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The Journal of Immunology, 1999, 162: 292-298.
Copyright © 1999 by The American Association of Immunologists

High Level Expression of the Xlr Nuclear Protein in Immature Thymocytes and Colocalization with the Matrix-Associated Region-Binding SATB1 Protein

Denise Escalier, Bénédicte Allenet, Anne Badrichani and Henri-Jean Garchon1

Institut National de la Santé et de la Recherche Médicale, Unit 25, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The X-linked lymphocyte-regulated (Xlr) protein is a 30,000 Mr nuclear protein bearing homology with meiosis-specific proteins and expressed in late stage B lymphoid cell lines. In the present study we investigated its expression in the T lymphoid lineage. In adults, a high level of expression was detected in CD4-CD8- thymocytes. Most remarkably, the peak of Xlr expression occurred early during thymus cell ontogeny, precisely on days 14–15 of gestation, and was associated with the first wave of pre-T cell differentiation. Its onset preceded the rearrangement of TCR genes, as Xlr expression was conserved in thymus cells from RAG10/0 mice. The lower expression of Xlr on day 13 of fetal development, the bright Thy1+ phenotype of Xlr-positive cells, their large size, and their absence from subcapsular areas suggest that Xlr expression must be turned on within the thymus and not in prethymic precursors. From day 16 of gestation, Xlr expression decreased markedly. At birth and later, Xlrhigh cells were mostly large cells scattered throughout the cortical area. As shown by confocal microscopy, expression of Xlr closely overlapped that of SATB1, which binds special AT-rich DNA sequences associated with the nuclear matrix and plays an important regulatory role for many genes. The remarkably regulated expression of Xlr in the lymphoid cell lineage and of its homologue Xmr in the germ cell lineage suggests that they might play an important role in chromatin metabolism at critical stages of differentiation during which the genome undergoes irreversible rearrangements.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The X-linked lymphocyte-regulated (Xlr)2 protein is a 30,000 Mr nuclear protein with stage-specific expression in terminally differentiated B lymphoid cells (1, 2, 3, 4). In mice it is encoded by a multigene family located on the proximal half of the X chromosome (1, 5, 6). Its function is not yet known. However, the recent identification of two homologous proteins expressed in the male germ cell line, including Xmr (X-linked, meiosis-regulated) (7) and SCP3/COR1 (8, 9), suggests a role for Xlr in chromatin conformation. Xmr, a 212-amino acid protein, is closely homologous to Xlr (94% identity) and thus also belongs to the Xlr family. Its expression is highly testis specific and occurs in spermatocytes in meiotic prophase I. Most strikingly, it is successively associated with autosome condensation and then with X and Y chromosome condensation in the XY body. SCP3 in the rat and COR1, its hamster equivalent, are more distantly related to Xlr (35 and 40%, respectively) and are structural components of the axes of synaptonemal complexes. A more distant homology of Xlr with the meiosis-specific MER2 gene product from Saccharomyces cerivisiae (10) has also been noted. Finally, in mice, the X chromosome carries another multigene family with sequence similarities to Xlr, designated Xlr3 and also transcribed in late stage B cells and in testis (11).

Expression of Xlr in the lymphoid lineage has been characterized mostly using lines of mouse lymphoid cells arrested at various stages of differentiation (1, 2, 3, 4). In man, its expression is known to be induced in mitogen-activated peripheral blood cells (12). The pattern of Xlr expression, however, remains to be characterized in detail. In the present study we investigated the ontogeny of Xlr expression in the T lymphoid cell lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and fetuses

C57BL/6 and RAG10/0 mice were bred in our animal facility under specific pathogen-free conditions. The age of fetuses was determined based on the day of the appearance of the vaginal plug and was verified by their relative sizes and development (13).

Abs and reagents

Affinity-purified anti-Xlr Abs were raised in rabbits immunized with recombinant Xlr protein (4). The RIK2D3 anti-Xlr mAb, an IgG1 {kappa}, was produced in the mouse (7, 12).

