ORIGINAL PAPER

Dominique Rocha ? Alice Carrier ? Marianne NaspettiGenevieve Victorero ? Elizabeth AndersonMark Botcherby ? Jean-Louis GuenetCatherine Nguyen ? Philippe NaquetBertrand R. Jordan

Modulation of mRNA levels in the presence of thymocytes and genome

mapping for a set of genes expressed in mouse thymic epithelial cells

Received: 16 December 1996

AbstractmModulation of gene expression in mouse thymicepithelium upon culture in the presence of thymocytes(coculture) was studied by comparison of hybridizationsignatures on a set of nearly 5000 mouse thymus cDNAclones. Forty-nine differentially expressed clones (usuallydown-regulated in coculture) were characterized by tagsequencing. Many of them corresponded to entities thathad not been described previously in the mouse, and werefurther characterized by genome mapping. This set of genesappears to be involved in growth regulation and differentia-tion within the thymus.

Introduction

Now that most human genes are at least partially character-ized by sequence information (for a recent review seeHoulgatte et al. 1995), a global understanding of cell andtissue function requires collection on a similar scale ofexpression data and genome mapping information. Map-ping of human ESTs is in progress on a large scale usingradiation hybrid technology (Boguski and Schuler 1995),and results for a first set of more than 16 000 sequenceshave been presented (Schuler et al. 1996). Expression dataare being obtained, again mostly in the human system,using primarily two methods: clone counting either in

specifically designed systems (Okubo et al. 1992) or asan afterthought (Adams et al. 1995), and collection ofhybridization signatures on high-density arrays of clonesor DNA (Zhao et al. 1995; Nguyen et al. 1995; Schena et al.1995; Pietu et al. 1996).

The mouse genome has so far been less extensivelystudied, although a detailed genetic map is now available(Dietrich et al. 1996); a mouse EST project has beeninitiated recently. We chose to concentrate on mousegenes expressed in the thymus, the major location for T-cell maturation, positive selection of T-cell clones thatdisplay adequate specificity and elimination of those thatare unreactive or autoreactive (for a recent review seeGodfrey and Zlotnik 1993). Thymocytes enter the thymusand progress through a series of stages during which theyexpress successively a number of surface markers andfinally emerge as mature T cells bearing either the CD4or the CD8 marker. Throughout this process, thymocytesinteract extensively with cortical and medullary epithelialcells via surface molecules and cytokines, in a bidirectionalprocess that influences both thymocyte and epithelial celldifferentiation (Ritter and Boyd 1993). Knock-out mice inwhich expression of the T-cell receptor is blocked displaynot only failure of thymocyte maturation but also a highlyabnormal thymus structure (Philpott et al. 1992; Malissenet al. 1995), indicating the extent to which thymocytesinfluence thymus development. In SCID mice, where T-celldevelopment is interrupted at an immature cortical stage,thymic medulla does not develop. Upon reconstitution bynormal bone marrow precursors, the entire medulla com-prising normal stromal cell type reappears (Shores et al.1991).

To further investigate the molecular events underlyingthese processes, thymic medullary epithelial cell lines havebeen derived and used in in vitro coculture assays (Naquetet al. 1989; Meilin et al. 1995). The growth characteristicsof epithelial cell lines are modified by contact with thymo-cytes, with an end result that can be either stimulation orinhibition depending on the coculture conditions (Meilinet al. 1995). In the case of the MTE-1D medullary epithelialcell line (Lepesant et al. 1995), this cell contact leads in

D. Rocha? A. Carrier ? G. Victorero? C. Nguyen? B.R. Jordan ( )Genome Structure and Immune Functions Laboratory,Centre d’Immunologie INSERM-CNRS de Marseille-Luminy (CIML),Case 906, 13288 Marseille, Cedex 9, France

M. Naspetti? P. NaquetLympho-stromal cell interactions Laboratory (CIML), Case 906,13288 Marseille, Cedex 9, France

E. Anderson? M. BotcherbyUK HGMP Resource Centre, Hinxton, Cambridgeshire, UK

J.-L. GuenetInstitut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15,France

Immunogenetics (1997) 46: 142–151 Springer-Verlag 1997

addition to morphological and phenotypic changes that canbe detected by monoclonal antibodies (M. Naspetti and co-workers, unpublished data). We therefore decided to in-vestigate this situation at the transcriptional level usingquantitative differential hybridization, which can providesignals directly proportional to the abundance of individualmRNA species (Nguyen et al. 1995), and to characterizegenes whose expression is modulated in this situation.

Materials and methods

Cell line, culture conditions, and RNA isolation

MTE-1D is a mouse thymic epithelium cell line established fromprimary stromal cell cultures from fetal day-16 thymuses of Swiss mice(Naquet et al. 1989). MTE-1D cells were grown in Dulbecco’smodified Eagle medium containing 10% fetal calf serum, 1 mMsodium pyruvate, 50µM 2-mercaptoethanol, and penicillin/streptomy-cin at 200µg/ml. For the coculture condition, MTE-1D cells weregrown in the presence of thymocytes, freshly extracted from thethymus of NMRI mice, 3–4 weeks old, added in the ratio of 25thymocytes for one MTE-1D cell. In both cases cells were grown toconfluence. Before harvesting, thymocytes were completely eliminatedfrom the adherent MTE-1D cells by washing. Total cellular RNAs wereextracted using Trizol reagent (Gibco-BRL, Bethesda, MD).

cDNA library and high-density filters

High-density colony filters (containing 1536 clones each) were pre-pared by spotting on Hybond-N filters (Amersham, Little Charlfont,UK) with a Biomek 1000 robot (Beckman, Fullerton, CA) cDNAclones from an adult mouse thymus cDNA library as described byNguyen et al. (1995).

