Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure.

Eils, Roland; Dietzel, Steffen; Bertin, Etienne; Schröck, Evelin; Speicher, Michael R.; Ried, Thomas; Robert‐Nicoud, Michel; Cremer, Christoph; Cremer, Thomas

doi:10.1083/jcb.135.6.1427

Cited by 214 publications

(193 citation statements)

References 55 publications

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“…Following DAPI counterstaining with 0.1 mg/ml in PBS for 5 min, the preparations were mounted in Vectashield (Linaris, Wertheim). Optical sectioning was carried out with a Leica TCS 4D confocal microscope as described in Eils et al (1996). To control for bleed through of FITC fluorescence through Cy3-detection filters, we used the 488 nm laser line for FITC excitation in combination with the filters used for Cy3 detection.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

Association of p53 and MSH2 with recombinative repair complexes during S phase

et al. 2002

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Our previous recombination and biochemical analyses have led to the hypothesis that the tumor suppressor p53 monitors homologous recombination, a function which was previously attributed to the mismatch repair protein MSH2. Here, we show that a certain fraction of p53 is concentrated within discrete nuclear foci of cells synchronized in G1 phase, a pattern which becomes even more pronounced in S phase, especially after g-ray treatment. p53 foci show some colocalization with MSH2 within distinct foci during G1 phase, while dots formed by BRCA1 display an independent localization pattern. In S phase nuclei, p53 foci almost completely colocalize with MSH2 foci and associate with the recombination surveillance factor BRCA1 in irradiated S phase cells. These p53 and MSH2 foci also show significant overlaps with foci of the recombination enzymes Rad50 and Rad51, which for the first time unveiled recombination-related functions of p53 in replicating cells. During S phase, p53 and MSH2 are maximally active in binding to early recombination intermediates, and coexist within the same nuclear DNA-protein complexes. Our data suggest that p53 is linked similarly to homologous recombination as MSH2 and provide further evidence for the new concept of a dual role of p53 in the regulation of growth and repair.

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Section: Immunofluorescence Microscopymentioning

confidence: 99%

Association of p53 and MSH2 with recombinative repair complexes during S phase

et al. 2002

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“…The appearance of the Barr body in the nucleus of female cells has led many to speculate that there might be increased chromatin condensation on the Xi (31). However, this idea was questioned by a study that reported equivalent volumes, and hence equivalent levels of overall chromatin condensation, of Xa and Xi (32). That study used X chromosome paints prepared from plasmid libraries and may have lacked the sensitivity to detect decondensed chromatin located at the periphery of the Xa.…”

Section: Discussionmentioning

confidence: 99%

Chromosome territory reorganization in a human disease with altered DNA methylation

Matarazzo

Boyle

D’Esposito

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Chromosome territory (CT) organization and chromatin condensation have been linked to gene expression. Although individual genes can be transcribed from inside CTs, some regions that have constitutively high expression or are coordinately activated loop out from CTs and decondense. The relationship between epigenetic marks, such as DNA methylation, and higher-order chromatin structures is largely unexplored. DNMT3B mutations in immunodeficiency centromeric instability facial anomalies (ICF) syndrome result in loss of DNA methylation at particular sites, including CpG islands on the inactive X chromosome (Xi). This allows the specific effects of DNA methylation on CTs to be examined. Using fluorescence in situ hybridization, we reveal a differential organization of the human pseudoautosomal region (PAR)2 between the CTs of the X and Y in normal males and the active X (Xa) and the Xi in females. There is also a more condensed chromatin structure on Xi compared with Xa in this region. PAR2 genes are relocalized toward the outside of the Y and Xi CTs in ICF, and on the Xi, we show that this can extend to genes distant from the site of DNA hypomethylation itself. This reorganization is not simply a reflection of the transcriptional activation of the relocalized genes. This report of altered CT organization in a human genetic disease illustrates that DNA hypomethylation at restricted sites in the genome can lead to more extensive changes in nuclear organization away from the original site of epigenetic change. X inactivation ͉ Y chromosomeT o fully understand the orchestration of gene expression, it is necessary to understand how chromatin is spatially organized within the cell nucleus. In particular, the position of a gene with respect to its chromosome territory (CT) and the chromatin condensation of its genomic region have been linked to gene activation and repression (1). Although individual genes can be transcribed from inside of CTs (2), some regions that have constitutively high gene expression (3,4) or are subject to coordinate gene activation during development (5, 6) or differentiation (7) or in response to physiological stimuli (8) locate at the edge or outside of their CT. This level of nuclear reorganization is often accompanied by a visible level of chromatin decondensation (5, 6, 9). The precise function, if any, of repositioning toward the outside of CTs remains unclear (1), but it may allow for genes to access a nuclear environment enriched in the components of the transcription (10) and/or mRNAprocessing machinery (4,11,12) and so enhance the efficiency of transcription.The interplay between this level of higher-order chromatin organization and epigenetic mechanisms that act at primary levels of chromatin structure, such as DNA methylation, is a little-explored area. In plants, changes in the architecture of CTs can be induced by treatment with inhibitors of DNA methylation (13). However, the genome-wide demethylation that follows 5-azacytidine treatment or genetic deficiency in DNA methyltransferase...