Abs against Thy-1, CD4, and CD8 were purchased from Becton Dickinson (Mountain View, CA). Rabbit anti-SATB1 Abs were provided by Dr. T. Kohwi-Shigematsu (Burnham Institute, La Jolla, CA). Biotinylated Abs against rabbit IgG (from donkey) or mouse IgG (from sheep) and Texas Red-conjugated anti-mouse IgG Abs (from sheep) were purchased from Amersham (Arlington Heights, IL). FITC-conjugated anti-rabbit IgG (from goat) were purchased from Biosys (Compiegne, France). Phycoerythrin- and peroxidase-conjugated streptavidin were obtained from Amersham.

Flow immunocytometry

Freshly isolated thymi were immersed in RPMI 1640 medium supplemented with 10% FCS and gently dissociated. Cell suspensions were passed through a nylon gauze and washed in PBS. For flow cytometric analysis, cells were fixed, permeabilized, and then stained with the RIK2D3 anti-Xlr mAb conjugated to FITC and with anti-Thy1, anti-CD4, or anti-CD8 coupled to phycoerythrin, exactly as described previously (12). The two-color fluorescence signal was analyzed with a FACScan using CONSORT 30 software (Becton-Dickinson, Sunnyvale CA).

Immunocytochemistry and quantitative image analysis

Cells were sedimented onto cover slides coated with poly-L-lysine. Alternatively, thymic prints were produced by applying transversal sections of freshly isolated thymi on glass slides precooled on ice. Cells and prints were fixed immediately for 15 min with 1% formaldehyde in PBS containing 3% sucrose, treated for 5 min with 0.1% Triton X-100 in PBS, washed, and incubated for 1 h in 5% nonfat milk in PBS. They were then incubated with anti-Xlr Abs or with control Igs. Peroxidase labeling was performed following the three-step technique using the biotin-streptavidin system and amino-ethyl-carbazole as the chromogen.

For quantitative image analysis, fields were examined under a Reichert Polyvar microscope (Reichert-Jung, Vienna, Austria) using a x25 objective. They were captured with a Sony 3-CCD color camera (Sony, Tokyo, Japan) and analyzed using a Matrox MVP-NP image processing card (Matrox Graphics, Quebec, Canada) implanted in a personal computer and the cell image processor Samba 2005 (Alcatel TITN, Data Systems Paris, France). This provided a video signal that was digitized into 512 x 512 pixels, 8 bits, corresponding to 256 gray levels. Each image was stored as a three-color component (red, green, and blue). Corrections were made for optical system, objective and illumination, distortions, and background noise. The peroxidase labeling was analyzed using immunoenzymatic reaction analysis software with a nuclear labeling program (version Immuno V3.100, Alcatel-TITN) (14). Overlapping or contiguous cells were rejected. Threshold values were established to distinguish nuclei from the preparation background (segmentation, 30; stained nuclei, 30; stained area, 170). Nuclear parameters for thymocyte nuclei were 4.2 µm for the minimal diameter (corresponding to a 13.85-µm2 area), 11 µm for the maximal diameter (corresponding to a 95.03-µm2 area), and 0.90 for the form factor (outline convexity). The color was adjusted between yellow and red. The results were averaged and were compared with the Mann-Whitney U test, using the Statistica package (Statsoft, Jandel, San Rafael, CA).

Confocal fluorescence microscopy

For immunofluorescence labeling for confocal laser microscopy, Texas Red- and FITC-conjugated anti-IgG Abs were used as the second reagents. Samples were studied with a Leica confocal microscope (Deerfield, IL) that uses an argon-krypton laser operating in multiline mode. Most of the preparations were sequentially analyzed. FITC was excited by the blue 488-nm line, and its emission was detected through an interferential narrow band filter centered at 535 ± 8 nm (Schott, Duryea, PA). Texas Red was selectively excited by the green 568-nm line. The red signal emission was detected through a long wave pass filter RG 590 (Schott).