Design of the experiment for quantitative differential screening

For each cDNA complex probe, two duplicate filters were used; thefilters were hybridized successively with the two complex probes. Toeliminate the effects of differential growth of clones between duplicatefilters and of filter stripping between the two complex probe hybridi-zations, new filters were first hybridized with an oligonucleotide proberecognizing the vector used, “softly” stripped (see below), and hybri-dized with the first cDNA complex probe. For the second complexprobe hybridization, the filters were “harshly” stripped, rehybridizedwith the oligonucleotide probe, softly stripped and hybridized with thesecond complex probe.

Oligonucleotide probe labeling and hybridization conditions

The oligonucleotide used for vector normalization has the followingsequence: 59-GCTTATCGAAATTAATACGACTCACTATAG-39. Oneµg of oligonucleotide was labeled with (gamma-32P)ATP using stan-dard methods (Sambrook et al. 1989). High-density filters wereprehybridized for 3 h at 42°C in 6 × Standard sodium citrate (SSC),5 × Denhardt’s reagent, 1% sodium dodecyl sulfate (SDS), 100µg/mlsheared denaturated salmon sperm DNA, and then hybridized withlabeled oligonucleotide (at 300 000 cpm/ml) and unlabeled oligonu-cleotide (at 200 ng/ml) overnight. Filters were washed twice for 2 minat room temperature and once for 5 min at 42°C with 5 × SSC, 0.1%SDS, then exposed with phosphor screens for 3 h.

Stripping conditions

High-density filters hybridized with the oligonucleotide probe werestripped twice for 2 h at 65°C with 0.1 × SSC, 0.1% SDS (softstripping). Filters hybridized with the cDNA complex probes and

Northern blots were stripped twice 3 h at 80°C with 0.1% SDS,1 mM ethylenediaminetetraacetate [(EDTA) (harsh stripping)].

cDNA complex probe preparation and hybridization conditions

Complex probes were prepared from total RNA essentially as de-scribed by Bernard and co-workers (1996). For each different complexprobe, 25µg total RNA, 8 µg poly(dT)25 and 300 ng poly(dT)12–18

were mixed, heated at 70°C for 6 min, and progressively cooled to42 °C to ensure annealing of poly(dT) with the poly(A) tail. First-strand cDNA synthesis was then performed: reverse transcription for1 h at 42°C in 25 µl with 40 units rRNasin (Promega, Madison, WI),50 µCi (alpha-32P) dCTP, 5µM dCTP, 0.8 mM each dATP, dGTP, anddTTP, and 200 units M-MLV SuperScript RNAse H– reverse tran-scriptase (Gibco-BRL). RNA was removed by treatment at 68°C for30 min with 1µl 10% SDS, 1µl 0.5 M EDTA, 3 µl 3 M NaOH, andthen equilibrated at room temperature for 15 min. Neutralization waswith 10 µl 1 M Tris-HCL plus 3 µl 2 N HCl. Unincorporatednucleotides were removed by purification on a Sephadex G-50 column.The probe (after 5 min denaturation at 100°C) was then incubated with5 µg poly(dA)80 in 1 ml of hybridization buffer (5 × SSC, 5 ×Denhardt’s reagent, 0.5% SDS, 100µg/ml sonicated salmon spermDNA) for 3 h at 65°C. High-density filters were pre-hybridized for16 h and hybridized with total probe overnight (6 filters in 50 ml ofhybridization buffer). After hybridization, filters were washed twice in0.1 × SSC, 0.1% SDS at 65°C for 2 h, and then exposed to phosphorscreens for 3 days.

Detection and quantification of hybridization signals

Hybridization filter images were obtained using a phosphor platesystem (Fujix Bas 1000; Fuji, Tokyo, Japan). Hybridization signalswere quantified with a modified version of the BioImage software(BioImage, Ann Arbor, MI) running on an Unix workstation (Gran-jeaud et al. 1996). Data reports were then exported into a microcom-puter and analyzed using Excel software (Microsoft).

Normalization of hybridization intensities

Two scaling steps were performed: a horizontal normalization ensuringreproducibility across the two duplicate filters and a vertical normal-ization minimizing experimental differences between the two differentcDNA complex hybridizations. The horizontal normalization consistsof dividing, for each clone, the intensity obtained with a complex probewith the intensity obtained previously with the oligonucleotide vectorprobe. To reduce variation due to differences in cDNA complex probelabelling, hybridization, washing, and filter exposure, a vertical nor-malization was done by dividing for each clone the “horizontallynormalized” intensity obtained with a complex probe by the mean ofhorizontally normalized intensities obtained for a series of elongationfactor (EF-1 alpha) cDNA clones present on the filters. We haveverified that mRNA levels for EF-1 alpha and actin are equivalent inthe two conditions relative to total RNA, as determined by quantifiednorthern blots normalized by hybridization of a 28S rRNA-specificoligonucleotide.

