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• W1997997534 abstract "Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate. Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate. X-chromosome inactivation (XCI) is the mechanism by which gene dosage equivalence is achieved between female (XX) and male (XY) mammals.1Lyon MF Gene action in the X-chromosome of the mouse (Mus musculus L).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2457) Google Scholar XCI occurs in individual cells early in embryonic development and is subsequently stably maintained in all daughter cells. Therefore, each tissue of an adult female is composed of cells that express either the maternal or the paternal X chromosome. Because which X chromosome will be silenced is random, in the general female population, XCI ratios have a normal distribution, with an average of ∼50:50, so that an equal number of cells express the maternal and paternal chromosomes.2Amos-Landgraf JM Cottle A Plenge RM Friez M Schwartz CE Longshore J Willard HF X chromosome-inactivation patterns of 1,005 phenotypically unaffected females.Am J Hum Genet. 2006; 79: 493-499Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Therefore, by chance, a small percentage of females (∼9%) have a skewed ratio (>80:20), where there is a bias toward cells expressing one or the other X chromosome, but extreme skewing (>95:5) is very rare (in <1% of females).2Amos-Landgraf JM Cottle A Plenge RM Friez M Schwartz CE Longshore J Willard HF X chromosome-inactivation patterns of 1,005 phenotypically unaffected females.Am J Hum Genet. 2006; 79: 493-499Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Whereas skewed XCI is rare in the normal population, it is relatively common in carriers of X-linked mutations and in cases of X-autosome translocations. In principle, heterozygosity for an X-linked mutation could cause skewed XCI, either by biasing the primary pattern of XCI (as is the case for the Xce locus in mice3Cattanach BM Isaacson JH Controlling elements in the mouse X chromosome.Genetics. 1967; 57: 331-346PubMed Google Scholar, 4Chadwick LH Pertz LM Broman KW Bartolomei MS Willard HF Genetic control of X chromosome inactivation in mice: definition of the Xce candidate interval.Genetics. 2006; 173: 2103-2110Crossref PubMed Scopus (41) Google Scholar, 5Chadwick LH Willard HF Genetic and parent-of-origin influences on X chromosome choice in Xce heterozygous mice.Mamm Genome. 2005; 16: 691-699Crossref PubMed Scopus (24) Google Scholar, 6Courtier B Heard E Avner P Xce haplotypes show modified methylation in a region of the active X chromosome lying 3′ to Xist.Proc Natl Acad Sci USA. 1995; 92: 3531-3535Crossref PubMed Scopus (97) Google Scholar) or through secondary selection in favor of cells expressing a particular X chromosome.7Belmont JW Genetic control of X inactivation and processes leading to X-inactivation skewing.Am J Hum Genet. 1996; 58: 1101-1108PubMed Google Scholar, 8Brown CJ Robinson WP The causes and consequences of random and non-random X chromosome inactivation in humans.Clin Genet. 2000; 58: 353-363Crossref PubMed Scopus (100) Google Scholar, 9Puck JM Willard HF X inactivation in females with X-linked disease.N Engl J Med. 1998; 338: 325-328Crossref PubMed Scopus (153) Google Scholar When skewed XCI is limited to specific tissues,10Hendriks RW Kraakman ME Schuurman RK X chromosome inactivation patterns in haematopoietic cells of female carriers of X-linked severe combined immunodeficiency determined by methylation analysis at the hypervariable DXS255 locus.Clin Genet. 1992; 42: 114-121Crossref PubMed Scopus (6) Google Scholar, 11Allen RC Nachtman RG Rosenblatt HM Belmont JW Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.Am J Hum Genet. 1994; 54: 25-35PubMed Google Scholar it can be assumed that this is because of cell selection, but, when XCI is skewed to a similar extent in many or all tissues, although this could result from cell selection due to a defect in a basic cellular function, this is not necessarily the case. Recently, it has been shown that several X-linked mental retardation (XLMR) disorders have a strong association with skewed XCI in carrier females.12Plenge RM Stevenson RA Lubs HA Schwartz CE Willard HF Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders.Am J Hum Genet. 