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Human Molecular Genetics Pages 1713-1727
A 94 kb genomic sequence 3' to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx
Introduction
Results
   Sequencing and assembly strategies
   Sequence features
   Homology with non-coding sequences
   Identification of new transcripts
   Isolation and analysis of the Tsx gene
   Gene conservation analysis
Discussion
   Search for chromosomal features
   Search for protein coding sequences
   Evolutionary studies
Materials And Methods
   Preparation of sequence templates
   Sequencing reactions
   Data searches and analyses
   Isolation of Tsx cDNA clones
   Hybridization
Acknowledgements
   DDBJ/EMBL/GENBANK ACCESSION NUMBERS
References

A 94 kb genomic sequence 3' to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx

A 94 kb genomic sequence 3 ' to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx Marie-Christine Simmler*, David B. Cunningham, Philippe Clerc, Thierry Vermat1, Bernard Caudron2, Corinne Cruaud3, André Pawlak4, Claude Szpirer5, Jean Weissenbach3, Jean-Michel Claverie6 and Philip Avner

CNRS URA1968, Génétique Moléculaire Murine, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, 1CNRS URA243, Laboratoire BGBP, Université Claude Bernard, Lyon 1, 69622 Villeurbanne Cedex, France, 2Service d'Informatique Scientifique, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, 3CNRS URA1922, Généthon, BP60, 91002 Evry Cedex, France, 4INSERM 99, Hopital Henri Mondor, 94010 Créteil Cedex, France, 5Laboratoire de Biologie du Développement, Université Libre de Bruxelles, B1640 Rhode-St-Genese, Belgium and 6CNRS EP91, Information Génétique et Structurale, IBSM, 13402 Marseille Cedex, France

Received May 2, 1996; Revised and Accepted August 7, 1996DDBJ/EMBL/GenBank accession nos X99946X99946, X9976X9976, X99797

X chromosome inactivation in both mouse and human requires the presence of a cis acting locus, the X inactivation centre. This locus is thought to be involved in the initiation and spreading of the inactivation signal in early development. In order to increase our understanding of the mouse X inactivation centre, a 94 kb region immediately distal to the Xist gene has been sequenced and analysed for the presence of transcription units and/or potential cis acting regulatory elements. We have identified a novel gene, Tsx, lying 40 kb 3' from Xist. Tsx is expressed specifically in the testis and shows no convincing homology to proteins currently in the databases. A rat homologue, also X linked, has been isolated. The mouse and rat Tsx sequences are highly divergent, suggesting that part of the X inactivation centre, including both Xist and Tsx are subject to relatively weak evolutionary constraints.

INTRODUCTION

Very early in development, one of the two X chromosomes in each somatic cell of mammalian females becomes transcriptionally silent, thus compensating for the dosage difference between females with two X chromosomes and males having a unique X.

The inactive X chromosome adopts the so called sex chromatin body configuration at the nuclear periphery of female cells (1 ). The active and inactive X chromosome can also be distinguished by late replication, methylation of the 5' end of constitutively expressed genes and resistance to DNA nucleases or association with hypoacetylated histones (2 ).

Studies concerning the inactivation pattern in females carrying X chromosome aberrations have given rise to the hypothesis that the initiation event is under the control of a unique region, the X inactivation centre (XIC/Xic) which must be present in cis for inactivation to occur (3 -7 ). At present, the human XIC candidate region is localized to an ~1 Mb interval within band Xq13.2, while the murine Xic maps to a homologous but less precisely mapped region of the mouse X chromosome (8 ).

XIC/Xic may also play a role in the counting of the number of X chromosomes. This mechanism apparently recognizes the ratio of the number of autosomes and X chromosomes: only a single X chromosome remains active for every two sets of autosomes. In triploids, there is evidence that either one or two X chromosomes are active (9 ,10 ) and in tetraploid mouse embryos, two X chromosomes are active (11 ). First hypothesized by Eicher (12 ), it has been proposed that one copy of XIC/Xic in each cell receives a trans acting signal which is present in limited supply and which blocks its inactivating function, thus marking this chromosome as the one that is to remain active (6 ,13 ). Moreover, at least two copies of XIC on different X chromosomes are needed for the process to initiate, since the duplicated XIC regions on a single X chromosome in males fail to inactivate (14 ,15 ).

XIC/Xic is also involved in the choice of X chromosome for activity: the inactivation of an X chromosome is thought to be random with either chromosome having an equal chance, on average, of becoming inactivated. However, in man, skewing of X inactivation has been described in studies of rare families (3 ,16 ), and in mouse, the choice of which chromosome is to be inactivated is under the control of the Xce locus (X chromosome controlling element) (17 ). Different Xce allelic variants skew the even inactivation pattern in the direction of the active X chromosome with the stronger allele (18 ). It has been postulated, but not yet proven, that Xic and Xce are, in fact, the same locus (19 ).

Finally, XIC/Xic has been implicated in the spreading of inactivation in both directions along the chromosome, the nature of which remains elusive although probably linked to a global heterochromatinization process (20 ). Recent evidence has shown that X inactivation is dependent on the expression of the Xist gene (21 ). Both thelocalization of XIST/Xist within XIC/Xic and its expression, in complete concordance with X chromosome inactivation, suggest that it is indeed involved in the process of X chromosome inactivation (22 -24 ). XIST/Xist codes for an RNA that remains intranuclear and specifically hybridizes to the inactive X in interphase nuclei (25 ,26 ). A role in chromatin packaging, through its action as a structural RNA, has been proposed (26 ). Although there is evidence for Xist being implicated in Xic function (21 ), genetic analysis currently excludes Xist as a candidate for the Xce locus (27 ) (Simmler, M.-C., Cattanach B. and Avner, P., unpublished data).