Data analysis and clone selection

After normalization, corrected data obtained for the two experimentalconditions were used to determine the coculture/mte ratio. For eachclone, the corrected intensity obtained for hybridization with the cDNAcomplex probe prepared from MTE-1D cells in culture with thymo-cytes (coculture condition) was divided by the intensity obtained withthe probe prepared from MTE-1D cells grown alone (mte condition).For each clone a comparison of the coculture/mte ratio obtained for thetwo duplicate filters was done and only cDNA clones with a ratio ofvariation between the duplicate filters lower than 2 were finallyselected.

D. Rocha et al.: Genes modulated in thymic epithelial cells 143

Cot-1 DNA and mitochondrial probe hybridization

High-density filters were hybridized with Cot-1 DNA to identifycDNA clones that contain repeated sequences. Prehybridization wasperformed in Church’s buffer (Church and Gilbert 1984) with sheareddenaturated salmon sperm DNA (at 100µg/ml) at 65°C. After 3h ofprehybridization, filters were hybridized in the same buffer with 100 ngof 32P-labelled mouse Cot-1 DNA (Gibco-BRL) at 500 000 cpm/mlovernight. Washes were performed two times with 0.1× SSC, 0.1%SDS at 65°C for 30 min and then filters exposed with phosphor platesovernight. Positive clones were scored manually and only clones thatfailed to hybridize with the Cot-1 probe were analyzed further. Toscore mitochondrial transcripts, hybridization was performed as de-scribed above with a 16.3 kb mitochondrial fragment (containing analmost complete mitochondrial genome) labelled by random priming(Feinberg and Vogelstein 1983).

Insert amplification and contamination control

Inserts were obtained by PCR amplification as described by Saiki andco-workers (1985), with the following modifications. To ensure am-plification directly from overnight bacteria cultures, aliquots of 800µlbacterial culture were centrifuged to pellet cells, medium discarded and400 µl sterile water added. Resuspended bacteria were mixed withPCR reaction buffer containing T7 and Sp6 primers. Usually, 3µlresuspended bacteria was dispensed into 50µl final reaction volume.Amplifications were performed in 96-well plate format using thePerkin Elmer 9600 thermocycler with the initial denaturation step at94 °C for 8 min, followed by 30 cycles of denaturation at 94°C for1 min, annealing at 51°C for 2 min, extension at 72°C for 1 min, andfinal elongation step at 72°C for 10 min. One tenth of the PCR productwas fractionated on an 0.8% agarose gel and insert size determined.Clones that gave multiple PCR products were discarded.

Determination of clone frequency by library screening

Eight different high-density filters (1536 clones each) were hybridizedwith probes prepared from an individual clone. These were preparedusing PCR products purified from 1% low-melting point agarose geland labelled by random priming (Feinberg and Vogelstein 1983). Thefilters were hybridized with the labelled probe (at 500 000 cpm/ml) inChurch’s buffer (Church and Gilbert 1984) with 100µg/ml sheareddenatured salmon sperm DNA at 65°C overnight after 3 h ofprehybridization in the same buffer. After hybridization the filterswere washed two times with 0.1× SSC, 0.1% SDS at 65°C for 30 mineach, and then exposed to phosphor plates for 30 min. Positive cloneswere scored manually and their frequency in the library sampledetermined.

Tag-sequencing and sequence comparison

cDNA clones were grown overnight in LB medium plus antibiotics.Plasmid DNA was prepared using the Qiawell 8 plasmid prep kit(Qiagen, Hilden, Germany). Cycle sequencing reactions were perform-ed with theTaqDye primer cycle sequencing kit (Applied Biosystems,Branchburg, NJ) in a Perkin Elmer (Branchburg, NJ) 9600 thermo-cycler using T7 or Sp6 fluorescent dye-labelled primers as suggestedby the manufacturer. The samples were analyzed on a 373A automatedDNA sequencer (Applied Biosystems). Sequence data outputs weremanually edited to remove vector and unreliable sequences. Theaverage length of reliable sequence was about 400 bases (range:100–644). Sequence comparison against nucleotide and protein data-bases maintained at the National Center for Biotechnology Informationwere performed using the Blast network server (Altschul et al. 1990).Partial sequences not yet reported in the mouse system have beensubmitted to dbEST, and their GenBank accession numbers areindicated in Table 1. The corresponding clones are available fromthe authors.

Northern blot analysis

Total cellular RNAs (25µg) extracted from cultured cells wereelectrophoresed on 1.2% agarose gels containing 3.7% formaldehyde,blotted to nylon Hybond-N membrane (Amersham) in 10× SSCovernight and rinsed in 2× SSC. RNA was cross-linked to themembrane by exposure to ultraviolet light (Stratalinker; Stratagene,La Jolla, CA) for 1 min. Probes were prepared and hybridizationperformed (at 500 000 cpm/ml) as described above for clone frequencydetermination. Filters were exposed to phosphor plates from 3 h toovernight, and subsequently hybridized with an actin probe to controlthe RNA quality and for standardization. Signal intensity measured foreach clone was divided by the intensity obtained with the actin probeafter measurement with the Fujix Bas 1000 software.

