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X-inactivation profile reveals extensive variability in X-linked gene expression in females.
Author: L. Carrel,H. F. Willard
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"28. Medico,E. et al. ThetyrosinekinasereceptorsRonandSeacontrol?scattering?andmorphogenesisof liver progenitor cells in vitro. Mol. Biol. Cell 7, 495?504 (1996). 29. Li, C. & Wong, W. H. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc. Natl Acad. Sci. USA 98, 31?36 (2001). 30. Oshima, M. et al. Chemoprevention of intestinal polyposis in the Apcdelta716 mouse by rofecoxib, a specific cyclooxygenase-2 inhibitor. Cancer Res. 61, 1733?1740 (2001). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We thank M. Risio, E. David and their staffs for pathology, M. Cilli for mouse surgery, R. Albano, A. Ferraro and R. Lo Noce for technical assistance, and M. Belluardo for computational analysis. We thank A.Cignetto for secretarial assistance. G.S. is the recipient of an AIRC fellowship. This research was supported by AIRC, CNR-MIUR, FIRB-MIUR, MIUR-PRIN and the Foundations CRTand ?Compagnia di San Paolo?. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to C.B.(carla.boccaccio@ircc.it). .............................................................. X-inactivation profile reveals extensive variability in X-linked gene expression in females Laura Carrel 1 & Huntington F. Willard 2 1 Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA 2 Institute for Genome Sciences & Policy, Duke University, Durham, North Carolina 27708, USA ............................................................................................................................................................................. In female mammals, most genes on one X chromosome are silenced as a result of X-chromosome inactivation 1,2 . However, some genes escape X-inactivation and are expressed from both the active and inactive X chromosome. Such genes are potential contributors to sexually dimorphic traits, to phenotypic varia- bility among females heterozygous for X-linked conditions, and to clinical abnormalities in patients with abnormal X chromo- somes 3 . Here, we present a comprehensive X-inactivation profile of the human X chromosome, representing an estimated 95% of assayable genes in fibroblast-based test systems 4,5 . In total, about 15% of X-linked genes escape inactivation to some degree, and the proportion of genes escaping inactivation differs dramati- cally between different regions of the X chromosome, reflecting the evolutionary history of the sex chromosomes. An additional 10%ofX-linkedgenesshowvariablepatternsofinactivation and are expressed to different extents from some inactive X chromo- somes. This suggests a remarkable and previously unsuspected degree of expression heterogeneity among females. Establishment of X-inactivation requires a key cis-acting master locus that includes the non-coding XIST gene 6,7 .HowXIST RNA and otherepigenetic modifications 8?10 aredirectedtosites along the inactive X chromosome (X i ), and how inactivation spreads in cis over the 155Mb X chromosome still remain poorly understood. That some X-linked genes should escape inactivation was first predictedfor pseudoautosomal genes on the Xand Ychromosomes that show equivalent dosage, with two active copies in both males and females 11 . Because genes that escape inactivation lack at least some of the epigenetic alterations that characterize the rest of the chromosome 12 , their identification and analysis are important for understanding the mechanics of the X-inactivation process. We have previously developed an X-inactivation assay system to detect gene expression directly from the X i in human female cellsby determining relative expression levels of polymorphic alleles in heterozygous fibroblasts that demonstrate non-random inacti- vation 5 . For this study, we developed a quantitative assay based on fluorescent, single-nucleotide primer-extension (Q-SNaPshot) to distinguish between alleles of expressed single-nucleotide poly- morphisms (SNPs) along the X chromosome (see Methods). For each heterozygous human fibroblast sample, complementary DNA was amplified, and the relative expression of each allele was determined after normalization to genomic DNA amplification products (Fig. 1). As all of the samples used show complete non- random inactivation 5 , genes that show biallelic expression must escape inactivation, with the normalized allelic ratios indicating the level of X i expression (relative to that on the active X chromosome, X a ), for example, HDHD1A and RIBC1 in Fig. 1. In contrast, monoallelic expression indicates that a gene is subject to inacti- vation (for example, the TMLHE gene in Fig. 1). Ninety-four