A Chicken Transferrin Gene in Transgenic Mice Escapes X-Chromosome Inactivation

Ma, Goldman; Stokes, KR; Idzerda, RL; McKnight, G. Stanley; Hammer, RE; Rl, Brinster; Gartler, S. M.

doi:10.1126/science.2437652

Cited by 42 publications

(22 citation statements)

References 33 publications

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“…The involvement of a common repetitive element is consistent with the observation that XCI can sometimes spread considerable distances into X-translocated autosomal segments (72). A few endogenous X-linked genes escape XCI (70), and in translocations its spread into autosomal regions is usually limited, suggesting that some autosomal segments either are resistant to inactivation or lack the elements required to stabilize and/or propagate the inactivating signal (29). However, X-autosome translocation studies have been mechanistically inconclusive because they represent only situations where XCI spread is not detrimental to the cell and thus selected against.…”

supporting

confidence: 58%

“…The fact that some normal X-linked genes escape inactivation suggests that control mechanisms act at the level of individual gene loci or chromosomal domains (70). Goldman et al (29) and Riggs (60) postulated that looped chromatin domains may be key elements of the X inactivation process (29) and that inactivation may not spread continuously along the DNA; rather, the key elements modified by XCI could be in or near the base of loops that are continuously or periodically in close contact (60). Most autosomal or noneukaryotic transgenes randomly integrated into the X chromosome are subject to XCI.…”

mentioning

confidence: 99%

“…The only example of complete escape of a transgene from XCI involves a chicken gene. Goldman et al (29) examined mice carrying 11 copies of a 17-kb chicken transferrin gene inserted into the D/C region of the X chromosome and found expression on the Xi to be the same as that on the Xa. It was suggested elsewhere that escape from X inactivation may be due to the large size (187 kb) of the transgene and/or the absence of special sequences (29).…”

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A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

Chong

Kontaraki

Bonifer

et al. 2002

Molecular and Cellular Biology

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To investigate the molecular mechanism(s) involved in the propagation and maintenance of X chromosome inactivation (XCI), the 21.4-kb chicken lysozyme (cLys) chromatin domain was inserted into the Hprt locus on the mouse X chromosome. The inserted fragment includes flanking matrix attachment regions (MARs), an origin of bidirectional replication (OBR), and all the cis-regulatory elements required for correct tissue-specific expression of cLys. It also contains a recently identified and widely expressed second gene, cGas41. The cLys domain is known to function as an autonomous unit resistant to chromosomal position effects, as evidenced by numerous transgenic mouse lines showing copy-number-dependent and development-specific expression of cLys in the myeloid lineage. We asked the questions whether this functional chromatin domain was resistant to XCI and whether the X inactivation signal could spread across an extended region of avian DNA. A generally useful method was devised to generate pure populations of macrophages with the transgene either on the active (Xa) or the inactive (Xi) chromosome. We found that (i) cLys and cGas41 are expressed normally from the Xa; (ii) the cLys chromatin domain, even when bracketed by MARs, is not resistant to XCI; (iii) transcription factors are excluded from lysozyme enhancers on the Xi; and (iv) inactivation correlates with methylation of a CpG island that is both an OBR and a promoter of the cGas41 gene.Functional chromatin domains are extended regions of chromatin containing a gene or gene cluster with all cis elements necessary for their appropriate expression. These domains are flanked by domain boundary elements, which may act to segregate the domain and protect against chromosomal position effects (5, 67). Chromosomal position effects are thought to be due to the influence of neighboring repressive chromatins, such as heterochromatin, a type of chromatin that appears more condensed during interphase, is late replicating, and is transcriptionally silent (73). Some of the proteins that affect the spreading and persistence of chromatin-mediated transcriptional repression are known for Drosophila melanogaster and Saccharomyces cerevisiae; these include heterochromatin protein 1 (HP1), members of the polycomb group, histone deacetylases, and histone methyltransferases (19,36,52,54). Much less is known for mammals, although it has been established elsewhere that methylation of histone H3 is correlated with an inactive chromatin state (7, 38, 46), as are hypoacetylation (39) and DNA methylation (25, 64). A remaining general question is whether a vertebrate functional chromatin domain can be resistant to the spreading effect of nearby heterochromatin.A special type of heterochromatin is the facultative heterochromatin comprising most of the mammalian inactive X chromosome (Xi). X chromosome inactivation (XCI) is a mammalspecific mechanism for equalizing the expression of X-linked genes between XX females and XY males. Early in embryogenesis one of the two X chromosomes in femal...