Identification of RFLPs and mouse chromosomal localization

High relative molecular mass DNA from organs of partial consomicmice (J.-L. Gue´net and co-workers, unpublished data) and parentalstrains C57BL/6 (Mus musculus) and SEG (Mus spretus) was digestedwith restriction enzymes under conditions recommended by the man-ufacturers. Agarose gel electrophoresis, Southern blot transfers, andhybridizations were performed with the cDNA probe as describedelsewhere (D. Rocha and co-workers, unpublished data). SEG-specificrestriction fragments were searched, partial consomic strains exhibitingsuch fragments were scored, and the pattern of SEG-specific fragmentsobtained for the different consomic strains was matched to previouslycollected data in order to obtain evidence for linkage.

Human chromosomal assignment

Chromosomal assignment of human genes homologous to mousecDNA clones was carried out using a set of 24 human-rodent mono-chromosomic somatic cell hybrids from the National Institute ofGeneral Medical Sciences (NIGMS), Coriell Institute (Dubois andNaylor 1993). Lines were grown locally under the conditions indicatedby NIGMS and DNA extracted by detergent lysis and phenol-chloro-form extraction. Tenµg of each DNA was digested withBamHI or HindIII restriction enzyme in parallel with human, hamster, and mouseDNAs, and Southern blotting and hybridization were performed asindicated above. Since mouse probes were used and detection of thehuman homologue was sought, washes were done at low stringency (2× SSC, 0.1% SDS for 30 min at 50°C).

Mouse location and human assignment analyses

Searches for human/mouse similarities were performed at the NationalCenter for Biotechnology Information (http://www. ncbi.nlm.gov/Ho-mology/) and using the Dysmorphic Human-Mouse Homology data-base (DHMHD) at the Institute of Child Health, University of London(http://www.hgmp.mrc.ac.uk/DHMHD/dysmorph.html). The lists ofmapped mouse mutations and mapped human genetic disorders wereaccessed through the Mouse Genome Database (MGD) at http://www.informatics.jax.org/mgd.html and the Online Mendelian Inheri-tance in Man database (OMIM) at http://www.ncbi.nlm.nih.gov/Omim/.

Results

Hybridization signature measurement and selectionof differentially expressed clones

Two duplicate sets of 4608 clones (two copies of threehigh-density filters containing each 1536 cDNA colonies)were hybridized successively (Fig. 1) to complex probes

D. Rocha et al.: Genes modulated in thymic epithelial cells144

prepared with RNA obtained from MTE-1D cells grownalone or in the presence of thymocytes (coculture condi-tion). Hybridization signatures were obtained after imagingplate acquisition of the data and quantification by thespecific BioImage software (Granjeaud et al. 1996).

The reproducibility of measurement (after vector correc-tion and normalization as described in Materials andmethods) was examined by comparison of data from tworeplica filters hybridized in parallel with the same complexprobe (Fig. 2A). Comparison of signals in the two experi-mental conditions (Fig. 2B) directed the choice of clonesfor further study. A set of EF-1 alpha reference clones wasused to normalize signals between the two conditions and toestimate confidence limits (Fig. 1C). We selected forfurther analysis clones for which signals were significantlyincreased or decreased under coculture conditions (ratio ofcorrected intensities greater than 1.8 or smaller than 0.55),

as well as those that gave a detectable signal only under oneof the two conditions. The analysis of 9216 hybridizationsignatures obtained on 4608 clones thus led us to select 167clones fulfilling these criteria (Fig. 3, top).

The rationale of using gridded libraries is that each bit ofinformation obtained on each clone is retrievable andusable at a later date. The results of systematic hybridiza-tion of the filters with Cot-1 DNA and with clonedmitochondrial DNA (see Materials and methods) werestored in the laboratory database, as well as insert ampli-fication results. This allowed us to eliminate clones con-taining repeated sequences, mitochondrial transcripts orcorresponding to wells with more than one colony, leaving58 clones. This set contained 49 different entities (as seen

D. Rocha et al.: Genes modulated in thymic epithelial cells 145

Fig. 1mHigh-density filter hybridizations. A high-density filter (F27)was hybridized successively with a vector oligonucleotide (VEC,top),a complex probe prepared from MTE-1D RNA (MTE,middle) and acomplex probe prepared from RNA of the same line under cocultureconditions (COC,bottom)

Fig. 2A–CmReproducibility and confidence limits. Quantified hybrid-ization signatures are represented in double logarithmic plots:A Reproducibility of signals on two duplicate filters (after vectorcorrection); correlation coefficient 0.812;B Signals with probes in thetwo different experimental conditions (MTE-1D alone or in coculture),displaying a certain degree of differential expression (correlationcoefficient 0.471);C Variation of expression ratios for a set of clonesall consisting of EF-1 alpha coding sequences. The ratio (coculture/mte) was determined individually for one filter (black) and an identicalreplica filter (white). The ratio is seen to vary from 0.61 to 1.3. Limitschosen to define differentially expressed clones (0.55 and 1.8) areindicated

by colony hybridization) that were retained for furtheranalysis (Fig. 3). Table 1 (right) shows the specificmRNA abundance for each of these 49 genes in normal

and cocultured MTE-1D cells, indicated relative to thevalue for the EF-1 alpha chain-encoding gene. The expres-sion of most of these genes is repressed under cocultureconditions. A number of northern analyses were performedusing the same two RNA preparations used for the complexprobe experiments (Fig. 4). These indicated the size of themouse mRNA, and the expression ratios thus obtained weregenerally in agreement with hybridization signature data(Table 2). Table 1 also shows in a number of cases theexpression level in whole thymus; this was determined bymeasuring clone frequency among a set of 12 000 clones(see Materials and methods) and is given, again, relative tothe EF-1 alpha clone frequency.

