Difference in Chromatin Packaging between Active and Inactive X Chromosomes by Fractionation and Allele-Specific Detection

Endo, Yoshiro; Watanabe, Takuya; Kuwabara, Katsuhiro; Tsunashima, K.; Mishima, Yukio; Arakawa, Masaaki; Takagi, Nobuo; Kominami, Ryo

doi:10.1006/bbrc.1998.8201

Cited by 6 publications

(8 citation statements)

References 22 publications

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“…In interphase, the inactive X chromosome (Xi) is found as a condensed heterochromatic Barr body, usually positioned at the nuclear or nucleolar periphery (1)(2)(3)(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).…”

mentioning

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.

194

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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mentioning

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.

194

192

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“…Both clones contained two B6-derived Xi chromosomes and two MSM-derived Xa chromosomes (Endo et al 1998). …”

Section: Methodsmentioning

confidence: 99%

“…The H/E assay was carried out as previously described (Endo et al 1998). In brief, the suspension of washed nuclear pellet in cation-free 0.25 M sucrose was sonicated at a maximum power of 200 watts for 10 min into smaller chromatin particles.…”

Section: Methodsmentioning

confidence: 99%

“…Our previous analysis of gene expression on X Chr in the cell lines showed that the X Chrs derived from MSM were active, while the B6 X Chrs were inactive (Endo et al 1998). In those cases, the MSM allele of Pgk-1 was supposed to take an open chromatin structure which tended to be more fractionated into the E fraction.…”

Section: Differential Chromatin Packaging Of Xist Between XI and Xamentioning

confidence: 97%

“…The heterochromatin (H) fraction of compact chromatin structure was obtained from isolated nuclei by sonication and subsequent centrifugation at a low speed. Further centrifugation of the supernatant at a high speed yielded the euchromatic (E) fraction enriched with the chromatin of open structure (Endo et al 1998). DNA in the two fractions was subjected to PCR analysis with primers on X Chr that distinguished the T16 and MSM alleles.…”

Section: Differential Chromatin Packaging Of Xist Between XI and Xamentioning

confidence: 99%

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Compact chromatin packaging of inactive X Chromosome involves the actively transcribed Xist gene

et al. 1999

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The Xist gene responsible for X inactivation may take a unique chromatin structure because of exceptional expression from inactive X Chromosome, (Chr). We have examined differential chromatin packaging of the Xist gene region between active and inactive X Chr with a novel method consisting of the chromatin fractionation and allele-specific detection. Analysis of F1 heterozygous female mice from T(X;16)16H x MSM crosses and two cell clones derived from inter-subspecific F1 female mice demonstrated that the packaging level of the transcribed Xist region on inactive X Chr was as tight as that of the repressed Pgk-1 allele on the same chromosome. On the other hand, restriction endonuclease sensitivity assay of chromatin showed that the promoter region, but not transcribed regions, of the transcribed Xist allele retained accessibility to nucleases. These results may suggest a cis-element(s) in a regulatory region of the Xist gene to prevent the transcriptionally inhibitory effect of the chromatin packaging.

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Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

Togashi¹,

Obata²,

Aoyagi³

et al. 2004

Biochemical and Biophysical Research Communications

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Difference in Chromatin Packaging between Active and Inactive X Chromosomes by Fractionation and Allele-Specific Detection

Cited by 6 publications

References 22 publications

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

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

Compact chromatin packaging of inactive X Chromosome involves the actively transcribed Xist gene

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

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