genes spanning the X chromosome were tested in a total of 40 human samples (Supplementary Table 1). Notably, only about 65% of the genes were subject to inactivation in all heterozygous samples (Fig.2);afurther20%wereinactivatedinsome,butnotallsamples, and 15% escaped inactivation in all samples. Surprisingly, among Figure 1 Q-SNaPshot assay of allelic expression for three X-linked genes. The assay measures expression by incorporating fluorescent dideoxy-nucleotides at polymorphic sites: G (blue), A (green), T (red), C (black). For each gene, DNA and cDNA amplification products are shown for three different cell lines (denoted a?i). For TMLHE, a randomly inactivated normal fibroblast line is included, whereas all other samples are non-randomly inactivated primary lines (that is, the same X chromosome is the X i in all cells). Relative expression of the less intense allele for each sample (normalized with DNA to compensate for biased fluorescence output) is indicated as a percentage of the more intense allele, and by inference reflects the X i expression level. letters to nature NATURE|VOL 434|17 MARCH 2005|www.nature.com/nature400 � 2005 Nature Publishing Group those genes that escape inactivation, most were not fully expressed from the X i , demonstrating that escape from inactivation, even for pseudoautosomal genes, is generally partial and incomplete. For example, although the FLJ39679 gene showed substantial X i expression (.75% of Xa expression) in some cell lines, other cell lines showed X i expression levels as low as ,25% of X a expression levels (Fig. 2). These data indicate that females have considerable heterogeneity in levels of X-linked gene expression. At this point, however, we cannot establish whether this allelic variation is a specific featureof X-inactivation or reflects genome-widevariation. To complement these data on a more comprehensive scale, we have established an X-inactivation profile in parallel with the complete sequence of the human X chromosome 13 . We determined the X-inactivation status of essentially all X-linked transcripts that are expressed in fibroblast-based assay systems 4,5 (see Methods and Supplementary Table 2), and conclude that the current survey of 624 transcripts represents ,95% of assayable genes on the X chromosome. X-inactivation status was assessed by analysing gene expression from human X a and X i chromosomes in an extensive panel of rodent/human somatic cell hybrids 4 . This method does not require distinction between X a and X i transcripts, and is applicable to all X-linked genes expressed in these cells. Each transcript was assayed using polymerase chain reaction (PCR) with reverse transcription (RT?PCR) to determine whether expression is detected only from hybrid lines retaining X a chromosomes (and is therefore a gene subject to X-inactivation) or from both X a hybrid lines and X i hybrid lines, indicating a gene that escapes X-inactivation. Genes wereassayed in up to nine rodent/human somatic cell hybrids, each containing a different, normal human X i chromosome (see Methods).ResultsforeachgeneineachX i hybridtestedaredepicted in Fig. 3a and aresummarized foreach transcript in Supplementary Table 3. A total of 401 X-linked transcripts gave completely concordant results in all hybrids; that is, they were either expressed (74 transcripts) or silenced (327 transcripts) in all X i hybrids tested. Considering only these transcripts (and excluding pseudoauto- somal genes), the frequency of escape from inactivation for X-specific transcripts is minimally 16%, essentially identical to Figure 3 X-inactivation profile of the human X chromosome. a, 624 genes were tested in nine X i hybrids. Each gene is linearly displayed. Blue denotes significant X i gene expression, yellow shows silenced genes, pseudoautosomal genes are purple, and untested hybrids remain white. Positions of the centromere (cen) and XIST are indicated. b, To determine whether heterogeneity is largely a property of a specific chromosome, three independently derived hybrids from one X i chromosome (denoted 10 a, b, c) and two hybrids carrying another X i chromosome (11 a, b) were isolated, and results for 19 genes are shown adjacent to results for the original nine X i chromosomes. Figure 2 X-inactivation as a measure of allelic expression in non-randomly inactivated primary fibroblasts. a, b, Gene expression levels from normal X i chromosomes (a) and from structurally abnormal X i chromosomes (b). Each gene is linearly arrayed and approximate correspondence to chromosome location is indicated. The gene order is as in Supplementary Table 1. Each gene was assayed in all heterozygous individuals. Uninformative samples remain uncoloured. Colour-coding reflects relative X i expression level as indicated. Grey