Tag sequencing, database comparison, and significanceof the observed changes

The selected clones were submitted to tag sequencing at the59 end and, for those unrelated to known genes, at the 39

end; results of the database comparisons are shown in Table1. The majority of the clones selected were related toknown sequences, although many had not been previouslydescribed in the mouse. As summarized in Table 1, most of

D. Rocha et al.: Genes modulated in thymic epithelial cells146

Fig. 3mSuccessive steps of theselection procedure

Fig. 4mNorthern blotting of two differentially expressed clones.Left,prothymosin alpha, clone MTA.B12.071 (repressed under cocultureconditions);right, CCT zeta subunit, clone MTA.D07.081 (stimulated).Twenty-five µg of total RNA from the MTE-1D line grown under thetwo conditions were used; the signal after rehybridization with an actinprobe (bottom) was used for normalization

D. Rocha et al.: Genes modulated in thymic epithelial cells 147

Table 1mSummary of information on differentially expressed clones

Clone Accessionnumber

Se-quenceLength

Identification Accessionnumber

Species Similarity Thy-mus

mte coc coc/mte

Known or homologous(Chromosome structure)MTA.H07.052 Gb:W91749 632 Histone H2A.Z Emb:X52316 Rat 95%/598 0.08 0.74 0.27 0.37MTA.D06.070 318 HMG-1 Emb:X80457 Mouse 97%/255 0.19 1.60 0.48 0.30MTA.G11.085 Gb:AA032310 380 XCAP-C Gb:U13673 Xenopus 78%/297 0.27 0.09 0.34MTA.G08.087 Gb:W91574 444 Inner centromere protein class I Emb:Z25420Gallus 73%/212 0.01 0.40 0.13 0.32

(Cell division. oncogenes and tumor-related)MTA.F01.052 492 unp Gb:L00681 Mouse 99%/284 0.05 0.73 0.00 0.00MTA.B12.071 360 Prothymosin alpha Emb:X56135 Mouse 98%/360 0.26 4.25 1.55 0.36MTA.G06.082 503 Ki-67 Emb:X82786 Mouse 99%/396 0.17 0.06 0.36MTA.C10.094 422 MHC class I tumor-

transplantation antigen P35BGb:M30128 Mouse 100%/192 0.01 0.32 0.13 0.40

(Signal transduction and kinases)MTA.B05.052 546 Ran GTPase Gb:L32751 Mouse 98%/279 0.97 0.28 0.29MTA.G05.081 276 Epsilon 14-3-3 eta isoform Gb:U57311 Mouse 98%/193 0.13 0.03 0.23MTA.H11.086 164 Myotonic dystrophy protein kinase Gb:S60312 Mouse 88%/144 0.02 0.05 0.00 0.00

(Transcription factors and developmental regulation)MTA.G11.055 439 CArG box binding factor Dbj:D90151 Mouse 100%/170 0.03 0.27 0.11 0.41MTA.A04.084 590 Putative transcriptional regulator

mEnx-1Gb:U52951 Mouse 99%/590 0.01 0.14 0.05 0.36

MTA.D07.086 644 Nuclear matrix attachment DNA-binding protein SATB1

Gb:U05252 Mouse 99%/637 1.12 0.52 0.46

(Transcription and translation machinery)MTA.H07.055 340 Nucleolar protein N038 Gb:M33212 Mouse 95%/204 0.65 0.15 0.23MTA.D03.064 Gb:W91743 509 Glutaminyl t-RNA synthase Emb:X54326 Human 98%/415 0.02 0.12 0.05 0.42

(Ribosomal proteins)MTA.G12.051 435 Ribosomal protein L27a Gb:X05021 Mouse 97%/365 0.12 0.84 0.26 0.31MTA.A07.053 222 Ribosomal protein S4 Gb:M73436 Mouse 98%/146 0.76 0.21 0.27MTA.A12.064 Gb:W91580 474 Ribosomal protein L18 Gb:L04128 Mouse 97%/407 0.04 0.15 0.06 0.40MTA.B07.066 Gb:AA032303 395 Ribosomal protein S26 Emb:X02414 Rat 85%/ 83 0.09 0.71 0.35 0.50MTA.H04.079 439 Ribosomal protein L30 Gb:K02929 Mouse 96%/433 1.43 0.49 0.34

(Heat shock proteins and chaperones)MTA.F12.050 275 CCT beta subunit Gb:Z31553 Mouse 95%/275 0.03 0.37 0.16 0.43MTA.G03.050 376 Phospholipase C alpha Gb:M73329 Mouse 90%/350 0.04 0.47 0.10 0.21MTA.D07.055 454 ERp99 Gb:J03297 Mouse 99%/318 0.86 0.30 0.35MTA.A10.072 301 Cyclophilin A Emb:X52803 Mouse 98%/301 0.69 0.30 0.43MTA.D07.081 351 CCT zeta subunit Gb:Z31557 Mouse 97%/329 0.17 0.97 3.08 3.17

