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

2018 ◽  
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.

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 XIST RNA: a window into the broader role of RNA in nuclear chromosome architecture

2017 ◽  
Vol 372 (1733) ◽  
pp. 20160360 ◽  
Author(s):  
K. M. Creamer ◽  
J. B. Lawrence
Keyword(s):  
X Chromosome ◽  
Chromatin Condensation ◽  
Higher Order ◽  
Chromosome Architecture ◽  
Genome Wide ◽  
Xist Rna ◽  
Architectural Proteins ◽  
Mary Lyon ◽  
Attachment Factor

XIST RNA triggers the transformation of an active X chromosome into a condensed, inactive Barr body and therefore provides a unique window into transitions of higher-order chromosome architecture. Despite recent progress, how XIST RNA localizes and interacts with the X chromosome remains poorly understood. Genetic engineering of XIST into a trisomic autosome demonstrates remarkable capacity of XIST RNA to localize and comprehensively silence that autosome. Thus, XIST does not require X chromosome-specific sequences but operates on mechanisms available genome-wide. Prior results suggested XIST localization is controlled by attachment to the insoluble nuclear scaffold. Our recent work affirms that scaffold attachment factor A (SAF-A) is involved in anchoring XIST , but argues against the view that SAF-A provides a unimolecular bridge between RNA and the chromosome. Rather, we suggest that a complex meshwork of architectural proteins interact with XIST RNA. Parallel work studying the territory of actively transcribed chromosomes suggests that repeat-rich RNA ‘coats’ euchromatin and may impact chromosome architecture in a manner opposite of XIST . A model is discussed whereby RNA may not just recruit histone modifications, but more directly impact higher-order chromatin condensation via interaction with architectural proteins of the nucleus. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

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.

Four-dimensional chromosome reconstruction elucidates the spatiotemporal reorganization of the mammalian X chromosome

2021 ◽  
Vol 118 (42) ◽  
pp. e2107092118
Author(s):  
Anna Lappala ◽  
Chen-Yu Wang ◽  
Andrea Kriz ◽  
Hunter Michalk ◽  
Kevin Tan ◽  
...  
Keyword(s):  
X Chromosome ◽  
Large Scale ◽  
Three Dimensions ◽  
Rna Molecules ◽  
Chromosome Architecture ◽  
Inactive X Chromosome ◽  
Mixing Dynamics ◽  
Chromosome Reconstruction ◽  
Slow Mixing ◽  
3D Information

Chromosomes are segmented into domains and compartments, but how these structures are spatially related in three dimensions (3D) is unclear. Here, we developed tools that directly extract 3D information from Hi-C experiments and integrate the data across time. With our “4DHiC” method, we use X chromosome inactivation (XCI) as a model to examine the time evolution of 3D chromosome architecture during large-scale changes in gene expression. Our modeling resulted in several insights. Both A/B and S1/S2 compartments divide the X chromosome into hemisphere-like structures suggestive of a spatial phase-separation. During the XCI, the X chromosome transits through A/B, S1/S2, and megadomain structures by undergoing only partial mixing to assume new structures. Interestingly, when an active X chromosome (Xa) is reorganized into an inactive X chromosome (Xi), original underlying compartment structures are not fully eliminated within the Xi superstructure. Our study affirms slow mixing dynamics in the inner chromosome core and faster dynamics near the surface where escapees reside. Once established, the Xa and Xi resemble glassy polymers where mixing no longer occurs. Finally, Xist RNA molecules initially reside within the A compartment but transition to the interface between the A and B hemispheres and then spread between hemispheres via both surface and core to establish the Xi.

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

2018 ◽  
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.

scholarly journals Megadomains and superloops form dynamically but are dispensable for X-chromosome inactivation and gene escape

2018 ◽  
Author(s):  
John E. Froberg ◽  
Stefan F. Pinter ◽  
Andrea J. Kriz ◽  
Teddy Jégu ◽  
Jeannie T. Lee
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Nuclear Localization ◽  
Functional Significance ◽  
Xist Expression ◽  
H3k27 Methylation ◽  
3D Genome ◽  
Inactive X Chromosome ◽  
Topologically Associated Domains ◽  
Escape From X Inactivation

ABSTRACTThe mammalian inactive X-chromosome (Xi) is structurally distinct from all other chromosomes and serves as a model for how the 3D genome is organized. The Xi shows weakened topologically associated domains and is instead organized into megadomains and superloops directed by the noncoding loci, Dxz4 and Firre. Their functional significance is presently unclear, though one study suggests that they permit Xi genes to escape silencing. Here, we find that megadomains do not precede Xist expression or Xi gene silencing. Deleting Dxz4 disrupts megadomain formation, whereas deleting Firre weakens intra-megadomain interactions. Surprisingly, however, deleting Dxz4 and Firre has no impact on Xi silencing and gene escape. Nor does it affect Xi nuclear localization, stability, or H3K27 methylation. Additionally, ectopic integration of Dxz4 and Xist is not sufficient to form megadomains on autosomes, further uncoupling megadomain formation from chromosomal silencing. We conclude that Dxz4 and megadomains are dispensable for Xi silencing and escape from X-inactivation.

X-Chromosome Inactivation: A Crossroads Between Chromosome Architecture and Gene Regulation

2018 ◽  
Vol 52 (1) ◽  
pp. 535-566 ◽  
Author(s):  
Rafael Galupa ◽  
Edith Heard
Keyword(s):  
X Chromosome ◽  
Current Knowledge ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Female Development ◽  
Barr Body ◽  
Chromosome Conformation ◽  
X Chromosomes ◽  
Chromosome Architecture ◽  
Inactive X Chromosome

In somatic nuclei of female therian mammals, the two X chromosomes display very different chromatin states: One X is typically euchromatic and transcriptionally active, and the other is mostly silent and forms a cytologically detectable heterochromatic structure termed the Barr body. These differences, which arise during female development as a result of X-chromosome inactivation (XCI), have been the focus of research for many decades. Initial approaches to define the structure of the inactive X chromosome (Xi) and its relationship to gene expression mainly involved microscopy-based approaches. More recently, with the advent of genomic techniques such as chromosome conformation capture, molecular details of the structure and expression of the Xi have been revealed. Here, we review our current knowledge of the 3D organization of the mammalian X-chromosome chromatin and discuss its relationship with gene activity in light of the initiation, spreading, and maintenance of XCI, as well as escape from gene silencing.

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.

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