2002; 71: 168-173Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar However, the mechanism underlying skewing in these cases is unknown. A clear example of this association is seen in the α-thalassemia XLMR syndrome (ATR-X syndrome [MIM 301040]). Affected males have profound psychomotor retardation and multiple congenital abnormalities, including facial dysmorphism and urogenital and skeletal defects.13Gibbons RJ Brueton L Buckle VJ Burn J Clayton-Smith J Davison BC Gardner RJ Homfray T Kearney L Kingston HM et al.Clinical and hematologic aspects of the X-linked alpha-thalassemia/mental retardation syndrome (ATR-X).Am J Med Genet. 1995; 55: 288-299Crossref PubMed Scopus (97) Google Scholar, 14Gibbons RJ Higgs DR Molecular-clinical spectrum of the ATR-X syndrome.Am J Med Genet. 2000; 97: 204-212Crossref PubMed Scopus (164) Google Scholar, 15Gibbons RJ Wada T ATRX mutations and X-linked alpha thalassaemia mental retardation syndrome.in: Epstein CJ Erickson RP Wynshaw-Boris A Inborn errors of development. Oxford University Press, Oxford2004Google Scholar ATR-X syndrome was originally identified via the unusual association of mental retardation with mild α-thalassemia, characterized by the formation of HbH inclusions (β-globin tetramers) in red blood cells.16Weatherall DJ Higgs DR Bunch C Old JM Hunt DM Pressley L Clegg JB Bethlenfalvay NC Sjolin S Koler RD et al.Hemoglobin H disease and mental retardation: a new syndrome or a remarkable coincidence?.N Engl J Med. 1981; 305: 607-612Crossref PubMed Scopus (96) Google Scholar ATRX is a ubiquitously expressed, chromatin-associated protein of the Snf2 family and is thought to affect the expression of α-globin and a wide range of other genes via epigenetic mechanisms.17Xue Y Gibbons R Yan Z Yang D McDowell TL Sechi S Qin J Zhou S Higgs D Wang W The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.Proc Natl Acad Sci USA. 2003; 100: 10635-10640Crossref PubMed Scopus (272) Google Scholar, 18Gibbons RJ McDowell TL Raman S O’Rourke DM Garrick D Ayyub H Higgs DR Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation.Nat Genet. 2000; 24: 368-371Crossref PubMed Scopus (408) Google Scholar, 19Picketts DJ Higgs DR Bachoo S Blake DJ Quarrell OW Gibbons RJ ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome.Hum Mol Genet. 1996; 5: 1899-1907Crossref PubMed Scopus (196) Google Scholar, 20Gecz J Pollard H Consalez G Villard L Stayton C Millasseau P Khrestchatisky M Fontes M Cloning and expression of the murine homologue of a putative human X-linked nuclear protein gene closely linked to PGK1 in Xq13.3.Hum Mol Genet. 1994; 3: 39-44Crossref PubMed Scopus (40) Google Scholar, 21Gibbons RJ Picketts DJ Villard L Higgs DR Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome).Cell. 1995; 80: 837-845Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 22Stayton CL Dabovic B Gulisano M Gecz J Broccoli V Giovanazzi S Bossolasco M Monaco L Rastan S Boncinelli E et al.Cloning and characterization of a new human Xq13 gene, encoding a putative helicase.Hum Mol Genet. 1994; 3: 1957-1964Crossref PubMed Scopus (52) Google Scholar Nearly all female carriers of ATR-X syndrome have highly skewed XCI in favor of cells expressing the normal ATRX allele and are essentially phenotypically normal.23Gibbons RJ Suthers GK Wilkie AO Buckle VJ Higgs DR X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.Am J Hum Genet. 1992; 51: 1136-1149PubMed Google Scholar In some carriers, a few red cells (∼1% of the frequency seen in affected males) contain HbH inclusions. In a unique carrier female with balanced XCI in the peripheral blood, the percentage of HbH cells (0.9%) was similar to that seen in affected males, consistent with skewed XCI being the mechanism by which such carriers are normally protected from the deleterious effects of an ATRX mutation.23Gibbons RJ Suthers GK Wilkie AO Buckle VJ Higgs DR X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.Am J Hum Genet. 1992; 51: 1136-1149PubMed Google Scholar Like ATRX, many XLMR genes are widely expressed and are predicted to regulate general nuclear processes, such as transcription, DNA repair, and cell proliferation.24Chelly J Mandel JL Monogenic causes of X-linked mental retardation.Nat Rev Genet. 