(Receptors and membrane-associated)MTA.F06.057 235 Calpactin heavy chain Gb:M33322 Mouse 100%/164 0.14 0.06 0.43MTA.A01.095 245 Interleukin receptor 4 alpha Gb:M64879 Mouse 100%/124 0.01 0.29 0.10 0.34MTA.B12.053 Gb:AA032305 313 Purine-specific Na+ nucleoside

cotransporterGb:M81884 Rat 95%/ 68 0.01 0.32 0.09 0.28

(Energy metabolism)MTA.C05.082 Gb:AA032307 428 ATP synthase coupling factor 6 Emb:X54510 Rat 91%/280 0.01 0.04 0.00 0.00

(Other metabolism and homeostasis)MTA.E03.058 Gb:AA032308 559 Thermosensitive glucokinase Sp:P39208E. coli 51%/ 49* 0.04 0.00 0.15410MTA.C12.096 Ferritin heavy chain** Mouse 0.62 0.28 0.45

EST-relatedMTA.H10.053 Gb:AA032313 509 clone, GEG-154, mouse fetal

ovaryEmb:X71642 Mouse 100%/505 0.51 0.06 0.12

MTA.D05.061 Gb:W91568 484 EST, human fetal liver spleen Gb:N58690 Human 87%/276 0.01 0.19 0.07 0.37MTA.B06.066 Gb:W91581 100 EST, mouse embryo Gb:AA007986 Mouse 97%/ 45 1.41 0.71 0.50MTA.A03.071 Gb:AA032303 416 EST, adult mouse Gb:W11912 Mouse 95%/250 0.01 0.58 0.30 0.52MTA.H04.083 Gb:W91575 450 EST, mouse embryo Gb:W66795 Mouse 100%/278 0.02 0.23 0.10 0.43MTA.B04.085 Gb:W91766 332 EST, mouse embryo Gb:W84138 Mouse 88%/115 0.01 0.03 0.00 0.00MTA.C01.085 Gb:W91679 246 EST, mouse embryo Gb:W77064 Mouse 99%/245 0.33 0.11 0.33MTA.C07.085 Gb:W91681 398 EST, mouse embryonal

carcinoma F9 cellDbj:D28719 Mouse 93%/123 0.35 0.10 0.28

MTA.A01.088 Gb:AA032302 409 EST, human infant brain Emb:F01633 Human 79%/ 49 0.20 0.08 0.40MTA.C02.091 Gb:W51668 571 EST, human fetal brain Dbj:D60571 Human 83%/ 62 0.01 0.27 0.08 0.30

them correspond to sequences involved in cell division andin the transcription/translation machinery.

The 49 clones studied were selected from a set of 4608thymus cDNA clones, on the basis of modulation uponcoculture of the MTE-1D cell line (representative of med-ullary thymic epithelium) with thymocytes. The variationof their expression level ranges from twofold to more thantenfold. Additional northern blots done with RNA prepara-tions from coculture experiments at much lower cell den-sity, or from MTE-1D cells under various growth condi-tions in the absence of thymocytes, gave different expres-sion ratios (data not shown), indicating that the modulationsof expression observed in the original experiments involveboth growth conditions and interaction with thymocytes.Tag sequencing shows that 32 (65%) of the sequences arerelated to genes known in the mouse or in other organisms,14 (29%) related only to mouse or human ESTs, and 3 (6%)unrelated to known sequences. The relatively small numberof new sequences is a reflection of the development of ESTprojects, and of the fact that hybridization signature meas-urement excludes genes expressed at very low levels (lessthan 0.01% abundance; Nguyen et al. 1995) that are the

most likely to be new. The nature of the molecules encodedsuggests that the modulation observed mainly reflects theeffect of thymocytes on growth of the epithelial cells.

Genome mapping in the mouse and human chromosomeassignment

Several of the selected genes related to known sequenceshad not been identified and/or mapped in the mousepreviously; we used a set of mouse strains correspondingto interspecific hybrids betweenM. spretusandM. muscu-lus (J.-L. Guenet and co-workers, unpublished data) todetermine their localization in the mouse genome. Thesepartial consomic mice contain large fragments ofM. spretuschromosomes in aM. musculusbackground. The approx-imate extent of these segments was determined with a set ofgenetically mapped probes. We first examined whether agiven clone was suitable for mapping by hybridizing a pre-localization Southern blot (Materials and methods; Fig. 5A)that indicated whether polymorphic fragments were ob-served between the two species and also whether cross-

D. Rocha et al.: Genes modulated in thymic epithelial cells148

Table 1mSummary of information on differentially expressed clones

Clone Accessionnumber

Se-quenceLength

Identification Accessionnumber

Species Similarity Thy-mus

mte coc coc/mte

MTA.C09.091 Gb:W91683 493 EST, human infant brain Gb:H29360 Human 87%/147 0.03 0.17 0.06 0.35MTA.C06.093 Gb:W91567 275 EST, human adult heart Gb:R41073 Human 81%/ 94 0.01 0.13 0.03 0.23MTA.G01.093 Gb:W91745 494 EST, mouse embryo Gb:W57035 Mouse 98%/447 0.34 0.08 0.23MTA.H09.096 Gb:W91576 430 EST, mouse thymus Emb:X83884 Mouse 98%/126 0.08 0.10 0.00 0.00

NewMTA.H09.053 Gb:W51703 316 0.39 0.17 0.43MTA.A05.071 Gb:W91638 421 0.01 0.10 0.00 0.00MTA.H07.094 Gb:AA03212 424 0.10 0.03 0.30