For each selected cell, six or eight optical sections separated by 0.5-µm steps were recorded through the x63/1.4 NA objective. Every picture resulted from four accumulated frames. The pixel size in the final image was 0.15 µm. Slides were printed from Ektachrome 100 film (Eastman Kodak, Rochester, NY) using a freeze-frame video recorder (Polaroid, Bois Darcy France).

Cell fractionation and immunoblot assay

Isolated thymocytes (see cell and tissue preparation section) were lysed in lysis buffer (10 mM HEPES (pH 7.6), 10 mM NaCl, 3 mM MgCl2, 1 mM ZnSO4, and 0.4% Nonidet P-40) (4). The cytoplasmic fraction was harvested. Nuclei were washed with PBS and resuspended in extraction buffer (10 mM HEPES (pH 7.6), 0.5 M NaCl, and 10 mM EDTA) and pelleted again. The supernatant (extractable nuclear fraction) was mixed with an equal volume of X2 reducing sample buffer (as in 7 . Electrophoresis and Western blotting were then performed as previously described (7).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Xlr in immature thymus cells

A preliminary study of Xlr expression by immunofluorescence labeling and examination of stained cells under the light microscope suggested that, compared with cells grown in continuous lines that had been previously studied, most adult thymocytes and peripheral lymphoid cells expressed Xlr faintly. This low level of expression could be discerned from the background by comparison with bone marrow cells, which yielded no staining (not shown). In addition, a small fraction of thymus cells was brightly labeled. To allow for a quantitative analysis, a flow cytometric assay of Xlr expression in fixed and permeabilized cells was set up. As shown in Fig. 1GoA, the mean peak of anti-Xlr fluorescence was approximately 5 times higher in adult thymus cells than in bone marrow cells. Although the distribution of fluorescence intensities was fairly homogeneous, about 10% of the cells stained brightly for Xlr, up to 5 times brighter than the mean peak. All Xlr-labeled cells were strongly positive for Thy1 and therefore belonged to the T lineage (not shown). We then focused our attention on the characterization of Xlrhigh cells.



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  		FIGURE 1. Flow cytometric analysis of Xlr expression in thymus cells. Cells were prepared from bone marrow (labeled BM in A) or from thymus (A–K; labeled T in A) at 8 wk of age (A–C), on day 18 (D–F), or on day 16 (G–I) of gestation or were a mixture (J–K) in equal amounts of fetal (day 16 of gestation, labeled F) and adult thymus cells (labeled A). In B and C, CD8+ and CD4+ cells were depleted by treatment with the appropriate mAb and rabbit complement. Cells were fixed, permeabilized, and immunolabeled with the anti-Xlr RIK2D3 mAb coupled to FITC and an anti-CD4 or anti-CD8 mAb coupled to phycoerythrin. Data are presented as histograms of anti-Xlr fluorescence intensity distribution (A, D, G, andJ) or as dot plots of anti-Xlr (x-axis) against anti-CD4 or anti-CD8 (y-axis) fluorescence intensity. The Xlr fluorescence cut-off shown in dot plots separates cells expressing high and low levels of Xlr.

 
Double labeling for Xlr and CD4/CD8 surface markers indicated that Xlrhigh cells were enriched in the double-negative thymus cell subset (not shown). This was confirmed by labeling of CD4- or CD8-depleted cells obtained by treatment with the appropriate mAb and complement (Fig. 1Go, B and C). Double-negative cells, which were enriched following such treatment, were stained homogeneously and brightly for Xlr, whereas single-positive cells showed heterogeneous labeling. This finding strongly suggested that Xlrhigh cells could be immature thymocytes, and that Xlr expression was related to early T cell ontogeny. This was fully confirmed by analysis of fetal thymocytes. On day 18 of gestation (Fig. 1Go, D–F) 40% of the cells were Xlrhigh, and on day 16 of gestation (Fig. 1Go, G–I) most cells were stained brightly for Xlr, as shown by labeling of a mixture of adult and day 16 fetal thymus cells in which adult cells were identified by CD4+ labeling (Fig. 1Go, J and K).

