augmented by the rise of XIST and the stable recruitment of specific histone modifications now classically associated with XCI.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Status of H3K27me3 enrichment and XIST-RNA accumulation on the inactive-X in distantly related mammals.
A. Examples of immunofluorescence for H3K27me3 (in green) on elephant cells (left panel), tammar wallaby cells (centre) and opossum cells (right panel). The top row shows a representative nucleus from female cell, the bottom row from male cell. Arrow heads point the inactive X chromosome enriched in H3K27me3. n = 100. B. Example of RNA FISH showing accumulation of XIST transcripts (in grey) on the DAPI-dense inactive X chromosome of female elephant cells. C. Immunofluorescence for H3K27me3 (in green) combined with RNA FISH for the UBE1 primary transcripts (in red) in female cells from tammar wallaby. Arrow head points the inactive X chromosome containing UBE1 transcripts and enriched in H3K27me3. Asterisk shows UBE1 transcripts from the active X chromosome. n = 30. D. Dual immunofluorescence for H3K27me3 (in green) and H3S10ph (in red) on female tammar wallaby cells showing that enrichment in H3K27me3 (arrow heads) on the Xi is restricted to the G1/S phase and the early G2 phase of the cell cycle. n = 90. E. Immunofluorescence for H3K27me3 (in green) combined with a cell proliferation assay to label replicating DNA (in red) showing H3K27me3 enrichment on the Xi in late S phase, at the time of Xi replication (enrichment of H3K27me3 (green) on the replicating Xi (red)). n = 200. DAPI is shown in grey. Scale bar = 1 µm.
Figure 2
Figure 2. Depletion of active marks and RNA polymerase II on the inactive-X of therian mammals.
Dual immunofluorescence with H3K27me3 (in green) combined with H3K4me2, H3K9ac, H3K27me1, H3K14ac, H3R17me2, H4Kac or with RNA polymerase II (in red) in elephant (top), tammar wallaby (bottom left) and opossum (bottom right) female cells. n = 100–200. DAPI is shown in grey. Scale bar = 1 µm.
Figure 3
Figure 3. Patterns of repressive marks on the inactive-X of distantly related mammals.
A. Dual immunofluorescence with H3K27me3 (in green) combined with H3K9me2, H3K9me3, H3K27me1, H4K20me1 or H4K20me3 (in red) in mouse (top left), elephant (top right), tammar wallaby (bottom left) and opossum (bottom right) female cells. n = 100. B. Line scans of H3K27m3, H4K20me3 (left) and H3K9me3 (right) intensities in the Xi territory. C. Sequential immunofluorescence for H4K20me3 (in green; left) or H3K9me3 (in green; right) followed with a DNA FISH for 3 X-linked genes UBE1, ATRX and MECP2 (in red) in tammar wallaby female cells. The arrow head indicates the inactive X chromosome enriched in H3K27me3, asterisks indicates the genes from the active X chromosome. n = 50. DAPI is shown in grey. Scale bar = 1 µm.
Figure 4
Figure 4. A model for the evolution of mammalian X-chromosome inactivation.
Arrows above the phylogeny show epigenetic features underlying X chromosome inactivation (solid arrows indicate stable components of XCI within the clades, whereas dashed lines indicate unstable repressive modification). We propose that in the ancestral therian mammal neighbouring centromeric constitutive heterochromatin histone modifications were exapted to reduce transcription from the paternal X chromosome in females. H3K27me3 and H3K9me2 were also recruited to the Xi in a cell-cycle and/or cell-lineage dependent manner. After the divergence of placental from marsupial mammals, the XIST gene evolved and H3K27me3 and H3K9me2 (along with H4K20me1) were stably recruited into the XCI machinery. In placental mammals the original silencing machinery was restricted, and organised in mutually exclusive domains with the Xi-specific facultative heterochromatin. In marsupials, much of the original silencing machinery that was recruited from constitutive heterochromatin remains in place.
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