2001; 2: 669-680Crossref PubMed Scopus (223) Google Scholar Therefore, it has been proposed that mutations in such genes should result in general defects influencing cell viability or proliferation and thus should result in skewed XCI (due to cell selection) in a wide variety of cell types.12Plenge RM Stevenson RA Lubs HA Schwartz CE Willard HF Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders.Am J Hum Genet. 2002; 71: 168-173Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Although most analyses of XCI in XLMR have been performed on peripheral blood, in ATR-X syndrome, skewed XCI has been demonstrated in buccal cells, hair roots, and peripheral blood (originating from endoderm, ectoderm, and mesoderm, respectively). As originally proposed,23Gibbons RJ Suthers GK Wilkie AO Buckle VJ Higgs DR X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.Am J Hum Genet. 1992; 51: 1136-1149PubMed Google Scholar this could result from cell selection prior to the differentiation of the embryo into separate tissue types, from separate selection events in different tissues, or from an effect of the mutation on the primary establishment of XCI. Which of these actually occurs in vivo has not yet been demonstrated for any form of XLMR in which carrier females have skewed XCI. Here, we have examined the pattern of XCI in female mice heterozygous for a null mutation in the highly conserved mouse orthologue of ATRX (Atrx [GenBank accession number NM_009530]).25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar, 26Picketts DJ Tastan AO Higgs DR Gibbons RJ Comparison of the human and murine ATRX gene identifies highly conserved, functionally important domains.Mamm Genome. 1998; 9: 400-403Crossref PubMed Scopus (55) Google Scholar The carrier female mice are viable and fertile, unlike males hemizygous for this mutation,25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar and they therefore provide a model in which XCI can be assessed in a range of tissues throughout development. We have shown that skewed XCI results from selection against Atrx-deficient cells at specific stages of development and differentiation in different tissues. These findings illustrate an important mechanism by which skewed XCI may occur in carriers of XLMR and provide insight into the normal role of ATRX in vivo. Atrx-mutant mice were generated, as described by Garrick et al.,25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar by homologous recombination in embryonic stem cells (E14, derived from 129/OlaHsd) and by injection of C57BL/6 blastocysts with the targeted cells. All wild-type mice used were F1 CBA/C57BL/6. Atrxwt/null females were obtained by crossing animals with a floxed Atrx allele and transgenic animals with Cre recombinase expressed from the Gata1 promoter.25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar, 27Mao X Fujiwara Y Orkin SH Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.Proc Natl Acad Sci USA. 1999; 96: 5037-5042Crossref PubMed Scopus (264) Google Scholar The Atrxwt/null embryos used were from crosses between these Atrxwt/null females and wild-type males. Mice with the floxed Atrx allele were also crossed with animals with a Cre recombinase transgene under the control of an inducible Mx1 promoter (Mx1Cre).28Kuhn R Schwenk F Aguet M Rajewsky K Inducible gene targeting in mice.Science. 1995; 269: 1427-1429Crossref PubMed Scopus (1472) Google Scholar Adult mice were genotyped at the Atrx locus by hybridizing SacI-digested DNA on Southern blots with an intron 17 probe, and embryos were genotyped by PCR with primers PPS1.17 (intron 17) and PPS1.28 (exon 19) (both methods described by Garrick et al.25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar). The Mx1Cre transgene was detected by PCR (forward primer 5′-GCGGAGCCAGCACTATTTA-3′; reverse primer 5′-CCGGCATCAACGTTTTCTTTT-3′).29Roebroek AJ Taylor NA Louagie E Pauli I Smeijers L Snellinx A Lauwers A Van de Ven WJ Hartmann D Creemers JW Limited redundancy of the proprotein convertase furin in mouse liver.J Biol Chem. 2004; 279: 53442-53450Crossref PubMed Scopus (93) Google Scholar To obtain