Quantitative analysis of Xlr expression in fetal thymocytes

Expression of Xlr was investigated at earlier time points of thymus ontogeny. Because of cell scarcity, immunoperoxidase labeling of fixed cells coated on glass slides was performed rather than immunofluorescence staining and flow cytometric analysis of cells in suspension. The staining was quantitated using Immuno-analysis software (Alcatel Data Systems, Paris, France). This package identifies cell nuclei, measures their surface, and, for each nucleus, determines the OD of the peroxidase reaction product at the pixel level. From the distribution of data thus collected for each nucleus, the mean OD is computed. For a given nucleus, this parameter is a reflection of the average concentration of Xlr protein within the nucleus, whereas the sum of the ODs relates to the total amount or quantity of Xlr protein expressed by this nucleus and corresponds to the fluorescence intensity determined by flow cytometry. Fig. 2GoA shows scatterplots of mean ODs against cell nucleus surface for each cell, measured on 3 consecutive days of fetal life (days 13, 14, and 15) and at birth. The corresponding histograms of the distributions of these two parameters are also shown in Fig. 2Go, B and C.



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  		FIGURE 2. Quantitative image analysis of Xlr expression in fetal thymus cells. Thymus cells were harvested on the indicated day of gestation (E13 to E15) or at birth (NB) in a single experiment and were immunoperoxidase labeled with the anti-Xlr RIK2D3 mAb (O) or with a control isotype-matched IgG1 {kappa} MOPC21 Ig (+). A, scatterplot representation of cell nucleus surface (in square microns, x-axis) against the mean OD of Xlr labeling (y-axis). B, Histogram representation of mean ODs of Xlr labeling. C, Histogram representation of cell nucleus surface distribution.

 
The average concentration of Xlr increased slightly, but very significantly, from days 13 to 15 of gestation and peaked on day 15 (p = 1 x 10-2 between days 13 and 14; p = 2 x 10-12 between days 14 and 15). It then decreased dramatically at birth (p < 10-20 between day 15 and birth). A separate experiment (not shown) indicated that this decrease initiated on day 16 (also compare Fig. 3Go, A and C). Remarkably, between days 13 and 15, the mean Xlr concentration was fairly uniform among the various cells, although their nuclear size varied markedly. In contrast, at birth the Xlr concentration was strongly dependent on the nuclear size; the larger the nucleus, the greater the concentration of Xlr. The nuclear size also evolved markedly during this period. On day 13, it varied up to fivefold, whereas at birth very few nuclei had a size exceeding twice the median size. Altogether between day 13 and birth the median size of the nuclei decreased by a factor of 3. As a result of the high variation in both the mean concentration of Xlr and the nuclear size, the total amount of Xlr per nucleus decreased about 10 times between days 14–15 and birth.



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  		FIGURE 3. High level of Xlr expression in fetal immature thymus cells. Thymus cells were harvested on day 15 of gestation (A,B, and E), on day 16 of gestation (C and F), or at birth (D) from normal C57BL/6 mice (A–D) or from RAG10/0 mice (E and F). Immunoperoxidase staining was then conducted with the anti-Xlr RIK2D3 mAb (A and C–F) or with the control isotype-matched IgG1{kappa} MOPC21 Ig (B). In A–D, bar = 15 µm; in E andF, bar = 10 µm.

 
Fig. 3Go visualizes the aspect of the Xlr labeling on different days. As mentioned above, Xlr staining intensity decreased markedly between days 15 and 16 (Fig. 3Go, A and C), and at birth most cells were only faintly labeled (Fig. 3GoD). Moreover, on days 15 and 16 of fetal development, expression of Xlr was not affected by inactivation of the RAG1 gene (Fig. 3Go, E and F). The product of this gene is an essential component of the recombinase complex that promotes the somatic rearrangement of TCR loci. Its inactivation blocks T cell development at an early stage (15). Our result therefore indicates that onset of Xlr expression precedes TCR gene rearrangement.