boxes in b indicate absent portions of the X chromosome due to deletions or translocations. letters to nature NATURE|VOL 434|17 MARCH 2005|www.nature.com/nature 401 � 2005 Nature Publishing Group the estimate derivedfrom studies in human cells. An additional 223 genes showed heterogeneity among different X i hybrids and were expressed in some, but not all, of the X i hybrids assayed (Fig. 3a), confirming and extending our observations in human cells (Fig. 2). Although this assay is inherently less quantitative then the SNP-based assay (see Methods), the majority of the genes escaping inactivation showed robust X i expression; a minority were expressed from the X i chromosome at relatively low levels (,10?15% that of the X a ). To assess the chromosomal basis for X i heterogeneity, we analysed the same X chromosome isolated inde- pendently in multiple hybrids (Fig. 3b). Although the data set is limited, no variability was observed between different hybrids carrying the same X i , supporting the idea that heterogeneity of X i expression is largely an intrinsic property of the X chromosome tested, rather than an artefact of the hybrid model system. Of these heterogeneous genes, the majority (161 transcripts) gave concor- dant expression patterns in all but one or two hybrids, and for classification purposes 4 , were considered to escape (30 genes) or be subject to (131 genes) X-inactivation. In total, of the 612 X-specific transcripts classified in this manner, 458 (75%) are subject to inactivation in most or all of the X chromosomes tested, and 94 (15%) escape inactivation; the remaining 10% are heterogeneous (Fig. 3a and Supplementary Table 3). Combined, these last two categories of genes indicate that at least 25% of X-linked genes escape inactivation in all or a significant subset of X chromosomes tested, a result fully consistent with the data obtained with human cells (Fig. 2). Importantly,giventhelimited number(,50)ofXchromosomes screened in the two assays, the results from the primary human cell assay were in agreement with the X i hybrid expression patterns in most instances. For example, 17 of 20 genes that escaped inacti- vation in X i hybrids were expressed from the X i in at least some of the human samples tested, although only 12 of these genes were expressed in all heterozygous lines tested (Supplementary Table 4). This comparison suggests that the total proportion of X-linked genes showing heterogeneous patterns of X i expression in the generalpopulationmaybeeven greater thanthe estimates provided here. The distribution of genes that escape inactivation is clearly not random along the chromosome 14 . Large blocks of transcripts expressed from the X i , apparent in both assays (Figs 2, 3), indicate that these genes are clustered and map primarily to the distal portion of the X chromosome short arm (Xp), perhaps related to their distance from the XIST gene, as recently proposed for the mouse 15 . Mammalian X and Y chromosomes are proposed to have diverged from an identical pair of ancestral autosomes 16 . Key to their evolution, structural rearrangements successively suppressed recombination, allowing independent evolution of each chromo- some and leading to the largely degraded Y chromosome, which contains very few genes 17 . Evolution of the X chromosome, on the other hand, has been heavily influenced by X-chromosome inacti- vation, and such stringent regulation requires that gene content remain highly conserved 16 . As a consequence, the long arm (Xq) of the human X chromosome and a portion of proximal Xp, retain a high degree of synteny even with marsupial and monotreme X chromosomes 18 . By using the boundary of this X-conserved region and calculating sequence divergence between X and Y homologues, thelocationsoftheseancestralrearrangementshavebeeninferred 19 . The X-specific portion of the chromosome can be partitioned into five evolutionary strata that show increasing levels of sequence divergence with increasing distance from the distal tip of Xp 13,19 .To explore an evolutionary influence on the X-inactivation profile, we compared X-inactivation patterns of genes mapping to each stratum (Fig. 4). Fulfilling the prediction that genes acquire the ability to be X-inactivated in response to the decay of Y-linked homologues 20 , the proportion of genes that is expressed from the X i in either the human samples (Fig.4a)or inmost hybrids (Fig.4b)is clearlyhighestintheyoungeststratum(stratum5)andlowestinthe oldest stratum (stratum 1). Even excluding pseudoautosomal genes, the majorityof X-linked genes with Y homology escape X-inactivation and are expressed in all X i hybrids tested (22 out of 35), and this proportion is even higher when considering X-linked genes with functional Y