As part of our ongoing interest in the mouse Xic, we have used a direct sequencing approach to analyse the transcriptional and structural contents of a 94 kb genomic region immediately distal to the Xist gene. In this report, we present a detailed analysis of the features found in this 94 kb genomic sequence. Within this region a new testis specific gene, Tsx, was identified 40 kb distal to Xist. We have isolated the rat homologue and found by mapping analysis that this gene is also X linked. Sequence comparison between the two rodent homologues showed that Tsx is among the most rapidly diverging genes.

RESULTS

Sequencing and assembly strategies

The 94 kb segment was contained in a series of overlapping lambda clones (28 ) extending from the 3' end of the Xist sequence (Fig. 1 ). M13 based shotgun libraries (29 ) were prepared from six lambda clones (A-C and E-G). A directed strategy using a nested set of deletion clones (30 ) deriving from the most terminal part of lambda clone D was used to fill the gap between lambda C and E. This approach was chosen to minimize redundancy, since lambda C and D were found to be highly overlapping (Fig. 1 ). Most of the DNA sequence data (over 95%) was obtained by the standard single stranded dideoxynucleotide chain termination procedure using fluorescence labelled primers (31 ). Vector trimming was carried out using the Ted editor (32 ) and quality trimming was estimated by visual inspection of the first ambiguity. A total of 2484 runs were incorporated into the assembly process. Each base was sequenced an average of six times. The sequence (94 459 bp) was assembled using the shotgun package of Roger Staden (33 ), except for lambda G for which we used the Seqman program (DNASTAR Inc., Madison, Wi 53715). Gaps in the assembled sequences were bridged by systematic sequence extension from PCR products using internal primers and fluorescence labelled dideoxynucleotides. The orientation of the contigs and the directed strategy was aided by a previously established EcoRI restriction map (unpublished data). Assembly of some regions was complicated by the presence of minisatellites and interspersed repetitive elements. Editing after visual inspection was employed to solve these problems. The error frequency was estimated to be 7 * 10-4 by comparison of independently assembled sequences derived from the overlapping segments of the lambda clones. We noted 14 nucleotide differences out of 1730 nucleotides when the 3' end sequence of Xist was compared to that previously published (34 ). Some of these differences may be due to DNA polymorphisms between the outbred ICR * Swiss Webster strain used by Brockdorff et al. (34 ) and the C3H strain present in the original YAC clone used by us (35 ).


Table 1 . Summary of primer sequences used to define markers.


Figure 1. Chromosomal localization and organization of the region containing the 94 kb sequence. Lambda clones are respectively: A = [lambda]1, B = IVC6, C = IB6, D = IIIF9, E = IVC3, F = IIIC2 and G = IG10, according to the nomenclature used by Rougeulle et al. (28). EcoRI fragment sizes are given above the lambda clones.

Sequence features

Seven new microsatellites were found (DXPas36-DXPas42, see Fig. 1 ) in addition to the previously identified DXPas29 marker (27 ). Table 1 lists the repeat containing sequences analysed by PCR and the size variations observed in various strains. One microsatellite lies within the Tsx gene (DXPas42). The DXPas42 marker will be useful for expression studies, for example in the case of transgenic animals where various donor/recipient strain combinations will be exploited (36 ). These new markers will be valuable given the well documented reduction in frequency of polymorphic markers on the X chromosome (37 ,38 ).

Since there is genetic evidence for the Xce locus being localized distally to the previously DXPas29 marker (27 ) (Simmler, M.-C., Cattanach, B. and Avner, P., unpublished data), we used those microsatellites which were localized beyond the DXPas29 marker for analysing linkage with the various inbred strains carrying one of the three allelic variants. None showed complete linkage with Xce in the various inbred mouse strains tested.

Three unique minisatellites, all located 20 kb downstream of the Xist sequence, were also identified (Fig. 1 ). The minisatellites are respectively composed of a 62 * 17mer, a 29 * 34mer and a 5 * 19mer (see consensus in Fig. 1 ) with the number of reiterations given according to the sequence obtained from the cloned DNA. The 34mer and the 17mer minisatellites show length polymorphism between Mus mus musculus and Mus mus domesticus strains (data not shown). None of these micro- or minisatellites is conserved in the human genome as determined by PCR and hybridization analysis.

The length of the 17mer array obtained from the cloned DNA is shorter than the length of the sequence found in the genomic C3H derived DNA as estimated by genomic restriction pattern. The observed number of repeats is 62 and the expected number estimated to be approximately 150. The 17mer array appears to be intact on YAC PA-2 (see Fig. 1 ), suggesting that deletion has occurred during the subcloning process. Interestingly, the G + A rich strand of the 17mer repeat unit is composed of numerous zeste protein binding sites (TGAGTG). These lie between position 9277 and position 8241 (on the reverse strand, defined according to the orientation of the contig 5' Xist-3' telomere). Zeste proteins are known to be involved in heterochromatinization in Drosophila (39 ,40 ).

The G + C rich 34mer minisatellite is composed of a number of tandem reiterations corresponding to the estimated fragment length (29 repeats). However, since the cloned minisatellite sequence is split across two lambda clones (A and B) with a very short overlap (two repeats), ambiguity at the level of plus or minus one repeat remains at the junction. This G + C rich 34 mer minisatellite and the nearby SalI site have previously been described as being associated with specific hypermethylation of the active X chromosome (41 ) (see below).