Interestingly, the nuclei that expressed the highest levels of Xlr had a heterogeneous pattern of labeling. Strong staining emerged from a background labeling; it predominated at the periphery close to the nuclear membrane with sinuous, cord-like expansions inside the nucleus. Computer-assisted image analysis confirmed this idea of a relationship between the intensity of Xlr staining and its heterogeneity, as among nuclei there was a good correlation between their mean OD and dispersion parameters (not shown).

Topology of Xlr expression in fetal thymus

The thymus is a highly compartmentalized organ through which developing T cells migrate in a centripetal manner as they reach successive stages of differentiation (16). To seek a possible relationship between the pattern of Xlr expression in maturing thymocytes and their position in the thymus, thymus prints were immunostained for Xlr. As shown in Fig. 4GoA on day 15 of fetal development, the most peripheral cells, which are at an earlier stage of development, were relatively weakly stained. So were the cells in the central region. The most intensely labeled cells lay in the intermediate layer of the thymus section print. This pattern was even more obvious on day 18 of gestation and after birth (Fig. 4Go, C and D), when there is greater heterogeneity of Xlr expression. Strongly Xlr-positive cells were scattered throughout the cortical region, whereas cells in the subcapsular and central areas were hardly labeled for Xlr. Importantly, stromal cells were negative for Xlr. They were surrounded by several lymphoid cells, of which most often one or two were strongly Xlr positive (Fig. 4GoA, inset).



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  		FIGURE 4. Xlr staining of thymus prints. Thymus prints were prepared on day 15 of gestation (A and B), on day 18 of gestation (C), or on day 5 after birth (D). Prints were incubated with affinity-purified anti-Xlr rabbit Abs (A, C, andD) or with preimmune rabbit IgG (B) followed by anti-rabbit IgG biotinylated Abs and streptavidin-peroxidase. The inset shows the lack of staining of epithelial cells. Bar = 50 µm.

 
Molecular characterization of thymic Xlr

Western blot analysis of day 15 thymus cell extracts disclosed a single molecular species of 30,000 Mr (Fig. 5GoA, lane 3). It migrated as the larger of the two molecular species previously recognized in myeloma cells (lane 2), the smaller of which migrates as the recombinant Xlr protein expressed in Escherichia coli (lane 1). Sequencing of the amplified cDNA synthesized from day 15 fetal thymus RNA revealed a unique sequence, identical with that previously determined in lymphoid cells. The thymus Xlr protein therefore very likely represents a post-translationally modified form of Xlr. The nature of the post-translational modification, however, is not known.



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  		FIGURE 5. Western blot detection of Xlr expression in thymus cells. Nuclear extracts were obtained from day 15 fetal thymus cells. Recombinant Xlr protein (1 ng; lane 1), SP2/0 myeloma cell extracts (lane 2), and thymus cell extracts (lane 3) were migrated on a 12% denaturing PAGE and blotted on a nylon membrane. The membrane was incubated with affinity-purified anti-Xlr rabbit Abs (A) or with preimmune rabbit IgG (B).

 
Colocalization of Xlr and SATB1

As shown above by immunoperoxidase labeling, the most intensely labeled Xlr thymus cells displayed a heterogeneous pattern of intranuclear labeling. A similar pattern was obtained by immunofluorescence staining (Fig. 6GoA). It was remarkably similar to that of SATB1 (Fig. 6GoB), another nuclear protein that is abundantly expressed in the thymus and binds special AT-rich DNA sequences associated with the nuclear matrix (17). Cells expressing high levels of Xlr stained brightly for SATB1 and reciprocally (compare Fig. 6Go, A and B). Moreover, in each nucleus, analysis of serial optical sections after signal normalization showed a perfect overlap of Xlr and SATB1 fluorescence signals (Fig. 6GoC, bottom) and a point for point correlation (Fig. 6GoC, top). Thresholding to select the most intensely labeled pixels indicated that the overlap was not coincidental (not shown). However, due to the marked instability of Xlr during cell extract preparation, coimmunoprecipitation experiments could not be performed to definitively demonstrate an association between Xlr and SATB1.