staged embryos, the morning on which a vaginal plug was noted was taken as embryonic day (E) 0.5. Mice were sacrificed by cervical dislocation. To obtain blood from E9.5 and E10.5 embryos, pregnant females were given an intraperitoneal heparin injection 5–10 min before they were sacrificed. Embryos were separated from the uterine wall and placenta, then were released from the yolk sac into a small volume of PBS/heparin, and were allowed to bleed out. Cre recombination was induced in mice with the floxed Atrx allele and Mx1Cre transgene by two intraperitoneal injections of 250 μg of the synthetic double-stranded RNA polyinosinic-polycytidylic acid (pI-pC) 2 d apart, within 3 wk after birth. Tissue analysis was performed at least 6 wk after pI-pC treatment, and the efficiency of recombination in hematopoietic samples was assessed by Southern blot after SacI digestion (see above). Phenylhydrazine treatment to induce hemolytic anemia consisted of three intraperitoneal injections of 0.04 mg 1-acetyl-2-phenylhydrazine (Sigma) per g of body weight at 17.00 on day 1 and at 09.00 and 17.00 on day 2.30Spivak JL Toretti D Dickerman HW Effect of phenylhydrazine-induced hemolytic anemia on nuclear RNA polymerase activity of the mouse spleen.Blood. 1973; 42: 257-266PubMed Google Scholar Mice were sacrificed on day 6. Staged embryos and adult mouse tissues were fixed in 4% paraformaldehyde overnight at 4°C. After a washing in PBS, samples were dehydrated through an ethanol series and xylene and were embedded in paraffin. Before the staining, 5-μm sections were treated with Histo-Clear (Fisher Scientific) and were rehydrated. When required, antigen unmasking was performed by heat treatment in Antigen Unmasking Solution (Vector Laboratories). Sections were stained for immunohistochemistry by use of the Rabbit ABC Staining System (Santa Cruz Biotechnologies), in accordance with the manufacturer’s protocol, after endogenous peroxidase activity was blocked with 3% H2O2. The antibodies used were rabbit polyclonal anti-ATRX H300 (Santa Cruz Biotechnologies) and normal rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnologies). Cells in suspension from peripheral blood, bone marrow, fetal liver, and methylcellulose colonies were allowed to settle onto poly-l-lysine–coated coverslips, were fixed in 4% paraformaldehyde for 1 h at 4°C, and were permeabilized with 0.5% Triton-X 100 (in PBS) prior to incubation with antibodies. For fluorescence microscopy, the secondary antibodies used were Alexa 488–conjugated anti-rabbit IgG (Molecular Probes), fluorescein isothiocyanate (FITC)–conjugated anti-rabbit IgG (Jackson Laboratories), and Cy3-conjugated anti-rabbit IgG (Jackson Laboratories), and slides were mounted in Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). To isolate erythroid cell populations by fluorescence-assisted cell sorting (FACS), single cell suspensions were incubated with a phycoerythrin (PE)–conjugated anti-TER119 antibody (BD Pharmingen) and an FITC-conjugated anti-CD71 antibody (BD Pharmingen). Gated erythroblast populations were as described by Zhang et al.31Zhang J Socolovsky M Gross AW Lodish HF Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system.Blood. 2003; 102: 3938-3946Crossref PubMed Scopus (307) Google Scholar Briefly, early precursors were taken as CD71 medium/high, Ter199 low; intermediate precursors were CD71 high, Ter119 medium; and intermediate/late precursors were CD71 medium/high, Ter119 high. Light and fluorescence microscopy was performed using Olympus BX61 microscopes. Images were collected using OpenLab software for light microscopy and MacProbe 4.3 for fluorescence microscopy. Further image annotation was performed using Adobe Photoshop 7.0. DNA from 11 Atrxwt/null (15 samples) and 4 Atrxwt/flox (6 samples) adult mice was digested with PstI and the methylation-sensitive enzyme SacII and was analyzed by Southern blot. The probe used to detect fragments, including the Atrx CpG island (5′ probe), was generated by PCR (forward primer 5′-TTGCCTAACATGCACAAAGC-3′; reverse primer 5′-ATTGGCTACGGAAATTCACC-3′). The relative intensity of bands resulting from methylated and unmethylated fragments was determined using a Typhoon PhosphoImager (Amersham Biosciences) and ImageQuant software. Hematopoietic colony-forming assays by use of E13.5 fetal liver and adult bone marrow were performed using MethoCult Media (Stem Cell Technologies 03434) in accordance with the manufacturer’s guidelines. Colony type was determined by cell morphology, as described by Stem Cell Technologies. Colonies were picked after 7 d of culture or after 14 d for mixed-lineage colonies. The targeted Atrx allele (Atrxflox), described previously by Garrick et al.,25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar enables conditional knockout of the full-length isoform of Atrx by Cre-mediated recombination. Male cells hemizygous for the recombined null allele (Atrxnull) or female heterozygous cells (Atrxwt/null) with the null allele on the active X chromosome lack expression of full-length Atrx. They can be distinguished from wild-type cells or heterozygous cells with the wild-type allele on the active X chromosome by staining with an antibody specific for full-length Atrx (H300).25Garrick D Sharpe JA Arkell R Dobbie L Smith AJ Wood WG Higgs DR Gibbons RJ Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.PLoS Genet. 2006; 2: e58Crossref PubMed Scopus (109) Google Scholar, 32Berube NG Mangelsdorf M Jagla M Vanderluit J Garrick D Gibbons RJ Higgs DR Slack RS Picketts DJ The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis.J Clin Invest. 2005; 115: 258-267Crossref PubMed Scopus (146) Google Scholar Although female Atrxwt/null mice are viable and fertile, some do exhibit mild behavioral abnormalities, including increased aggression and hyperactivity (J.A.S., unpublished data). Preliminary data from behavioral studies showed that a proportion of heterozygotes performed poorly in a T-maze test, suggesting impaired learning and/or memory (P.M.N., unpublished data). We initially assessed the XCI pattern in adult Atrxwt/null females, by immunohistochemical analysis for Atrx expression, to determine whether skewed XCI is present in mice as it is in humans. Tissues were chosen for analysis in which the majority of cells (90%–100%) expressed Atrx in wild-type samples (fig. 1). If XCI were balanced, in Atrxwt/null females, one would expect ∼50% of the cells to lack Atrx expression due to the null allele being on the active X chromosome. In heterozygotes, the percentage of cells lacking Atrx expression was higher than in wild-type cells but remained <20% in the cerebral cortex and the granular layer of the cerebellum, as well as in the crypts of Lieberkuhn and epithelia of intestinal villi (fig. 1). Villi develop as clonal populations of cells, so each is either Atrx positive or Atrx negative. In a range of other tissues, including kidney, ovary, and muscle, a similar bias in favor of cells expressing Atrx was observed in heterozygous samples (data not shown). The observation of <20% Atrx-negative cells in adult tissues indicates that Atrxwt/null mice do have skewed XCI in favor of cells with the wild-type allele on the active chromosome, similar to the situation in human female carriers of ATR-X syndrome. This analysis shows that this is a suitable model to examine the causes of skewed XCI and confirms that XCI is skewed in tissues that are inaccessible in humans. To determine whether skewed XCI in ATR-X carrier females is likely to be because of an effect of ATRX mutation on the primary pattern of XCI or secondary selection events during development, we assessed the XCI pattern in E8 embryos. At E8, Atrxwt/null embryos are mosaics of Atrx-expressing and -nonexpressing cells (fig. 2A), with Atrx-negative cells present in all three primary germ layers (endoderm, ectoderm, and mesoderm) at ∼40% of the total cell number. Because Atrx-negative cells were found throughout the embryo, it seems that there is no strong selection against these cells during the establishment of XCI or in the designation of the germ layers. The reduced percentage of negative cells (∼40%) could be explained either by a weak competitive disadvantage of the null cells or by a bias in the original XCI ratio due to heterozygosity at the Xce locus, which can cause a bias in the choice of XCI.3Cattanach BM Isaacson JH Controlling elements in the mouse X chromosome.Genetics. 