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supporting

confidence: 58%

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confidence: 99%

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confidence: 99%

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A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

Chong

Kontaraki

Bonifer

et al. 2002

Molecular and Cellular Biology

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“…In humans at least, this escape appears to operate at the level of chromosomal domains possibly demarcated by boundary elements (19,20). The role for chromosomal domains in escape from X inactivation is supported by the demonstration that a 17-kb chicken transferrin gene randomly integrated as multiple copies on the X chromosome is expressed whether the transgene is on the Xi or the active X chromosome (Xa) (21). However, in two separate transgenic studies, neither the human ␤-globin locus control region, nor a functional DNA fragment containing matrix attachment regions, resisted silencing on the Xi (22,23), indicating that these boundary elements are not sufficient to form escape domains on the Xi.…”

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confidence: 89%

A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation

Ciavatta¹,

Kalantry

Magnuson

et al. 2006

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

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Some genes on the inactive X chromosome escape silencing. One possible escape mechanism is that heterochromatization during X inactivation can be blocked by boundary elements. DNA insulators are candidates for blocking because they shield genes from influences of their chromosomal environment. To test whether DNA insulators can act as boundaries on the X chromosome, we inserted into the mouse X-linked Hprt locus a GFP transgene flanked with zero, one, or two copies of a prototypic vertebrate insulator from the chicken ␤-globin locus, chicken hypersensitive site 4, which contains CCCTC binding factor binding sites. On the active X chromosome the insulators blocked repression of the transgene, which commences during early development and persists in adults, in a copy number-dependent manner. CpG methylation of the transgene correlated inversely with expression, but the insulators on the active X chromosome were not methylated. On the inactive X chromosome, insulators did not block random or imprinted X inactivation of the transgene, and both the insulator and transgene were almost completely methylated. Thus, the chicken hypersensitive site 4 DNA insulator is sufficient to protect an X-linked gene from repression during development but not from X inactivation. CCCTC binding factorE ukaryotic genomes contain interspersed domains of transcriptionally active euchromatin and inactive heterochromatin. Heterochromatin can encroach into transcriptionally active euchromatin and silence adjoining genes, as illustrated by position effect variegation in Drosophila (1, 2). Maintaining these chromosomal domains and preventing the spread of heterochromatin implies the existence of boundary elements that act as barriers (3). One class of boundary element candidates consists of DNA insulators because they can block the positive effects of an adjacent enhancer and protect a gene from negative position effects (4, 5).One of the best-characterized vertebrate DNA insulators is from the chicken ␤-globin locus. In chicken erythrocytes, DNaseI hypersensitive site 4 upstream of the ␤-globin locus marks the transition between a euchromatic region that contains the ␤-globin genes and upstream heterochromatin (6, 7). Chicken hypersensitive site 4 (cHS4), a 1.2-kb fragment that encompasses hypersensitive site 4, has both insulator properties: it can block an enhancer in an enhancer blocking assay, and it has barrier activity in a position effect assay (8). A binding site for the transcription factor CTCF (CCCTC binding factor) within cHS4 is necessary and sufficient for its enhancer blocking activity, but is not necessary for its barrier function (9, 10). Flanking copies of cHS4 without the CTCF binding site can still protect randomly integrated transgenes from position effects. In a position effect assay the cHS4 insulator itself and transgenes flanked with it are associated with acetylated histones, histone H3 methylated at lysine 4, and reduced CpG methylation relative to the methylation of uninsulated transgenes (11-13).A specialized for...

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“…Hemizygous female embryos displayed a mosaic pattern of transgene expression in about 50% of the somatic cells, most probably due to random inactivation of one X-chromosome per cell (Tagaki & Sasaki, 1975;Tan et al 1993). However, it has also been reported that a transgene inserted into the X_chromosome might completely escape X-chromosome inactivation (Goldman et al 1987). This could be due to integration in the X-Y pairing region, which normally escapes X-inactivation.…”

Section: Position Effects On Transgene Expressionmentioning

confidence: 99%