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“…4, 6, 7, 11, cells were prepared for in situ hybridization according to our standard protocol (62)(63)(64). For all other experiments, an alternate technique with no air or ethanol dehydration was used (21,50). Cells were fixed before extraction in 4% paraformaldehyde͞PBS for 10 min at room temperature (RT), rinsed in PBS, incubated in 0.1 M HCl for 10 min at RT, extracted in 0.5% triton-X-100͞0.5% saponin in PBS for 10 min at RT and then equilibrated for 20 min in 20% glycerol͞PBS at RT.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were fixed before extraction in 4% paraformaldehyde͞PBS for 10 min at room temperature (RT), rinsed in PBS, incubated in 0.1 M HCl for 10 min at RT, extracted in 0.5% triton-X-100͞0.5% saponin in PBS for 10 min at RT and then equilibrated for 20 min in 20% glycerol͞PBS at RT. Cells were further permeabilized through a freeze-thaw cycle consisting of dipping the coverslip quickly into liquid nitrogen (21). The cells were allowed only to thaw and, if not used immediately, were stored in PBS at 4°C.…”

Section: Methodsmentioning

confidence: 99%

The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences

Clemson

Hall

Byron

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

193

192

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We investigated whether genes escape X chromosome inactivation by positioning outside of the territory defined by XIST RNA. Results reveal an unanticipated higher order organization of genes and noncoding sequences. All 15 X-linked genes, regardless of activity, position on the border of the XIST RNA territory, which resides outside of the DAPI-dense Barr body. Although more strictly delineated on the inactive X chromosome (Xi), all genes localized predominantly to the outer rim of the Xi and active X chromosome. This outer rim is decorated only by X chromosome DNA paints and is excluded from both the XIST RNA and dense DAPI staining. The only DNA found well within the Barr body and XIST RNA territory was centromeric and Cot-1 DNA; hence, the core of the X chromosome essentially excludes genes and is composed primarily of noncoding repeat-rich DNA. Moreover, we show that this core of repetitive sequences is expressed throughout the nucleus yet is silenced throughout Xi, providing direct evidence for chromosomewide regulation of ''junk'' DNA transcription. Collective results suggest that the Barr body, long presumed to be the physical manifestation of silenced genes, is in fact composed of a core of silenced noncoding DNA. Instead of acting at a local gene level, XIST RNA appears to interact with and silence core architectural elements to effectively condense and shut down the Xi.Barr body ͉ chromosome territory ͉ nuclear organization ͉ XIST ͉ noncoding RNA X inactivation in mammalian females is a prominent example of the formation of facultative heterochromatin during early development, which prevents the deleterious effects of overexpression of X-linked genes. In interphase, the inactive X chromosome (Xi) is found as a condensed heterochromatic Barr body, usually positioned at the nuclear or nucleolar periphery (1-4). Because many genes have been identified that escape X inactivation, packaging differences of sequences at some level within the Xi is presumed (5, 6). Although it is generally assumed that the Barr body is condensed DNA comprising genes normally expressed on the active X chromosome (Xa), the specific makeup of the Barr body has not been investigated.X inactivation is a multistep process initiated by XIST (7-9). Just after XIST RNA sweeps across the chromosome, a defined pattern of chromatin changes including histone modifications, recruitment of macroH2A, and methylation occurs (for review see refs. 10 and 11). We have shown that XIST RNA remains in the nucleus, functionally associated with the inactive chromatin, forming an interphase territory coincident with Xi (12, 13). We incorporated our results into two models for the higher-level organization of the inactive X chromosome (12). In each alternate view, XIST RNA would induce differences in the packaging of the chromatin to facilitate inactivation. In the first model, all genes, regardless of whether they escape from or are subject to X inactivation, would be interspersed throughout the chromosome territory and would not be cytologically disti...

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Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure.

Cited by 214 publications

References 55 publications

Association of p53 and MSH2 with recombinative repair complexes during S phase

Association of p53 and MSH2 with recombinative repair complexes during S phase

Chromosome territory reorganization in a human disease with altered DNA methylation

The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences

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