ortho- logues. Notably, however, two such genes?TSPYL2 and RBMX? were subject to inactivation in all hybrids; these genes belong to the previously defined ampliconic class of XY-homologous genes 17 , suggesting that they may have less stringent dosage controls. We further examined the distribution and frequency of genes with Y homology,specificallyamonggenesthatwerefullysubjecttoorthat fully escaped from inactivation (Supplementary Fig. 1). This com- parison confirmedthat youngerstratacontainasignificantly higher proportion of genes with Y homology, but moreover, that the propensity of a gene with Y homology to escape inactivation diminishes in older strata, consistent with the idea that these genes lose the ability to escape inactivation over time 20 . The clustering of genes that escape inactivation, particularly evident in strata 4 and 5, supports the idea that control is at the level of chromosome domains, and is consistent with the premise that many genes escape inactivation because they lie within an epigenetic domain containing at least one X-linked gene with a Y homologue. However, this possibility cannot explain all genes that escape inactivation, because there are several other transcripts (particularly in stratum 1) that are expressed from the X i even Figure 4 X i expression data in primary fibroblasts and X i hybrids correlate with location on X. Transcripts were subdivided according to X i results and chromosomal region. a, The proportion of samples assayed in primary fibroblasts that show similar X i results are indicated for the pseudoautosomal region PAR1, and for each X-specific evolutionary strata (S1?S5). b, For each chromosomal region, the percentage of genes showing similar X i hybrid results (out of the nine X i hybrid lines tested) are also shown. c, Location of each evolutionary strata on the X chromosome 13,19,21 . letters to nature NATURE|VOL 434|17 MARCH 2005|www.nature.com/nature402 � 2005 Nature Publishing Group though they lack Y homology and map quite far from other expressed transcripts. One intriguing cluster of X i -expressed genes maps within a gene-rich region of Xq28 that, on the basis of comparison with the chicken genomic sequence, has different evolutionary origins than the rest of Xq 21 . Using comparative mapping, this region is proposed to be included in stratum 2 (designated stratum 2b) 21 , potentially explaining the number of genes in this region of Xq28 that are expressed from all or at least a high percentage of the X i chromosomes tested (Figs 2, 3). ModelstoexplainhowinactivationspreadsacrosstheXchromo- some postulate that specific sequences along the chromosome function as cis-acting ?booster elements? 22 . Lyon has proposed that longinterspersedrepeatelements(L1)couldservesuchafunction 23 , in part because of their overall twofold enrichment on the X chromosome relative to autosomes 13,24 . L1 concentration is inversely correlated with the proportion of genes that escape inactivation 13 (Supplementary Fig. 2). Stratum 5 has the highest proportion of genes that escape inactivation, but also the lowest L1 and the highest Alu repeat concentrations. In fact, L1 levels in the pseudoautosomal region and in strata 4 and 5 are below the genome average 13 . Locally, however, differences in L1 concentra- tion that correlate with X-inactivation patterns are not apparent (SupplementaryFig.3).Attheveryleast,thesedataindicatethatthe genomic and epigenomic determinants are likely to be more complex than repeat content alone. Anotherlocalfactor thatcouldinfluenceinactivationstatusisthe presence of a CpG island, as DNA methylation is well known to be involved in the maintenance of X i silencing 2,25 . In contrast to a previous report using a small number of genes 26 ,thewhole chromosome profile shows that the distribution of island- associated genes does not differ between genes that escaped X-inactivation in all X i hybrids and those that were completely silenced (Supplementary Fig. 4). However, the proportion of island-associated genes that are subject to or escape from inacti- vation does differ significantly from the proportion of genes containing CpG islands that demonstrate heterogeneous patterns of inactivation (P , 0.001 and P , 0.025 respectively). These data suggest that the presence of an island by itself does not determine whether a gene is inactivated; however, it seems that the absence of an island may contribute to the variability of X i expression among different X chromosomes, as seen both in human cells and hybrids. The large number of genes that escape inactivation, and their non-random distribution on the chromosome, has implications for counseling individuals with X chromosome abnormalities, estimated at 1 in 650 live births 3 . The evolutionary and genomic patterns of