GENOMICS

11,

374-378

(1991)

The Gene Encoding Human TFE3, a Transcription Factor That Binds the lmmunoglobulin Heavy-Chain Enhancer, Maps to Xpl 1.22 PAULA5. HENTHORN,* CHRISTINE C. STEwART,t TOM KADESCH,$ AND JENNIFER M. PucKt *Section of Medical Genetics, University of Pennsylvania School of Veterinary Medicine; tDepartment of Pediatrics, University of Pennsylvania School of Medicine and Children’s Hospital of Philadelphia; and *Howard Hughes Medical Institute and Department of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19704 Received

March

18, 1991;revised

June 3, 1991

al., 1989; Brown et al., 1990). In fact, a 1;19 translocation associated with 30% of pediatric pre-B-cell acute lymphoblastic leukemias has been shown to directly create a novel gene, encoding a chimeric transcription factor (Kamps et al., 1990; Nourse et aZ., 1990). Nonneoplastic phenotypes have also been linked to transcription factors. For example, the transcription factor Pit-l has been implicated in the Snell form of dwarfism in the mouse (Camper et al., 1990; Li et al., 1990). The correlation of a disease with alterations in a given transcription factor serves not only to supply insights into the molecular basis of the disease, but also to reveal genetic phenotypes for the transcription factor itself. To characterize the TFE3 gene more completely and to gain more insight into its in vivo functions and potential involvement in disease states, we determined its chromosomal location. We used a TFE3 cDNA fragment as a probe against a human/rodent somatic cell hybrid line and in linkage analysis. A frequent restriction fragment length polymorphism (RFLP) in the TFE3 gene allowed us to map the TFE3 locus to Xp11.22, between DXS7 and DXS14.

TFE3, a member of the helix-loop-helix family of transcription factors, binds to the pE3 motif of the immunoglobulin heavy-chain enhancer and is expressed in many cell types. We have localized human TFE3 to the proximal short arm of the X chromosome using a somatic cell hybrid panel. A frequent RsaI RFLP detected by the TFE3 cDNA was found and used to confirm this location by linkage analysis in 20 pedigrees. Two-point and multipoint lod scores place TFE3 near markers in Xpll.22 with the most likely order DXS7-DXS255-TFE3-DXSl46-DXS14. o 1991 Academic Press. Inc.

INTRODUCTION

TFE3 is a ubiquitous DNA-binding protein that binds to the pE3 motif within the immunoglobulin heavy-chain enhancer. It has been shown to function as a positive-acting transcription factor (Beckmann et al., 1990). The TFE3 cDNA encodes a 59kDa protein that contains a helix-loop-helix motif (Murre et aZ., 1989a) adjacent to a putative leucine zipper (Landschulz et al., 1988). In other proteins, these motifs have been shown to mediate protein-protein interactions (Murre et al., 1989a,b; Davis et al, 1990; Benezra et al., 1990; Landschulz et al., 1989; Voronova and Baltimore, 1990; Hu et al., 1990, and references therein). Despite extensive characterization of the TFE3 gene product (Beckmann and Kadesch, 1991), its physiologic function is not understood. A well-established, yet still growing body of evidence has linked altered expression of transcription factors to a variety of disease states. Most notably, many virally encoded oncogenes and their chromosomal counterparts encode transcription factors (for review, see Bishop, 1987). Additional genes encoding proteins with the structural hallmarks of DNA-binding proteins have been shown to be deregulated in hematopoietic neoplasias as the result of nearby translocation events (Mellentin et al., 1989; Begley et 08&3-7543/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

MATERIALS

METHODS

Subjects and DNA Preparation Genomic DNA was prepared from individuals in 6 previously described (Puck et al., 1989) and 14 additional (Puck et al., 1990, and unpublished) well-studied pedigrees segregating for X-linked severe combined immunodeficiency. Genomic DNA was isolated using an automated DNA extractor (Applied Biosysterns, Inc., Foster City, CA), digested, and subjected to Southern blot analysis using standard techniques (Maniatis et al., 1982).

Probes The probe used for the TFE3 gene was a 1.9-kb EcoRI fragment from the 3’ end of the TFE3 cDNA, 374

Inc. reserved.

AND

ASSIGNMENT

designated pTFE3-1.9 (Puck et al., 1991). Probes that detect the following polymorphic loci from the pericentric region of the X chromosome (locations are indicated in parentheses) were used: DXS7 (p11.4p11.3), DXS255 (p11.22), DXS146 (p11.22), DXS14 (p11.21), DXSl (q11.2-q12), DXS159 (q12), and PGKl (q13) (Mandel et al., 1989).

Cell Hybrid Lines A commercially available panel of hamster/human hybrid cell line DNAs was used for an initial chromosomal assignment (Bios, New Haven, CT). The hamster/human cell line 4.12 contains only the human X chromosome (Nussbaum et al., 1983). The cell line 88H5 (kindly provided by Dr. David Ledbetter) contains a single human X chromosome deleted for most of the short arm. The long arm is intact. The Sin176 cell line contains an X chromosome that is deleted for the region Xp11.23 to Xp22.11, including the DXS7 locus (Ingle et al., 1985; Paulsen et al., 1986).