CpG islands found within the promoter regions of some genes (42 ) can be defined by two criteria: a high G + C content and a high CpG/GpC index (43 ). Clustering of rare cutting restriction sites is often also found (44 ). We have identified two putative CpG islands at positions 16 373-16 803 (430 bp) and positions 32 860-33 120 (220 bp) (Fig. 2 A). The 430 bp CpG island is found at the edge of the G + C rich 34mer minisatellite (14 957-15 978) which has itself a high CpG/GpC index due to the presence of a CCGG HpaII restriction site within the repeat units (see Fig. 3 A). No rare cutting restriction sites with the exception of a nearby SalI site (see Fig. 3 A) were identified in the proximity of the 34mer minisatellite. The core of the 34mer repeated sequence contains several E-a-H-box sites (EaHboxrev, CAGGTCC) found in the Major Histocompatibility Complex class II A[alpha] gene (45 ).


Figure 2. Sequence features within the 94 kb sequence using the task manager Champagne (121). (A) Predictions of CpG islands zooming the region containing the two putative CpG islands. A green spot means a CpG island defined by having a moving average of observed over expected CpG > 0.6 and a moving average of percent G + C > 0.5. (B) Predictions of interspersed repetitive sequences B1, B2 and L1, in each orientation. (C) Compositional profile representing the frequencies of A, C, G and T.


Figure 3. Sequence features within the 94 kb sequence using Xnip (121). (A) Positions of restriction sites. (B) Positions of simple repeats. (C) Positions of potential modular elements (59) (on the direct or the reverse `-rev' strand): SARa1/AATAAAYAAA//SARa2/WADAWAYAWW//; SARt1/TTWTWTTWTT//SARt2/TWWTDTTWWW//; MAR/AATATTTTT//; TopoII/GTNWAYATTNATNNG/RNYNNCNNGYNGKTNYCY//; ArsCs/WTTTAYRTTTW//; PurBinding/GGNNGAGGGAGARRRR//. (D) Positions of constitutive and inducible promoter consensus sequences (60) (on the direct or the reverse `-rev' strand): GCBox/KRGGCGKRRY/KGGGCGGRRY//; CAATBox/GGYCAATCT/AGCCAAT/GCCAATGA//; NFE4/RAGAGGRGG//; EaHbox/GGACCTG//; Bglobin /CCACACCC//; Pyr/TTTCCTTTCCTTTC//; Ant/TAATAATAATAATAA//; zeste/TGAGTG//.

Computer analysis identified a number of known repetitive elements interspersed within the region. Overall, 1.8% of the sequence shows homology to LINE-1 (Long Interspersed Elements) or partial LINE-1 elements and 8.5% shows homology to SINEs (Short Interspersed Elements) or B1 and B2 repeats. There is no clear tendency for clustering of B2 repeats in either the same or a reverse orientation. A slight tendency for clustering of B1 elements in a reverse orientation is visible within the central part of the contig in the vicinity of the Tsx transcription unit (Fig. 2 B). Overall, these results are compatible with in situ hybridization data showing that LINE and SINE sequences are not enriched in specific chromosomal bands on the mouse X chromosome, as they are on autosomes (46 ).

The nucleotide composition of the region is 58.1% A + T (A = 30.4%, T = 27.7%) and 41.8% G + C (G = 21.1%, C = 20.7%). This is slightly higher than the predicted A + T content for the mouse genome overall (55% A + T and 45% G + C) and approaching that of the human genome (60.7% A + T and 39.3% C + G) (47 ,48 ). The observed dinucleotide frequencies were in close agreement with the expected frequencies with the exception of the well established underrepresentation of CpG dinucleotides in higher vertebrates (49 -51 ). A composition profile is presented in Figure 2 C.

It has been recognized that very long oligopyrimidine and oligopurine tracts are well represented in mammalian genomes (52 ,53 ): we identified numerous mononucleotide repeats (Fig. 3 B). All are potential sources of length variants (54 ). There is no evidence of clustering of simple repeats, although there is a tendency for An or Tn to be overrepresented towards the telomeric end of the contig. The positions of simple purine, pyrimidine and alternating purine/pyrimidine stretches are indicated on Figure 3 B. Interestingly, one type of polypyrimidine tract (CCTTT)n with n = 15 on the forward strand at position 71 896 (Pyr, see Fig. 3 D) has also been described in the [beta]-globin complex (47 ,55 ) as well as in the intronic sequences of numerous genes.

The presence of characteristic bends or folds in the proximal region of the inactive X chromosome at metaphase has previously been reported (56 -58 ). However, sequence analysis with the GCG program Findpatterns (Genetics Computer Group, Inc., Madison, WI 53711) looking for the presence of potential modular sequence elements (59 ) showed no obvious concentration of the consensus sites usually associated with bending domains or functionally defined open chromatin regions (Fig. 3 C).

Finally, Figure 3 D shows the localization of some constitutive and inducible promoter sites using the TFD resource (Release 7.4) (60 ,61 ). The most notable of these is a group of four GC boxes (Sp1 binding sites) within a 211 bp sequence at position 58 996 on the reverse strand (GCboxrev). Northern blot analysis using adult and embryonic tissues (see Materials and Methods) of all predicted coding sequences on either strand in the proximity of this putative promoter region has yet to identify a transcribed sequence.