Clones are grouped according to broad functional categories, or asEST-related or new. New accession numbers (left) are given for thosethat represented sequences not yet reported in the mouse system.Expression information (right part of the table) includes clone redun-dancy in the thymus library (expressed relative to the redundancy ofelongation factor 1 alpha sequences), expression levels in the MTE-1D

cell line without (mte) and with (coc) thymocytes (again relative to theexpression level of EF-1 alpha), and expression ratio under these twoconditions* Similarity at the amino acid level** Identification by hybridization

Table 2mComparison of expression ratios from hybridization signature and northern blot experiments

Clone name Identification Ratio coc/mte (*)assayed by quantitative differentialscreening

Ratio coc/mteassayed by northern blot analysis

MTA.F12.050 CCT beta subunit 0.43 N. D.MTA.H07.052 Histone H2A.Z 0.37 0.66MTA.B07.066 Ribosomal protein S26 0.50 0.59MTA.D06.070 HMG-1 0.30 0.53/0.38 (**)MTA.B12.071 Prothymosin alpha 0.36 0.28MTA.G12.071 Prothymosin alpha 0.37 0.46MTA.A10.072 Cyclophilin A 0.43 0.55MTA.H04.079 Ribosomal protein L30 0.34 0.23MTA.D07.081 CCT zeta subunit 3.16 9.10MTA.D07.086 DNA-binding protein SATB1 0.46 N. D.

* Mean value computed from two duplicate filters** Two transcripts were detected with this probeN. D. Ratio not determined, no clear band detected by northernanalysis

The correlation coefficient between the results of the two methods is0.99.

hybridization with human DNA was readily observed. Themost common difficulty was the observation of complexhybridization patterns indicating the existence of a genefamily or of pseudogenes (Fig. 5B). An example of suc-cessful regional mapping is given in Figure 5C. Of eleven

clones for which mapping was attempted using this proce-dure, eight could be located and three gave complexpatterns with mouse DNA (Table 3) that could not beresolved.

For those clones that cross-hybridized well with humanDNA, and in which the human-specific band could bedistinguished from the mouse and hamster bands, we usedhybridization to a panel of monochromosomal hybrids eachcontaining a single human chromosome in a mouse orhamster background. An example of successful chromo-some assignment is shown in Figure 5D. A summary of theresults is displayed in Table 3.

Discussion

Genes that are down-regulated under coculture conditions

Almost all genes selected were found to be expressed at amuch higher level in the MTE-1D epithelial cell line than inwhole thymus, and most of them were down-regulated fromtwo to tenfold upon coculture. Prothymosin alpha, almosttwenty times more abundant in MTE-1D than in thymus, isa particularly striking example. This ubiquitous molecule,strongly expressed in the thymus, is involved in cell growth(Sburlati et al. 1991). The IL-4 receptor gene is alsostrongly expressed in the epithelial line with respect towhole thymus; it is known (Ritter and Boyd 1993) thatthymocytes express both IL-4 and its receptor, while thymicepithelial cells synthesize only the receptor. Another mem-ber of this class of moderately down-regulated genes isSATB1, a DNA-binding protein identified in human (Dick-inson et al. 1992) and mouse (Nakagomi et al. 1994) that ispredominantly expressed in the thymus and is involved innuclear matrix attachment. Finally, a clone of this groupwas found to correspond tomENX-1, a newly identifiedmouse homologue of theDrosophila Enhancer of zestePolycomb group gene whose expression is restricted tothe thymus in the late embryo (Hobert et al. 1996). Wesuggest that this molecule is likely to play a role in thymicepithelial cell development.

A few of the identified genes display extreme down-regulation upon coculture, giving in fact no detectableexpression under this condition (as verified in duplicateexperiments, see Materials and methods). The most strikingof these isUnp, a mouse gene related to the humantreoncogene (Gupta et al. 1993) and which codes for a nuclearprotein (Gupta et al. 1994). It is tumorigenic in nude mice,and probably involved in the regulation of cyclins throughbinding to elements of transcription factors. It is totallyextinguished under coculture conditions.

Genes up-regulated under coculture conditions

Two sequences display increases in mRNA level undercoculture conditions. The entity labeled thermosensitiveglucokinase is a clone whose sequence overlaps that of a

D. Rocha et al.: Genes modulated in thymic epithelial cells 149

Fig. 5A–DmGene localization in the mouse genome using partialconsomic mice, and chromosome assignment in human.A, B Exam-ples of a workable (MTA.D07.081, CCT zeta subunit) and unworkable(MTA.H07.055, nucleolar protein N038) clone, as seen by hybridiza-tion to a Southern blot containingM. musculus(B6), M. spretus(SEG),human (4XY) and Chinese hamster (CHO) DNA cleaved by severalenzymes. The arrows (A) show the polymorphic fragments used insubsequent localization of this clone. The complex pattern observed inthe other case (B) makes localization impossible by this method.CResult after hybridization of clone MTA.D07.081 (CCT zeta subunit)to the set of DNAs from partial consomic strains (Taq I digest). One ofthem (in addition to the parentalM. spretusstrain SEG) displays thediagnostic SEG-specific band (arrow); this information allows local-ization of the corresponding sequence to mouse chromosome 7(15–51 cM interval).D Assignment of clone MTA.G11.055 (CArGbox binding factor) to human chromosome 5 (arrow). DNAs frommonochromosomal hybrids were digested withHin dIII in this case

