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

2019 ◽  
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.

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

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.

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

2017 ◽  
Author(s):  
G. Bonora ◽  
X. Deng ◽  
H. Fang ◽  
V. Ramani ◽  
R. Qui ◽  
...  
Keyword(s):  
Long Range ◽  
X Chromosome ◽  
3D Structure ◽  
Chromatin Accessibility ◽  
Ctcf Binding ◽  
Partial Restoration ◽  
Multiple Loci ◽  
Inactive X Chromosome ◽  
Repeat Locus ◽  
Contact Distribution

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.

scholarly journals Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

1989 ◽  
Vol 9 (4) ◽  
pp. 1635-1641 ◽  
Author(s):  
S G Grant ◽  
R G Worton
Keyword(s):  
Cell Lines ◽  
X Chromosome ◽  
Gene Activation ◽  
Chinese Hamster ◽  
Fibroblast Cell ◽  
Dna Demethylation ◽  
Hprt Gene ◽  
Primary Fibroblast ◽  
Chinese Hamster Cells ◽  
Inactive X Chromosome

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.

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

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.

Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells

1989 ◽  
Vol 9 (4) ◽  
pp. 1635-1641
Author(s):  
S G Grant ◽  
R G Worton
Keyword(s):  
Cell Lines ◽  
X Chromosome ◽  
Gene Activation ◽  
Chinese Hamster ◽  
Fibroblast Cell ◽  
Dna Demethylation ◽  
Hprt Gene ◽  
Primary Fibroblast ◽  
Chinese Hamster Cells ◽  
Inactive X Chromosome

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.

scholarly journals Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture

2016 ◽  
Vol 113 (31) ◽  
pp. E4504-E4512 ◽  
Author(s):  
Emily M. Darrow ◽  
Miriam H. Huntley ◽  
Olga Dudchenko ◽  
Elena K. Stamenova ◽  
Neva C. Durand ◽  
...  
Keyword(s):  
Genome Editing ◽  
X Chromosome ◽  
Structural Features ◽  
Higher Order ◽  
Genome Architecture ◽  
3D Mapping ◽  
Barr Body ◽  
Inactive X Chromosome ◽  
Binding Factor

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the “Barr body.” Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called “superdomains,” such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called “superloops.” DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

scholarly journals Escape from X Chromosome Inactivation and the Female Predominance in Autoimmune Diseases

2021 ◽  
Vol 22 (3) ◽  
pp. 1114
Author(s):  
Ali Youness ◽  
Charles-Henry Miquel ◽  
Jean-Charles Guéry
Keyword(s):  
Autoimmune Diseases ◽  
X Chromosome ◽  
Gene Dosage ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Female Sex Hormones ◽  
Inactive X Chromosome ◽  
Immune Related Genes ◽  
Female Specific

Women represent 80% of people affected by autoimmune diseases. Although, many studies have demonstrated a role for sex hormone receptor signaling, particularly estrogens, in the direct regulation of innate and adaptive components of the immune system, recent data suggest that female sex hormones are not the only cause of the female predisposition to autoimmunity. Besides sex steroid hormones, growing evidence points towards the role of X-linked genetic factors. In female mammals, one of the two X chromosomes is randomly inactivated during embryonic development, resulting in a cellular mosaicism, where about one-half of the cells in a given tissue express either the maternal X chromosome or the paternal one. X chromosome inactivation (XCI) is however not complete and 15 to 23% of genes from the inactive X chromosome (Xi) escape XCI, thereby contributing to the emergence of a female-specific heterogeneous population of cells with bi-allelic expression of some X-linked genes. Although the direct contribution of this genetic mechanism in the female susceptibility to autoimmunity still remains to be established, the cellular mosaicism resulting from XCI escape is likely to create a unique functional plasticity within female immune cells. Here, we review recent findings identifying key immune related genes that escape XCI and the relationship between gene dosage imbalance and functional responsiveness in female cells.

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