Homology with non-coding sequences

The 94 kb genomic sequence, masked for SINEs and LINEs was compared with the Primate division of Genbank (release 94, April 15, 1996) containing 55 318 entries for a total of 54 427 930 nucleotides. We used Blastn 1.4 (62 ) in a `sensitive' mode (W = 5) run in parallel on a pool of local workstations (63 ). Alignments with minimal scores of 200 (S = 200, S2 = 200, p >10-6) were retained for further analysis. Matches with a total of 1936 different Genbank entries were reported. Using a set of filtering tools (63 ), we determined that the vast majority of those matches were due to microsatellite sequences (e.g. CACACA...) or middle repetitive elements. Four matches with unique target sequences were retrieved: three of them were found within exon VI of the human XIST gene (accession no. M97168; at positions: 15 761-15 827, 77% identity; 15 490-15 616, 76% identity; 14 691-14 747, 84% identity) and the fourth was identified within an intron of the human coagulation factor VII gene (accession no. J02933). The latter was found to be a minisatellite marginally homologous to the 17mer array.

Identification of new transcripts

We searched for homology to previously identified coding sequences in dbEST (64 ) using Blastn (62 ). The search yielded a 245 nucleotide long perfect match with a mouse EST (accession no. R74734, Fig. 4 A). This EST is localized 2873 bp from the end of the existing mouse Xist sequence (accession no. U41394) (23 ) and was clearly homologous to the eighth and last exon of the human XIST gene (accession no. X56196). This EST also contains a canonical polyadenylation signal site. Figure 4 shows the alignment of all the three sequences: the 94 kb contig (94 kb), the murine EST R74734 (EST) and the human XIST-d clone X56196 (XIST-d). Sequencing the original cDNA clone (MDB0905) reveals that the identity with our 94 kb sequence extends over 104 nucleotides lying beyond the EST sequence itself, which are also partly homologous to the human XIST-d sequence. Although we were not able to precisely define the 5' limit of the murine eighth exon of the Xist gene, this extension of the EST sequence generates a 349 nucleotide long sequence, partially matching with the 377 nucleotide long human XIST-d sequence. We therefore consider this sequence to be a strong candidate for the eighth exon of the murine Xist gene (34 ). We have not been able to definitively identify the seventh exon of the murine Xist gene using data bases search or cDNA sequencing, suggesting this exon may be poorly transcribed.


Figure 4. Alignments of our 94 kb, the mouse EST and the human XIST-d sequences. (A) Blastn matches between the mouse EST (Accession No. R74734)(EST) and the 94 kb contig (94 kb) sequences (occurrences of N undetermined nucleotides in the EST sequence accounted for an identity score of 95%). (B) Blastn matches between the 94 kb contig (94 kb) and the HUMXIST-d (accession no. X56196) (XIST-d) sequences. Boxes represent homologous regions in all three sequences.

Candidates for internal protein coding exons were identified using various programs: Grail (65 ) (Version 1a and 2), xGrail (Version 1.2) (66 ) and a combined approach using hexamer statistics and splice site consensus search (67 ). In parallel, exon identification by similarity search was attempted using Blastx (62 ,68 ) to scan public protein sequence databases. No significant match was found when the ubiquitous and low entropy repeats were properly masked (69 ). Sixteen original candidates were retained. PCR products generated from these 16 potential coding sequences were tested as probes on northern blots containing RNA from various mouse tissues including embryonic and adult material.

Two PCR products from the same strand (corresponding to exons 1 and 5, see below) detected a unique testis specific 1 kb mRNA (Fig. 5 A). No other expressed sequences were revealed with the remaining potential exons, by this method.


Figure 5. Expression analysis and gene organisation of Tsx. (A) A mouse multiple tissue (on the right of the figure) northern blot (Clontech) was hybridized with a mouse PCR product corresponding to exon 1. Schematic EcoRI restriction and exonic maps of the 94 kb contig are given on the left of the figure. The length in kb is indicated on a schematic scale. (B) Genomic organization of Tsx (RI: EcoRI sites).

Isolation and analysis of the Tsx gene

A mouse testis cDNA library constructed by Mitchell et al. (70 ) was screened with the PCR probes derived from the two positive predictions and three independent clones were isolated and sequenced. This generated 663 bp of sequence corresponding to the 3' end of the transcript. No cDNA clone was isolated from the 5' end due to the presence of an EcoRI site within the transcript unit which corresponds also to the cloning site used to construct the cDNA library. However, we were able to identify the 5' end of the gene by 5' RACE (Rapid Amplification of cDNA ends). The sequence of the 5' end RACE product was found to be identical to the genomic sequence. The compiled sequence spans 794 bp including a 432 bp ORF. Since testis specific expression has been observed for this gene, we have called this gene Tsx, for testis specific X linked gene.

Analysis of the sequence surrounding the potential translation initiation site showed poor homology to the Kozak consensus sequence (71 ). Moreover, the predicted 71 bp 5' untranslated region (5' UTR) does not contain any stop codons. We used Promoterscan (72 ) to search for a recognizable promoter signature within the genomic sequence 5' to the first exon of the Tsx transcript, but none was found. However, we noted a GC box like structure (Sp1 binding site) 76 bp upstream of the transcription start site. A potential polyadenylation signal (AATAAA) is found at position 794 bp, preceding the poly(A) tail. The transcription of the Tsx gene was found to be in the reverse orientation to the Xist gene (Fig. 5 A).