mouse EST expressed in testis and indicated as homologous(at the amino-acid level) toEscherichia colithermosensi-tive glucokinase. Its protein translation is indeed closelyrelated to this protein as reported in Table 1. This protein is,however, different from the (known) mouse glucokinase,involved in glucose metabolism (Iynedjian 1993). The otherup-regulated gene is the CCT zeta subunit, expressed atquite a high level in the epithelial cell line and undergoing afurther threefold stimulation upon coculture. This increasehas been confirmed by northern blotting. CCT (for chaper-onin-containingt-complex polypeptide 1) is a chaperonin(for a review see Kubota et al. 1995) that contains multiplesubunits and acts on a variety of proteins including actinand tubulin (Kubota et al. 1994); it is believed that thedifferent subunits target the chaperonin to different proteins(Kim et al. 1994). The beta subunit of the same chaperoninwas seen to be repressed under coculture conditions,although northern experiments were not conclusive in thiscase. Thus, the data on the CCT zeta clone suggest that thisform of the chaperonin is involved in folding proteinssynthesized as a response to the coculture conditions, andis potentially involved in thymic epithelial cell differentia-tion.

Indications from genome mapping

Regional mapping in the mouse genome was obtained foreight of the genes not previously located in this organism.The partial consomic system used provided localizationwith a resolution of approximately 30 cM using a relativelysmall number of DNA preparations (eighteen includingcontrols). Cross-hybridization with human DNA made itpossible to use human/mouse and human/hamster somaticcell hybrids for assignment of the human homologue in sixcases. In particular, the CCT beta and zeta subunits wereregionally localized in the mouse and assigned to humanchromosomes; they are found in quite different regions ofthe genome, as the recently assigned CCT delta subunit(Nabetani et al. 1996). In five cases the human and mouselocations did not correspond to previously reported con-

served syntenies, and may indicate new syntenic relation-ships. For the CArG box binding factor gene, taking intoaccount known syntenies would restrict the region to 14.8to 33 cM on mouse chromosome 11 and to the q23-q24regions on human chromosome 5. The mouseKi-67 gene(related to cell proliferation) was mapped in a region ofchromosome 2 known to contain a colon tumor suscepti-bility gene (Scc2;Moen et al. 1996) and may be considereda candidate gene, although more precise mapping is needed.

This paper presents initial results of exploring aspects ofthymus function using an integrated approach that com-bines quantitative differential hybridization for expressioninformation, tag sequencing for structural data, and genomemapping for potential correlation with mutations and dis-eases. It indicates how information of many different kindscan be integrated to gain knowledge about a complexbiological event. The general down-regulation of genesinvolved in proliferation that we observed is consistentwith the onset of differentiation induced by the presenceof thymocytes and with previous observations on thisprocess (Meilin et al. 1995). Further study of differentiallyregulated clones not related to known genes may revealnew structures involved in growth control and/or thymo-cyte-epithelial cell interactions.

AcknowledgmentsmWe thank colleagues at CIML for discussions andhelp, particularly K. Bernard for development of complex probe pro-tocols and S. Granjeaud for computer expertise. A. Rotig (INSERMU12, Paris) provided the mitochondrial probe, and L. Maltais (JacksonLaboratory) gave helpful advice on genetic nomenclature. This re-search was supported by institutional grants to CIML from CentreNational de la Recherche Scientifique (CNRS) and Institut National dela Sante´ Et de la Recherche Me´dicale (INSERM), as well as by specificgrants from Groupement de Recherches et d’Etudes sur les Ge´nomes(GREG) and from Association Franc¸aise contre les Myopathies(AFM). D.R. was supported by a Ph.D. fellowship from the Ministe`rede l9Enseignement Supe´rieur et de la Recherche.

D. Rocha et al.: Genes modulated in thymic epithelial cells150

Table 3mSummary of gene localizations and assignments in the mouse and human genomes

Clone Gene Localizationof mouse gene

Assignmentof human gene

MTA.F12.050 CCT beta subunit (Cct2) Chr. 15 (4.8–33.9 cM) Chr. 12MTA.G03.050 Phospholipase C alpha (Plca) Chr. 2 (41–67 cM) Chr. 1MTA.B12.053 Purine specific Na+ nucleoside cotransporter (Spnt) Chr. 2 (41–67 cM) No cross-hybridizationMTA.H10.053 GEG-154 (EST) (D9Cim11) Chr. 9 (13.2–64.2 cM) Chr. 8MTA.G11.055 CArG Box binding factor (Cgbfa) Chr. 11 (2.2–43.5 cM) Chr. 5MTA.H07.055 Nucleolar protein N038 (Npm1) Complex pattern Complex patternMTA.B12.071 Prothymosin alpha (Ptma) Complex pattern Chr. 2 (1)MTA.A10.072 Cyclophilin A (Ppia) Complex pattern Chr. 7p11.2–13 (2)MTA.D07.081 CCT zeta subunit (Cct8) Chr. 7 (15–51 cM) Chr. 8MTA.G06.082 Ki-67 (Mki-67) Chr. 2 (0–41 cM) Chr. 10q25–qter (3)MTA.G08.087 Inner centromere protein class I (Incenp1) Chr. 19 (0–20 cM) No cross-hybridization

(1) Szabo et al. (1993)(2) Willenbrink et al. (1995)(3) Fonatsch et al. (1991)

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