genes that escape inactivation suggest that aneuploidy for Xp is more severe than aneuploidy for Xq 4 . The profiles presented here provide a database of candidate genes to explain the clinical findings in such cases, although it is important to emphasize that the current profile has been obtained in fibroblast cells and may or may not reflect the situation in other tissues relevant to specific disease phenotypes. Owing to different levels and different subsets of genes escaping X-inactivation, females will be even more variable with respect to X-linked gene expression than previously recognized. Because of these heterogeneous genes and the ,15% of genes that escape inactivation, the female genome differs from the male genome in at least four ways. First, the Y chromosome endows the male with at least several dozen genes that are absent in the female 17 . Second, the incomplete nature of X-inactivation means that at least 15% of X-linked genes are expressed at characteristically higher (but often variable) levels in females than in males. Third, a minimum of an additional 10% of genes show heterogeneous X-inactivation and thus differ in expression levels among females, whereas all males expressasinglecopyofsuchgenes.Andfourth,thelong-recognized random nature of X-inactivation indicates that females, but not males, are mosaics of two cell populations with respect to X-linked gene expression 1,3 . Notwithstandingthegenomicandbiologicalsignificanceofthese sex-specific differences, their general clinical and phenotypic impli- cations remain unexplored. However, as many of the genes that escape from inactivation do not have Y-linked homologues, strict dosage compensation may not be necessary for all genes on the chromosome. Such characteristic genomic differences should be recognized as a factor for explaining sex-specific phenotypes both in complex disease as well as in normal, sexually dimorphic traits 27 . A Methods X-linked genes included in this study Out of 931 X-linked genes annotated in NCBI build 34.3 (Supplementary Table 2), 267 were excluded from analysis because (1) they mapped to multiple locations, (2) they were computationally-predicted proteins that lacked significant expressed-sequence-tag (EST) support,or(3)theyweretranscriptswithrestrictedexpressionpatterns;forexample,10% of genes on the X chromosome are cancer-testis antigen genes that are not expressed in fibroblasts 13 (Supplementary Table 2 and Supplementary Note 1). Primers were designed for the remaining genes, and expression was initially tested in DNA and cDNA from human and mouse fibroblasts and mouse/human hybrid cell lines carrying X a chromosomes.Thispreliminaryscreeningidentified471transcriptsthatwereexpressedin primary fibroblasts and could therefore be tested for X-inactivation. This frequency of expressed transcripts (71%) is consistent with microarray studies of fibroblasts 28 .An additional 153 transcripts (including full-length mRNAs and ESTs) that remain unassociated with currently annotated genes were also included in the profile. The transcripts analysed are listed in Supplementary Table 3. Allelic expression in primary fibroblast cell lines Apanel of 40 primary fibroblastcelllines fromfemales with non-random inactivation has been previously characterized 5 . These lines show complete skewing of inactivation, primarily as a result of secondary cell selection that maintains dosage despite structural rearrangements involving the X chromosome 3 . Confirmation that these lines were non- randomly inactivated was established by multiple assays 5 . Cell lines were maintained and DNA and cDNA isolated as described 5 . Several assays were used to determine relative allele frequencies 5 . A novel quantitative assay (Q-SNaPshot, a modification of the commercial SNaPshot assay (Applied Biosystems))wasestablishedthatusesprimerextensiontoincorporateasinglefluorescent dideoxy nucleotide at the polymorphic site and quantitates products on an ABI 3100 (L.C., unpublished). Samples were normalized by comparison to heterozygous DNA samples with a known 1:1 allele ratio (Fig. 1). Quantitative conditions were confirmed by correctly predicting allele ratios for known mixtures of homozygous samples (data not shown). All assays were performed in duplicate. Expression of transcripts from active and inactive X hybrids Somatic cell hybrids, cell culture conditions, and nucleic acid purification methods have previously been reported 4 . Initial studies established that many genes have very low levels of X i expression (Supplementary Fig. 5), and we therefore adopted a protocol that would allow us to identify significant X i expression levels in hybrids. Large-scale reverse transcription reactions were performed as described (but volumes were increased