The largest ORF predicts an acidic protein of 144 amino acids with a molecular mass of 15 873 daltons. The small size difference between that estimated by northern blot hybridization analysis for the mRNA length, and the 794 bp deduced cDNA sequence may be accounted for by a long poly(A) tail. The size difference is unlikely to be due to the presence of another unknown exon given that the homologous rat gene spans the same size (see next section). A database search found no significant identity with known genes.

The genomic organization of Tsx is shown in Fig. 5 B. It is composed of seven exons and covers approximately 10 kb, with the 3' end situated 40 kb from the last exon (see above) of the Xist gene. The average size of the exons is 82 bp, varying from 36-165 bp. All the splice site junctions are good matches with the donor/acceptor splice consensus sequences with one exception. The non-consensus splice junction (underlined in Table 2 ) has a GC instead of a GT at its donor site (73 ) and on the acceptor site, a lower number of pyrimidines than expected for a gene encoding a protein (74 ).


Table 2 . 5' and 3' splice sites detected in the Tsx transcript

To rule out any X linked regional gene duplication, hybridization experiments using enzyme restricted genomic DNA or nested, deleted YAC fragments derived from the same region (75 ) were carried out. No duplication of the Tsx gene was detected (data not shown).

Gene conservation analysis

Northern blot analysis of rat and human testis mRNAs using the murine Tsx cDNA as a probe under low stringency conditions revealed no human counterpart, although a 1 kb band was detected in rat uniquely in the testis (Fig. 6 ). This confirms the tissue specificity of Tsx at least in rodents. An additional faint 1.5 kb band was observed in lanes corresponding to the rat, human and mouse testis RNAs, indicating either an additional Tsx related mRNA or cross hybridization to an unrelated transcript due to the low stringency conditions used in order to preserve cross-species hybridization.


Figure 6. Conservation analysis of Tsx expression. Northern blot analysis of Tsx expression in various species. The northern blot was hybridized with a 663 bp mouse cDNA. Blot contains 3 [mu]g of an equal mixture of brain, liver, and kidney mRNAs from rat (lane 1) and mouse (lane 5) and 3 [mu]g of rat (lane 2), human (lane 3) and mouse (lane 4) testis poly(A)+ mRNAs.

Screening of two different human testis cDNA libraries [(76 ) and Clontech HL1161x] failed to detect a human homologue. Since the mammalian X chromosome is highly conserved (77 -79 ), we tried to ascertain whether the absence of signal observed by hybridizations on human northern blots and cDNA libraries was due to the high level of sequence divergence or to an absence of transcription in human. No X chromosome specific signal was obtained on Southern blots containing human genomic DNAs either from 48,XXXX, 46,XX and 46,XY individuals or from YACs derived from the XIC candidate region (80 ,81 ).

Murine Tsx cDNA clones were used to screen a rat testis cDNA library (Clontech). Two clones were isolated using the 663 bp 3' end murine cDNA as a probe, and a single clone was isolated using a 138 bp murine PCR product located 5' to the EcoRI unique site. Southern blot hybridization analyses of somatic cell hybrid DNA using the 3' end of the rat cDNA clone as a probe have shown that the rat homologue of the mouse Tsx gene is situated on chromosome X: the two rat specific bands (at 7 kb and 5.2 kb using an XbaI restriction digest) are only absent in the two hybrids missing the rat X chromosome (LB330TG3 and LB1040TG1) (data not shown).

The GC frequencies within exons of the rat and murine Tsx genes were 47.6 and 47.7%, respectively. The GC levels of the third codon position of these two genes were 45 and 46.5% whereas the GC levels of the second codon position were 31.2 and 35.4%. These values lie within the lowest window of GC content in the compositional distributions of rodent coding sequences and positions (82 ).

At the nucleotide level, the rat and mouse cDNAs share 79% identity. In the coding region, the identity is 84%. The 5' UTR represents the most conserved region (89%), whereas a lesser degree of identity was observed in the 3' UTR (67%). The 5' UTR of the rat sequence is composed of 71 bp containing an in-frame stop codon upstream of the first translational start site (Fig. 7 ). Several sequence differences were apparent between the Tsx genes of the two species (Fig. 7 ), the most notable of which was a 3 bp deletion in the rat sequence corresponding to a single codon, compensated downstream in the murine sequence by an in-frame 3 bp deletion. All differences between the murine and the rat cDNA have been confirmed by sequencing both strands of independent clones.


Figure 7. Comparison of the rat (RatTsx) and mouse (MoTsx) Tsx sequences at the nucleotide (A) and amino acids (B) levels.

In genes encoding highly conserved proteins, amino acid sequence identity exceeds nucleotide identity due to silent (synonymous) nucleotide substitutions. In poorly conserved proteins, nucleotide similarity may, however, exceed protein similarity. The predicted 144 amino acid protein sequence shows 72% identity and 82% similarity to the mouse protein. There is no particular region which shows a higher percentage of homology in the predicted protein sequences of the two species. Using the equations of Li et al. (83 ), we computed Ks = 0.215 synonymous substitutions over 81 sites and Ka = 0.159 non-synonymous substitutions over 348 sites (Ks/Ka = 1.35). This suggests a rapid evolutionary rate and, therefore, only a moderate level of selective pressure imposed on the protein structure.