1967; 57: 331-346PubMed Google Scholar, 4Chadwick LH Pertz LM Broman KW Bartolomei MS Willard HF Genetic control of X chromosome inactivation in mice: definition of the Xce candidate interval.Genetics. 2006; 173: 2103-2110Crossref PubMed Scopus (41) Google Scholar, 5Chadwick LH Willard HF Genetic and parent-of-origin influences on X chromosome choice in Xce heterozygous mice.Mamm Genome. 2005; 16: 691-699Crossref PubMed Scopus (24) Google Scholar, 6Courtier B Heard E Avner P Xce haplotypes show modified methylation in a region of the active X chromosome lying 3′ to Xist.Proc Natl Acad Sci USA. 1995; 92: 3531-3535Crossref PubMed Scopus (97) Google Scholar However, the skewed XCI observed in adults must result from secondary selection events that occur later in embryonic development. We performed a systematic, detailed analysis of the XCI pattern in a range of tissues at different stages of development. Atrx can be detected in virtually all cells of wild-type embryos throughout gestation (fig. 3), but skewed XCI (ratio >80:20) was apparent in most tissues by late gestation (E17.5) in Atrxwt/null mice (fig. 2B). However, although the overall impression for the embryo as a whole is of a gradual decline in Atrx-null cells during development, at the level of individual tissues, the degree of cell selection varied significantly. For example, at E14.5 (fig. 2B and 2C), the percentage of negative cells remained 30%–40% in the forebrain and dermis (mesenchyme) but had declined to <20% in the epidermis and dorsal root ganglia. These data indicate that skewed XCI is a result of secondary cell selection but, interestingly, suggest that the selection pressure varies between cell lineages. We next considered the possibility of whether the high proportion of Atrx-positive cells could alternatively be explained by reactivation of the silent wild-type allele. Many gene promoters on the inactive X chromosome are characterized by methylated CpG islands, whereas these loci are unmethylated on the active X. Therefore, females normally have an ∼50:50 ratio of methylated:unmethylated X-chromosome CpG island alleles. Reactivation of the silent allele would be expected to result in loss of methylation of that allele, detectable by a deviation from the ∼50:50 methylation ratio. We assessed the ratio of methylated:unmethylated Atrx alleles in Atrxwt/null mice and females without Cre recombination (Atrxwt/flox), using a methylation-sensitive restriction digest (fig. 4), and found an ∼50:50 ratio overall in both genotypes. Although CpG island methylation is an indirect assay for gene silencing, this suggested that the XCI status of Atrxwt/null cells is stable. This conclusion is also supported by the observation that, within the clonal cell populations of intestinal villi, all cells are either Atrx negative or Atrx positive (fig. 1), which would be unlikely if spontaneous reactivation of the silent allele occurred. Therefore, we can be confident that cell selection is the mechanism that results in the increase in the proportion of Atrx-expressing cells. Having determined that cell selection underlies skewed XCI in carriers of an Atrx mutation, we next addressed the question of whether selection is continual (i.e., due to a general cellular defect affecting viability or proliferation, as has been proposed for XLMR mutations) or limited to particular stages of differentiation or development. We initially analyzed skewing in the hematopoietic system, because blood lineages are highly skewed in carriers of ATR-X syndrome mutations.23Gibbons RJ Suthers GK Wilkie AO Buckle VJ Higgs DR X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.Am J Hum Genet. 1992; 51: 1136-1149PubMed Google Scholar Embryonic hematopoiesis is divided into two phases, both of which were analyzed: the transient production of erythroblasts in the yolk sac (“primitive” erythropoiesis), followed by the emergence of hematopoietic stem cells (HSCs) and establishmen" @default.
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• W1997997534 title "Defining the Cause of Skewed X-Chromosome Inactivation in X-Linked Mental Retardation by Use of a Mouse Model" @default.
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