tenfold) 4 . RNA samples were treated with DNaseI before reverse transcription, to eliminate any contaminating genomic DNA. Each large-scale cDNA synthesis panel was normalized by amplification of the pseudoautosomal gene MIC2, which is equally expressed from X a and X i (Supplementary Fig. 5 and Supplementary Note 2). Each gene was tested at two concentrations of cDNA, corresponding to ,50ng and 250ng of total RNA (Supplementary Fig. 6). Cycles were minimized to ensure that, in particular,thedilutecDNAsampleswereamplifiedintheexponentialrange.Althoughnot strictly quantitative, this protocol allowed us to estimate the relative abundance of X a and X i transcripts. Faint amplification products that disappeared in the diluted samples were not considered positive. Samples that showed heterogeneous expression among different X i chromosomes,ordiscordantpatternsbetweencDNAdilutions,wererepeatedtoensure that scored positives were significantly expressed. Because of the semiquantitative nature of the RT?PCR assay used, the X i expression levels in samples scored as positive are estimatedtobeatleastat10?15%ofX a levels(SupplementaryFigs5,6andSupplementary Note 2). To assess the reproducibility of results within the same gene, ,15% of genes were assayed independently and blindly using at least two primer pairs. Virtually all (97%) of the X i hybrid samples scored werein completeagreement; those that did not agreeusually had faint bands that were called positive in one case but not another. For three genes, discordant results from more than one hybrid suggest that the assayed transcripts are incorrectly linked in the Unigene database and belong to distinct transcripts. A small number of genes included in this profile havebeen tested byother laboratories (for example, see ref.29). Most were re-analysed in our more extensive set of hybrids. The data here include and extend data previously reported from our laboratory 4 . Transcript analysis Transcripts were mapped physically along the X chromosome using the May 2004 assembly from the UCSC genome browser (http://genome.ucsc.edu). CpG islands, as letters to nature NATURE|VOL 434|17 MARCH 2005|www.nature.com/nature 403 � 2005 Nature Publishing Group annotated in the UCSCgenomebrowser,wereassignedtoeach geneif theylaywithin 2kb upstream or downstream of the 5 0 end of the RefSeq transcript or Unigene cluster. ESTs and most single exon transcripts were excluded from this analysis (Supplementary Table 3). Evolutionary stratawereassigned as described 13,19,21 , and specific gene designations are indicated in Supplementary Table 3 and Supplementary Note 3. 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Heterogeneous gene expression from the inactive X chromosome: an X-linked gene that escapes X inactivation in some human cell lines but is inactivated in others. Proc. Natl Acad. Sci. USA 96, 7364?7369 (1999). 6. Brown, C. J. et al. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349, 38?44 (1991). 7. Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. & Brockdorff, N. Requirement for Xist in X chromosome inactivation. Nature 379, 131?137 (1996). 8. Rougeulle, C. et al. Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome. Mol. Cell. Biol. 24, 5475?5484 (2004). 9. Chadwick, B. P. & Willard, H. F. Barring gene expression after XIST: maintaining facultative heterochromatin on the inactive X. Semin. Cell Dev. Biol. 14, 359?367 (2003). 10. Chadwick,B.P.&Willard,H.F.Multiplespatiallydistincttypesoffacultativeheterochromatinonthe human inactive X chromosome. Proc. Natl Acad. Sci. 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Graves,J.A.Mammalsthatbreaktherules:geneticsofmarsupialsandmonotremes. Annu. Rev. Genet. 30, 233?260 (1996). 19. Lahn,B.T.&Page,D.C.FourevolutionarystrataonthehumanXchromosome. Science 286,964?967 (1999). 20. Jegalian, K. & Page, D. C. A proposed path by which genes common to mammalian X and Y chromosomes evolve to become X inactivated. Nature 394, 776?780 (1998). 21. Kohn, M., Kehrer-Sawatzki, H., Vogel, W., Graves, J. A. & Hameister, H. Wide genome comparisons reveal the origins of the human X chromosome. Trends Genet. 20, 598?603 (2004). 22. Gartler, S.M. & Riggs, A. D. Mammalian X-chromosome inactivation. Annu. Rev. Genet. 17,155?190 (1983). 23. Lyon, M. F. X-chromosome inactivation: a repeat hypothesis. Cytogenet. Cell Genet. 80, 133?137 (1998). 24. Bailey, J. A., Carrel, L., Chakravarti, A. & Eichler, E. E. Molecular evidence for a relationship between LINE-1 elements and X chromosome inactivation: the Lyon repeat hypothesis. Proc. Natl Acad. Sci. USA 97, 6634?6639 (2000). 25. Mohandas, T., Sparkes, R. S. & Shapiro, L. J. Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation. Science 211, 393?396 (1981). 