DISCUSSION

In order to increase our understanding of the X chromosome inactivation mechanism in the mouse, we have undertaken the genomic sequencing of a selected 94 kb region from the mouse X chromosome inactivation centre. The definition of the primary structure of the DNA in this region may provide insights concerning the function, regulation and gene evolution which could not be determined simply by isolating genes using currently available techniques such as cross-species hybridization, exon amplification or cDNA selection (84 ). The 94 kb region was targeted for three reasons. Firstly, to search for genes in close proximity to Xist which have a discordant X inactivation profile and, therefore, identify sequences potentially responsible for allowing Xist to escape activation on the active X chromosome; secondly, to allow us to search for structural features that might modulate chromatin conformation or epigenetic DNA modification; and finally, to search for new coding or non-coding sequences possibly corresponding to the Xce locus.

The chromosome-based repression of the inactive X chromosome and its transmission from one cell generation to the next in a stable manner are thought to rely partly on chromatin compaction (1 ,84 -88 ). There is also convincing evidence for a role of methylation in maintaining X-linked gene inactivity, at least in mammals (89 ). In addition, a major role in the stable repression of transcriptional activity is attributed to the chromosomal compartmentalization of the inactive X chromosome within the cell nucleus. Many important nuclear functions are well recognized to take place within specific nuclear compartments (90 ,91 ). The XIST/Xist RNA is of particular interest as it is the first gene to be isolated with transcriptional activity within a non-transcribing, compartmentalized X chromosome (25 ,92 ). A direct role for the Xist RNA as an architectural nuclear component has recently been proposed on the basis of FISH analysis (26 ). Although recent results show that the initiation of inactivation over the X chromosome is dependent upon the expression of the Xist gene (21 ) and a 450 kb transgene centered around the Xist gene displays properties of the Xic (94 ), the identity and the nature of the components of the initiation, propagation and memory functions of X chromosome inactivation are still unknown. Therefore, the identification and characterization of all conserved non-coding (93 ) and coding sequences possibly involved in this process remains a central issue in X chromosome inactivation studies.

Search for chromosomal features

We systematically identified nucleotidic signatures potentially related to the regulation of the X chromosome inactivation such as compaction, compartmentalization, long range cis acting regulation or methylation functions. Computer based searches for similarities with known regulatory elements revealed three potential candidates which are currently being tested for functional activity. One long polypyrimidine tract was revealed on the forward strand (Pyr). A similar element has been identified within the [beta]-globin complex known to be partly regulated at the chromatin level (47 ,55 ). In mammals, polypyrimidine.polypurine tracts have been shown in particular to form unusual secondary structures (95 ,96 ). Although there is at present no direct proof of a role in a long range effect on chromatin structure, numerous other genes containing such polypyrimidine.polypurine tracts in their intronic sequences were also identified by computer search of the sequence data bases. One cluster of binding sites for the zeste protein was also identified, on the reverse strand (zesterev). It is known that in Drosophila, clustering of zeste protein binding sites is capable of inducing co-operative interactions between proteins bound to a given promoter site or bound at independent sites, resulting in the joining of two separate DNA molecules known as transvection (39 ,40 ). Although a mammalian zeste homologue has not been identified, there is some evidence for possibly related somatic pairing in mammals as shown by the study of malignant diseases in humans (97 ). Four GC boxes characteristic of Sp1 binding sites have been found on the reverse strand (GCboxrev). Multiple Sp1 elements are known to be associated with promoter regions and to play a key role in preventing histone H1 repression of chromatin (98 ) or in protecting a CpG island from methylation (99 ). Efforts have therefore been undertaken to identify exons in the vicinity of this Sp1 site cluster. However, no coding sequences have so far been found by northern blot analysis.

The presence of characteristic folds in the inactive X chromosome in the Xic region has been previously reported by cytological and in situ analysis (56 -58 ). Examination for putative structural elements such as modular sequences associated with the unwinding of duplex DNA showed that there was no evident gathering of consensus sites usually associated with open chromatin or bent DNA (59 ).

Apart from the previously identified stretch of sites specifically methylated on the active X chromosome, lying within the 34mer minisatellite (41 ), no other clusters of methylation sensitive sites were detected. However, it is known that there are a lower number of rare cutting restriction sites found in mouse DNA as compared to human DNA (100 -103 ). These results confirm both the physical mapping data in the mouse (104 ) and the paucity of CpG islands in the human homologous region (80 ). Interestingly, we have noted within the 34mer minisatellite the occurrence of multiple E-a-H-box (EaHboxrev) previously identified in the conserved promoter region of the Major Histocompatibility Complex class II A[alpha] gene expressed in lymphocytes in a developmentally regulated manner (44 ). It is therefore possible that a transcription unit lies in the vicinity of the 34mer minisatellite. Neumann et al. (105 ) have hypothesized that a hallmark of imprinted genes is the presence of CG-rich sequences that display monoparental methylation and are closely associated with arrays of direct repeats. Deletion by site specific recombination should provide an approach for verifying the role of such sequences.

Finally, several polymorphic markers were identified in the course of the sequencing assembly. None showed linkage disequilibrium with Xce in the inbred mouse strains tested. These results, therefore, did not allow us to reduce the genetic interval containing Xce (27 ). There is no evidence so far for non coding sequences which might correspond to the allelic variants at the Xce locus, within the 94 kb contig.

Search for protein coding sequences

Apart from the identification of the last exon from the murine Xist gene, only one new gene, located 40 kb from the Xist sequence, has been definitively identified within the 94 kb contig. We are presently testing new predictions using a more recent version of the xGrail program (Version 1.3) and searches with updated databases. The structure of the new gene comprises seven small exons and it spans approximately 10 kb of genomic sequence. This gene encodes a 1 kb mRNA and its transcription orientation is opposite to that of the Xist gene.

