Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

AbstractThe mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts separated by a boundary or hinge region. Using in situ DNase Hi-C in mouse cells with deletions or inversions within the hinge we show that the conserved repeat locus Dxz4 alone is sufficient to maintain the bipartite structure and that Dxz4 orientation controls the distribution of long-range contacts on the Xi. Frequent long-range contacts between Dxz4 and the telomeric superdomain are either lost after its deletion or shifted to the centromeric superdomain after its inversion. This massive reversal in contact distribution is consistent with the reversal of CTCF motif orientation at Dxz4. De-condensation of the Xi after Dxz4 deletion is associated with partial restoration of TADs normally attenuated on the Xi. There is also an increase in chromatin accessibility and CTCF binding on the Xi after Dxz4 deletion or inversion, but few changes in gene expression, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of a bank of CTCF motifs at Dxz4, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

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Trans- and cis-acting effects of Firre on epigenetic features of the inactive X chromosome

Nature Communications ◽

10.1038/s41467-020-19879-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

He Fang ◽

Giancarlo Bonora ◽

Jordan P. Lewandowski ◽

Jitendra Thakur ◽

Galina N. Filippova ◽

...

Keyword(s):

X Chromosome ◽

3D Structure ◽

Ectopic Expression ◽

Ctcf Binding ◽

Cell Type ◽

Cis Acting ◽

Gene Dysregulation ◽

Inactive X Chromosome ◽

Cell Type Specific ◽

Polycomb Repressive Complexes

AbstractFirre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.

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Trans- and cis-acting effects of the lncRNA Firre on epigenetic and structural features of the inactive X chromosome

10.1101/687236 ◽

2019 ◽

Cited By ~ 7

Author(s):

He Fang ◽

Giancarlo Bonora ◽

Jordan P. Lewandowski ◽

Jitendra Thakur ◽

Galina N. Filippova ◽

...

Keyword(s):

X Chromosome ◽

Ectopic Expression ◽

Fibroblast Cell ◽

Structural Features ◽

Ctcf Binding ◽

Functional Link ◽

Cis Acting ◽

Inactive X Chromosome ◽

Contact Distribution ◽

Allelic Deletion

AbstractFirre encodes a lncRNA involved in nuclear organization in mammals. Here we find that Firre RNA is transcribed from the active X chromosome (Xa) and exerts trans-acting effects on the inactive X chromosome (Xi). Allelic deletion of Firre on the Xa in a mouse hybrid fibroblast cell line results in a dramatic loss of the histone modification H3K27me3 and of components of the PRC2 complex on the Xi as well as the disruption of the perinucleolar location of the Xi. These features are measurably rescued by ectopic expression of a mouse or human Firre/FIRRE cDNA transgene, strongly supporting a conserved trans-acting role of the Firre transcript in maintaining the Xi heterochromatin environment. Surprisingly, CTCF occupancy is decreased on the Xi upon loss of Firre RNA, but is partially recovered by ectopic transgene expression, suggesting a functional link between Firre RNA and CTCF in maintenance of epigenetic features and/or location of the Xi. Loss of Firre RNA results in dysregulation of genes implicated in cell division and development, but not in reactivation of genes on the Xi, which retains its bipartite structure despite some changes in chromatin contact distribution. Allelic deletion or inversion of Firre on the Xi causes localized redistribution of chromatin contacts, apparently dependent on the orientation of CTCF binding sites clustered at the locus. Thus, the Firre locus and its RNA have roles in the maintenance of epigenetic features and structure of the Xi.

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Faculty Opinions recommendation of Smchd1 regulates long-range chromatin interactions on the inactive X chromosome and at Hox clusters.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733846835.793570002 ◽

2020 ◽

Author(s):

Anton Wutz

Keyword(s):

Long Range ◽

X Chromosome ◽

Chromatin Interactions ◽

Inactive X Chromosome ◽

Hox Clusters

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Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

Nature Communications ◽

10.1038/s41467-018-03694-y ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 27

Author(s):

G. Bonora ◽

X. Deng ◽

H. Fang ◽

V. Ramani ◽

R. Qiu ◽

...