26. Ke, X. & Collins, A. CpG islands in human X-inactivation. Ann. Hum. Genet. 67, 242?249 (2003). 27. Committee on Understanding the Biology of Sex and Gender Differences. In Exploring the Biological Contributions to Human Health?Does Sex Matter? (eds Wizemann, T. M. & Pardue, M.) (National Acadamies Press, Washington, DC, 2001). 28. Iyer, V. R. et al. The transcriptional program in the response of human fibroblasts to serum. Science 283, 83?87 (1999). 29. Esposito, T. et al. Escape from X inactivation of two new genes associated with DXS6974E and DXS7020E. Genomics 43, 183?190 (1997). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We thankM. Ross for sharing informationbeforepublication and gratefully acknowledgetechnicalassistancefromG.Nickel,K.Trevarthen,J.Dunn,A.CottleandM.Moon. This work was supported in part by a National Institutes of Health research grant to H.F.W. and L.C. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to L.C. (lcarrel@psu.edu). .............................................................. The vacuolar Ca 21 -activated channel TPC1 regulates germination and stomatal movement Edgar Peiter 1 , Frans J. M. Maathuis 1 , Lewis N. Mills 2 , Heather Knight 3 , Je�ro?me Pelloux 1 , Alistair M. Hetherington 2 & Dale Sanders 1 1 Biology Department, Area 9, University of York, PO Box 373, York YO10 5YW, UK 2 Biology Department, Lancaster Environment Centre, University of Lancaster, Lancaster LA1 4YQ, UK 3 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK ............................................................................................................................................................................. Cytosolic free calcium ([Ca 21 ] cyt ) is a ubiquitous signalling component in plant cells 1 . Numerous stimuli trigger sustained or transient elevations of [Ca 21 ] cyt that evoke downstream stimulus-specific responses. Generation of [Ca 21 ] cyt signals is effected through stimulus-induced opening of Ca 21 -permeable ion channels that catalyse a flux of Ca 21 into the cytosol from extracellularor intracellularstores.ManyclassesofCa 21 current have been characterized electrophysiologically in plant mem- branes 2 . However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show thattheTPC1(?two-porechannel1?)geneofArabidopsisthaliana encodes a class of Ca 21 -dependent Ca 21 -release channel that is known from numerous electrophysiological studies as the slow vacuolar channel 3?5 . Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca 21 ] cyt . We show that a tpc1 knockout mutant lacks functionalslowvacuolarchannelactivityandisdefectivein both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca 21 - release channels in the physiological processes of plants. In A. thaliana the TPC1 gene (AGI code At4g03560) is the only representative of the TPC gene family 6 that is also present with one or two members in other dicotyledonous plants 7 , monocotyledo- nous plants 8,9 , gymnosperms (Pinus pinaster, GenBank accession numberBX252040),mosses(Physcomitrella patens,GenBankacces- sionnumberBQ039582),andanimals 10 .Thecorrespondingprotein comprises two fused Shaker-like units, each with six transmem- brane spans, a number of basic residues in the fourth transmem- branedomainofeachShaker-likeunit,acytosoliclinkerregionwith two EF hands?that is, helix-loop-helix domains?suggestive of a roleinCa 2� binding(Fig.1a),andseveralputativephosphorylation sites. Expression of plant TPC1 proteins in a yeast mutant lacking endogenous channel activity results in complementation of two phenotypic properties of the mutant?mating-induced cell death and low basal Ca 2� influx?suggesting that TPC1 might form a Ca 2� -permeable channel 6?9 . To determine the intracellular location of the TPC1 protein in planta, we constructed a TPC1?green fluorescent protein (GFP) fusion protein and introduced it into Arabidopsis mesophyll protoplasts. Figure 1b and c show images of protoplasts expressing GFP alone. In intact protoplasts, fluorescence appears as a clear band surrounding most of the cell, but is also present in a cytosol- rich region that surrounds the nucleus and chloroplasts. Fluores- cence rapidly disperses after disruption of protoplasts expressing GFP alone, suggesting a cytosolic location for the GFP (Fig. 1c). Images of cells expressing the TPC1?GFP fusion protein are shown inFig.1dande.TheintactprotoplastexhibitsGFPfluorescencethat is distinct from the plasma membrane and strongly suggestive of an association with intracellular membranes (Fig. 1d). Intact vacuoles letters to nature NATURE|VOL 434|17 MARCH 2005|www.nature.com/nature404 � 2005 Nature Publishing Group "
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Genetics
Gene Inheritance and Transmission
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