We also report the cloning and analysis of the rat homologue of Tsx. The DNA sequence comparison with mouse revealed a higher degree of identity at the nucleotide level than at the amino acid level. Moreover, a high rate of substitutions in non-silent positions and a low Ks/Ka ratio suggest that Tsx encodes a protein with a fast rate of evolution as compared with a group of 363 nuclear genes analysed in the rat and mouse genomes (106 ). It is possibly the RNA rather than the protein that is important for function, as for the XIST/Xist gene. Interestingly, it has been previously hypothesized that the rapid evolution of sex determining genes such as the SRY/Sry gene (107 -109 ) and the DAX1/Dax1 gene (110 ) or of the less well characterized DAM genes whose function is still currently unknown (111 ), are related to natural selection due to genomic conflict and speciation (112 ,113 ).

No human or murine EST corresponding to Tsx or a similar sequence has yet been described and no significant identity of mouse/rat Tsx to known proteins has been found. The best match (S = 70, Blosum 62, P = 0.27, Blastp, but not significant) of mouse Tsx is with nucleolin, which might suggest a RNA/DNA binding role. It is mainly due to a large number of acidic amino acids. This homology is not found when using the rat protein sequence.

Evolutionary studies

The sequenced region of 94 kb distal to the Xist gene appears to have a poor gene content with, on average, one gene every 50 kb (114 ). Other properties such as an overall A + T rich composition, occurrences of tissue specific regulated genes, lack of restriction site defined CpG islands, a fast rate of evolution of the Tsx gene, along with the poor sequence conservation of the nearby Xist gene, suggest that these sequences are located in a region that is relatively free of evolutionary constraints (115 ,116 ). This may be a general property of genes within Giemsa positive bands or may be characteristic of the region surrounding the Xist gene.

The known genes found within a larger 300 kb region, encompassing our sequence, show variable inactivation status, with Cdx4 (117 ) and Bpx (81 ) being subject to inactivation and Xist being expressed exclusively from the inactive X chromosome (22 ,24 ). Several regions containing genes mapped in close proximity, which differ in X chromosome inactivation status, have been described in man (117 -120 ). Results presented in this report could be therefore useful as a step in the search for possible boundary elements, governing the transition from genes active on the inactive X chromosome to genes inactive on the inactive X chromosome, in this region of the murine X chromosome (121 ).

There is no evidence of an X linked Tsx homologue in human using Southern blot hybridization of DNA from a YAC contig spanning 1 Mb on the syntenic region of the human X chromosome (80 ) or hybrid cell lines containing several copies of the human X chromosome. In contrast, using very low stringency hybridization conditions, signals attributed to an autosomal location could be observed. Several efforts to isolate the human homologue by PCR from human DNA, using primers designed from the rodent sequences at low annealing temperature, yielded no X-specific product. Taken together, these results suggest that the human homologue for the rodent Tsx gene is likely to be so divergent that it will be difficult to identify. Alternatively, it is possible that it does not exist. Confirmation of one or other hypothesis awaits sequencing of the homologous region in man or the use of gene isolation methods which do not rely on expression such as exon trapping.

Our analysis of the genetic content at the nucleotide level of a 94 kb region immediately distal to the Xist gene in the mouse extends the data concerning the evolution of the homologous XIC/Xic region between man and mouse. A subregional inversion within the X inactivation centre between man and mouse has previously been described within the otherwise well conserved central part of the X chromosome (80 ). This inversion covers at least 600 kb in man and 300 kb in mouse (81 ). At the moment, the limits of the inverted segment on the human X chromosome are defined by the BPX gene on the proximal side and the XIST gene on the distal side; on the mouse X chromosome, the limits of the inverted segment aredefined by the Xist gene on the proximal side and the Bpx gene on the distal side. Figure 8 presents the localization of genes currently known in the region surrounding XIST/Xist. Taking the man/mouse chromosomal inversion into account, the human homologue of the mouse Tsx gene should be localized between CDX4 and XIST. The apparent absence of an X-linked Tsx homologue in man may suggest that the rearrangement within the XIC/Xic region is more complex than previously hypothesized and that this region may be associated with other properties such as chromosomal rearrangements, as in the case of the distal human Xp22.3 region (122 -124 ). Identification of the eighth exon of the mouse Xist gene suggests that the rearrangement or deletion does not include Xist. A comparison of the sequence surrounding the XIST/Xist gene with the homologous sequence in man would extend our knowledge of the evolution and the function of the area.


Figure 8. Schematic diagram of the currently known chromosomal rearrangements within the region surrounding XIST/Xist on the human and mouse X chromosomes. Open boxes indicate the location of genes in the area (the Xpct gene is not yet localized in the mouse). The black box indicates the location of the mouse Tsx gene (the Tsx homologue has not been identified in the human).

MATERIALS AND METHODS

Preparation of sequence templates

Lambda clone DNA (lambdas A-C and E-G) was sonicated, blunt ended by filling-in with T4 DNA polymerase and the Klenow fragment of DNA polymerase I, subcloned in an HincII-cut M13mp18 vector and transformed into competent E.coli SURE cells (Stratagene). White M13 plaques were picked into the wells of microtitre plates containing 150 [mu]l of 2YT broth and grown at 36.5oC for 6 h. The supernatant was recovered by filtration through a millipore filtration plate (PALL). Approximately 2000 recombinant clones per sonicated lambda were isolated for amplification. Screening using random primed lambda and mouse insert DNAs was carried out on ordered phage DNAs immobilized on N+ Hybond membrane (Amersham) after treatment of 20 [mu]l of each supernatant with 1% SDS. DNA sequencing templates were prepared using the trapping magnetic beads method (125 ) (Amersham). Templates for the most distal part of lambda D DNA were generated with the double stranded nested deletion kit (Pharmacia).