Keyword(s):

X Chromosome ◽

3D Structure ◽

Inactive X Chromosome

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Position effect of translocations involving the inactive X chromosome: physical linkage to XIC/XIST does not lead to long-range de novo inactivation in human differentiated cells

Cytogenetic and Genome Research ◽

10.1159/000015065 ◽

1998 ◽

Vol 82 (1-2) ◽

pp. 58-66 ◽

Cited By ~ 5

Author(s):

J. Surrallés ◽

A.T. Natarajan

Keyword(s):

Long Range ◽

X Chromosome ◽

De Novo ◽

Position Effect ◽

Differentiated Cells ◽

Inactive X Chromosome ◽

Physical Linkage

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The non-canonical SMC protein SmcHD1 antagonises TAD formation on the inactive X chromosome

10.1101/342147 ◽

2018 ◽

Cited By ~ 2

Author(s):

Michal R Gdula ◽

Tatyana B Nesterova ◽

Greta Pintacuda ◽

Jonathan Godwin ◽

Ye Zhan ◽

...

Keyword(s):

X Chromosome ◽

Gene Activation ◽

Higher Order ◽

Partial Restoration ◽

Chromosome Architecture ◽

Inactive X Chromosome ◽

Key Factor ◽

Topologically Associated Domains ◽

Unique Chromosome ◽

Cpg Hypermethylation

AbstractThe inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), and formation of two mega-domains. In this study we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 null cells revealed the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred in an acute SmcHD1 knockout model, but in this case, independent of Xi gene de-repression. We conclude that SmcHD1 is a key factor in antagonising TAD formation on Xi.

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Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells

10.1101/2021.02.25.432827 ◽

2021 ◽

Author(s):

Hegias Mira-Bontenbal ◽

Beatrice Tan ◽

Cristina Gontan ◽

Sander Goossens ◽

R.G. Boers ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Rett Syndrome ◽

X Chromosome ◽

Neurodevelopmental Disorder ◽

Ctcf Binding ◽

Neural Cells ◽

Reporter System ◽

Inactive X Chromosome ◽

Phenotypic Reversal

AbstractRett Syndrome is a neurodevelopmental disorder in girls that is caused by heterozygous inactivation of the chromatin remodeler gene MECP2. Rett Syndrome may therefore be treated by reactivation of the wild type copy of MECP2 from the inactive X chromosome. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model system for monitoring Mecp2 reactivation that is more sensitive and versatile than any bioluminescent and fluorescent system currently available. The model consists of neural stem cells derived from female mice with a dual reporter system where MECP2 is fused to NanoLuciferase and TdTomato on the inactive X chromosome. We show by bioluminescence and fluorescence that Mecp2 is synergistically reactivated by 5-Aza treatment and Xist knockdown. As expected, other genes on the inactive X chromosome are also reactivated, the majority of which overlaps with genes reactivated early during reprogramming of mouse embryonic fibroblasts to iPSCs. Genetic and epigenetic features such as CpG density, SINE elements, distance to escapees and CTCF binding are consistent indicators of reactivation, whereas different higher order chromatin areas are either particularly prone or resistant to reactivation. Our MeCP2 reactivation monitoring system thereby suggests that genetic and epigenetic features on the inactive X chromosome affect reactivation of its genes, irrespective of cell type or procedure of reactivation.

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Long-range chromatin interactions on the inactive X and at Hox clusters are regulated by the non-canonical SMC protein Smchd1

10.1101/342212 ◽

2018 ◽

Cited By ~ 2

Author(s):

Natasha Jansz ◽

Andrew Keniry ◽

Marie Trussart ◽

Heidi Bildsoe ◽

Tamara Beck ◽

...

Keyword(s):

Gene Regulation ◽

Long Range ◽

X Chromosome ◽

Chromatin Structure ◽

Short Range ◽

Higher Order ◽

Chromatin Interactions ◽

Inactive X Chromosome ◽

Hox Clusters

AbstractThe regulation of higher order chromatin structure is complex and dynamic; however we do not yet understand the full suite of mechanisms governing architecture. Here we reveal the non-canonical SMC protein Smchd1 as a novel regulator of long-range chromatin interactions, and add it to the canon of epigenetic proteins required for Hox gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes dependent on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we find increased short-range interactions and ectopic enhancer activation. By contrast, the inactive X chromosome is transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. There we observe spreading of H3K27me3 domains into regions not normally decorated by this mark. Together these data suggest Smchd1 has the capacity to insulate the chromatin, thereby limiting access to other chromatin modifying proteins.

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