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  		FIGURE 6. Colocalization of Xlr and SATB1 in thymus cell nuclei analyzed by confocal microscopy. Day 14 fetal thymus cells were doubly labeled for Xlr (A) using an anti-Xlr mouse mAb followed by Texas Red-conjugated sheep anti-mouse Ig Abs, and for SATB1 (B) using a rabbit anti-SATB1 immune serum (provided by Dr. Kohwi-Shigematsu) followed by FITC-conjugated goat anti-rabbit IgG Abs. C, Overlap of normalized SATB1 and Xlr fluorescence signals. Top, Correlation at the pixel level between SATB1 (x-axis) and Xlr (y-axis) staining in a representative nucleus. Bottom, Serial sections of the same nucleus. Overlap of Xlr (red) and SATB1 (green) generates a largely predominant yellow color.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have characterized a remarkably regulated and high level expression of the nuclear Xlr protein in immature fetal thymocytes. The peak of expression was on days 14–15 of gestation, both in terms of the proportion of cells expressing Xlr and of the amount of Xlr protein detected in their nuclei. At this stage of thymus development, thymus lymphoid progenitors expand rapidly and differentiate into pre-T cells in an ordered and synchronous manner (16, 18, 19). They are characterized by the lack of the three T cell surface markers, CD3, CD4, and CD8. At the molecular level, the most remarkable event is the initiation of TCR gene rearrangement to produce functional loci. In mice deficient for RAG genes, T cell development is arrested due to lack of TCR ß-chain locus rearrangement (20). The timing of Xlr expression in normal fetal thymus cells and its conserved expression in RAG10/0 mice therefore indicate that Xlr expression is associated with the first wave of T cells, at the pre-T cell stage, before the rearrangement of TCR loci.

This pre-T cell differentiation stage itself is complex, however, and intermediate steps have been defined, based on the down-regulation of CD117 (c-Kit) and CD44 and on the transient expression of CD24 (heat-stable Ag) and CD25 (IL-2R {alpha}-chain) (21). Thus, in RAG10/0 mice, thymocyte development is blocked at the CD44-CD117-CD25+ stage (20). To situate Xlr expression along this differentiation pathway more precisely, the study of mice bearing other mutations, such as an inactivated TCRß locus, or the scid mutation (affecting the DNA-dependent protein kinase, which is a component of the recombinase complex), that arrests early T cell development (20, 22) should be most instructive. Also, the time of onset of Xlr expression remains to be characterized. The lower expression of Xlr on day 13 of gestation and its marked heterogeneity compared with those observed on days 14 and 15 suggest that its onset must occur approximately at this time of thymus cell development and not much earlier, for example in prethymic precursors. The high level of Thy1 expression on Xlr+ cells and their topographical distribution in the thymus, being exclusive of subcapsular areas, also support the idea that Xlr expression must be turned on in thymus cells. However, this remains to be formally demonstrated.

From day 16 of gestation, the proportion of cells expressing high levels of Xlr decreases dramatically, and after birth these represent only a minor fraction of the whole thymus cell population. Remarkably, however, these Xlrhigh cells are among the largest cells, and reciprocally the largest cells are Xlr positive. They are scattered throughout the thymus. This topographical distribution is reminiscent of that described for large CD25+ cells (23, 24) and seems to be related to the cell proliferation pattern, as suggested by experiments of thymus repopulation after irradiation (25). Thus, in adult thymus, Xlrhigh cells are likely to be a qualitatively important, even though numerically minor, subset.