Sequencing reactions

The fluorescence labelled modified version of the dideoxy chain termination method was used (126 ). Sequencing reactions were carried out by cycle sequencing (Amersham) in a thermocycler (Perkin Elmer) using the following programme: 95oC for 30 s, 55oC for 30 s, 70oC for 1 min (15 cycles) followed by 15 cycles at 95oC for 30 s, 70oC for 1 min. Gaps in the sequence were closed by selective PCR amplification. Oligonucleotide primers were made from unique sequence on either side of the gap and used in the PCR reaction with either lambda or genomic DNA as template (sequences available on request). The PCR was performed at 94oC for 4 min, followed by 35 cycles at 94oC for 1 min, 55oC for 1 min and 2 min extension at 72oC. After excision of the PCR product from an agarose gel, DNA was purified with the Wizard PCR prep DNA purification system (Promega) or the Prep A Gene DNA purification matrix (Biorad). Cycle sequencing of these products (Perkin Elmer) in a thermocycler (Perkin Elmer) was carried out using the following programme: 96oC for 15 s, 50oC for 1 s and 60oC for 4 min (25 cycles). All reactions were electrophoresed on ABI 373A DNA sequencers (Applied Biosystems Inc.). Raw sequence data was processed using the Staden shotgun package assembly program (xdap and xbap) (33 ) except for lambda G for which we used the DNASTAR's Macintosh version of Seqman (DNASTAR Inc., Madison, Wi 53715). The Trace editor, Ted, incorporated into the sequence assembly program from Staden (32 ), was used for manual trimming of the head and tail from the sequence and for output of the edited sequence.

Data searches and analyses

Nucleotide and dinucleotide frequencies were obtained with the Staden composition program (127 ). The compositional profile represented the frequencies of A, C, G and T nucleotides using a 1000 bp sliding window with window shifts of 100 bp. The CpG islands were predicted with Gardiner-Garden and Frommer method (43 ) using a 200 bp window with window shifts of 100 bp. The profiles representing the nucleotide frequencies, the prediction of CpG islands and the presence of low complexity repetitive sequences were produced using Champagne, a task manager for molecular biometry (128 ). Simple repeats as well as consensus sites for modular elements (59 ) or for promoters (60 ,61 ) were identified using the pattern matching program in the Staden package (127 ).

Isolation of Tsx cDNA clones

Two PCR products that included the putative exon 1 and 5 sequences of Tsx were used to isolate clones from a 129/Sv derived mouse strain testis cDNA library (70 ). Hybridization of mouse Tsx clones to a testis rat cDNA library (Clontech RL3004a) was carried out under cross-species hybridization conditions as described below. The mouse 663pb and the rat 676pb cDNA clones are referenced as pMoTsx and pRaTsx, respectively. For cloning the 5' end of Tsx, we used a Marathon RACE kit (Clontech). All the primer sequences used are available on request.

Hybridization

Standard procedures for hybridization of Southern blots were utilized (129 ), except that transfers were carried out using alkaline conditions, the hybridization buffer consisted of 1 M NaHPO4 (pH 7.0) and 7% SDS and the wash buffer consisted of 0.04 M NaHPO4 (pH 7.0) and 1% SDS at 65oC in 2 * SSC, 0.1% SDS. Southern blot of XbaI-digested DNAs from mouse (BWTG3), rat (HRSD) and mouse * rat hybrids (LB series) and a 694 bp rat cDNA probe were used for the localization of the rat Tsx gene as described elsewhere (130 ). Northern blot containing polyA+ mRNAs from heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis adult tissues (Clontech 7762-1) and from embryonic tissues corresponding to different developmental stages (Clontech 7763-1) were used. Northern blot hybridizations were carried out using the Express Hyb solution (Clontech) as recommended by the manufacturer. For cross species northern blot analysis and cDNA library screening, low stringency conditions for prehybridization and hybridization were as follows: 42oC in 25% formamide, 1% SDS, 1 M NaCl, 5 mM EDTA, 50 mM phosphate buffer (pH 7.2). The blots were washed at 42-60oC in 1-2 * SSC, 0.1% SDS. Genomic PCR derived product or cDNA probes were random prime labelled using the Megaprime kit (Amersham).

ACKNOWLEDGEMENTS

We thank J. Levilliers for kindly providing the human Southern blots, Philippe Millasseau and Simon Nguyen for helpful advice, Angelos Kalogeropoulos for kindly performing statistical analysis, Michele Riviere for technical help, David Beier for kindly providing the MDB0950 cDNA clone and Evie Melanitou, Edith Heard, Claire Rougeulle, Emmanuel Debrand, Amanda Stafford and Georges Guellaën for helpful comments on the manuscript. This work was supported by the CNRS, AFM, GREG 68/94, ARC (France) and the FRSM (Belgium). D.B.C. was supported by NRSA Postdoctoral Fellowship no. 5F32GM17021. M.-C. S. and D.B.C. have contributed equally to this work.

DDBJ/EMBL/GENBANK ACCESSION NUMBERS

The EMBL accession number for the 94454 bp sequence reported in this paper is X99946. The EMBL accession numbers for the mouse Tsx cDNA and rat Tsx cDNA are X99796 and X99797, respectively.

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*To whom correspondence should be addressed

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