The significance of the heterogeneity of Xlr nuclear distribution in Xlrhigh cells is still unknown. However, the colocalization of Xlr and SATB1 provides potential clues. SATB1 is an abundant component of the nuclear matrix, originally identified in adult thymus cells (17) but also present in fetal thymocytes, as shown for the first time by our study. It has 763 amino acids and a Mr of 90,000-110,000. It binds selectively to special AT-rich sequences with high base-unpairing propensity that are associated with the nuclear matrix (26). Binding of SATB1 to these matrix-associated regions of DNA requires a 150-amino acid domain and is also enhanced by an atypical homeodomain of SATB1 (27, 28). The regulatory regions of several genes, including A{gamma}-globin, CD8, and the long terminal repeat of mouse mammary tumor virus, have been shown to contain matrix-associated regions that bind SATB1 (29, 30, 31). Experiments in transgenic mice and in transfected cells suggest that SATB1 generally exerts a negative regulatory effect on gene expression (27, 30).

This colocalization of Xlr with a component of the nuclear matrix exerting a regulatory effect on gene expression and its peak of expression coincident with the initiation of TCR gene rearrangement during thymus cell ontogeny take on particular significance in light of its sequence similarity with three proteins involved in meiosis, including SCP3/COR1 (8, 9), Mer2 (10), and Xmr (7). We propose to group these proteins in a novel superfamily, designated the Xlr superfamily after its first described member. SCP3 has been characterized both biochemically and at the ultrastructural level as a component of the synaptonemal complexes in rodent spermatocytes. The MER2 gene has been identified in yeast. In MER2 null mutants, meiotic interchromosomal gene conversion, crossing over, and intrachromosomal recombination are abolished (32). Consequently, formation of meiosis-specific double-stranded breaks is prevented, and alignments of homologous chromosomes are significantly reduced.

Finally, the Xmr protein is the testis-specific homologue of Xlr. Its expression is precisely regulated during meiotic prophase I in spermatocytes. It begins at the preleptotene stage and is first associated with autosome condensation. At midpachytene and subsequently, when autosomes decondense, Xmr expression is repressed, except in the area of the XY body, in which sex chromosomes condense in a delayed manner relative to autosomes, from midpachytene to diplotene. In this restricted area of the spermatocyte nucleus, Xmr was found associated with the chromosomal axes (7). Modification of the chromatin condensation state during the meiotic prophase is related to chromosome pairing and formation of synaptonemal complexes with ensuing recombination events (33).

Thus, in both the lymphoid and the germ cell lineages, proteins of the Xlr superfamily are expressed at high levels at stages of differentiation during which, most remarkably, the genome undergoes irreversible rearrangements. Not surprisingly, recent progress in the dissection of the molecular machinery underlying these processes has disclosed genes used in common in each lineage, notably ATM (34) and RAD51 (35, 36). Our data strongly suggest that Xlr/Xmr may belong to this category of gene products playing a role at similar steps affecting DNA metabolism or chromatin conformation in each lineage. The evidence for this hypothesis, however, is still indirect, and functional studies are now needed to support it.


   Acknowledgments
 
We thank Dr. T. Kohwi-Shigematsu (Burnham Institute, La Jolla, CA) for generously providing the anti-SATB1 Ab, Dr. D. Schoevaert (Laboratoire de Microscopie Quantitative, CHU Bicêtre, Kremlin-Bicêtre, France) for guidance with the automated quantitative image analysis program, and Mr. R. Hellio (Institut Pasteur, Paris, France) for help with the laser confocal microscope.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Henri-Jean Garchon, Institut National de la Santé et de la Recherche Médicale, Unit 25, Hôpital Necker, 161 rue de Sèvres, 75743 Paris Cedex 15, France. E-mail address: Back

2 Abbreviations used in this paper: Xlr, X-linked lymphocyte-regulated; Xmr, X-linked, meiosis-regulated; RAG, recombinase-activating gene. Back

Received for publication July 15, 1998. Accepted for publication September 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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