Linkage Analysis Lod scores were calculated under the assumption of no interference with the computer programs MLINK, ILINK, and LINKMAP of the computer program package LINKAGE, version 4.9 for the VAX (Lathrop and Lalouel, 1984; Lathrop et al., 1985). Allele frequencies for the RFLP loci were as published in the report of the Tenth Human Gene Mapping Conference (Mandel et al., 1989). RESULTS

To determine the chromosomal location of the human TFE3 locus, we used a TFE3 cDNA EcoRI subclone, referred to as pTFE3-1.9 (Puck et al, 1990), to probe a commercially available mapping panel. Hybridization patterns from the panel indicated that the TFE3 locus was on the X chromosome (data not shown). This assignment was confirmed by hybridization of pTFE3-1.9 to DNA from somatic cell hybrid lines containing all or portions of the X chromosome. The TFE3 probe hybridized to 4.12, a line containing an intact human X chromosome as its only human material (see Table l), confirming the result obtained with the mapping panel. Hybridizations to DNAs from two other cell lines, Sin176 and 88H5, were particularly informative in localizing TFES. The TFE3 probe did hybridize to the cell line Sin176, which has an X chromosome deleted for the interval Xp11.23 to Xp22.11 (Ingle et aZ., 1985; Paulsen et uZ., 1986). It did not hybridize to hybrid line 88H5, the human content of which is an X chromosome cytogenetically deleted for almost all of Xp. Additional probes from previously ordered marker loci from the proximal long and

OF

TFE3

TO

375

Xp11.22

TABLE Hybridization Lines Containing

1

of TFE3 to Somatic Cell Hybrid Portions of the X Chromosome Cell hybrid

line

4.12

Sin176

88H5

DXS7 (L128) DXS255 (M278) DXS146 (pTak8) DXS14 (58.1) DXSl (~8) DXS159 (cpX73)

+ + + + + +

+ + + + +

+ + +

TFE3

+

+

-

Locus

(probe)

(pTFE3-1.9)

Note. A + indicates

hybridization;

- indicates

absence

of hybrid-

ization.

short arms (Greer et al., 1990; Keats et cd., 1989; Lafreniere et al., 1989; Mahtani et al., 1989; Mandel et al., 1989) were hybridized to the cell lines to confirm their breakpoints and to further characterize the deleted X chromosome in line 88H5 (Table 1). The X chromosome in 88H5 was deleted for loci DXS146 and DXS255 (in Xp11.22) as well as for more distal probes including DXS7. At least some portion of Xp11.21 is present in this hybrid, however, as indicated by the positive hybridization with probe 58.1 at the DXSl4 locus. The Sin176 hybridization pattern is consistent with a proximal deletion boundary in Xp11.23, proximal to DXS7. Taken together, these data place TFE3 in the interval Xp11.21-Xp11.23 or distal to Xp22.11. To further localize the TFE3 gene, genetic linkage studies were undertaken. These studies were possible because of a frequent two-allele RsaI restriction fragment length polymorphism which was detected with the pTFE3-1.9 probe (Puck et al., 1991). This polymorphism consists of l.l- and 1.8-kb alleles seen in 53 and 47%, respectively, of 132 X chromosomes tested. A faint 1.4-kb constant band is also detected. Twenty extensive, well-studied families segregating for SCIDXl were genotyped for the pericentromeric polymorphic loci DXS7, DXS255, DXS146, DXS14, DXSl, DXS159, DXS106, and DXS132, as well as for TFE3. Eight particularly informative, phase-known meiotic recombinational events are diagrammed in Fig. 1. Closed circles represent alleles derived from the grandpaternal X chromosome, while open circles represent grandmaternal alleles. Crossover events between TFE3 and DXS7, shown in meioses 6,7, and 8, demonstrated that TFE3 is proximal to DXS7 and therefore is proximal to Xp11.4. Combined with the Sin176 data, this indicated that TFE3 is located in the interval Xp11.21-Xp11.23. Meioses 1, 2, and 3 also

376

HENTHORN

DXS7 DXSP55

11.3 11.23

DXS146

11.22 11.21 11.1

DXS14

DXS159

12 DXS106

13.1

DXS132

13.2

FIG. 1. Meiotic crossover events in maternally derived X chromosomes, showing grandmaternal (0) or grandpaternal (0) alleles from data collected from multigeneration pedigrees of families with phase-known X chromosomes. Polymorphic loci and their locations on the pericentric region of the X chromosome are shown on the left. Horizontal dashes at a locus represent maternal homozygosity. The relative order of the Xq12 marker loci DXS159, DXS106, and DXS132 has not been confirmed, as indicated by the vertical line.

DXS7-0.08

for Increasing

Recombination

Fraction

M-

M-DXS14-0.001 DXSl-0.074

TABLE Scores

M-DXS255-0.042

DXS146-0.0385

confirmed that TFE3 is either on Xp or proximal to DXS106, DXS132, and DXS159 in Xq12 (Mandel et al., 1989). Meioses 4 and 5 provided the narrowest genetic boundaries for TFE3. In meiosis 5, a crossover placed TFE3 centromeric to DXS255, while a crossover in meiosis 4 placed TFE3 distal to DXS146. Since these two loci are both in Xp11.22, TFE3 is therefore also likely to be located in Xp11.22. Two-point linkage analysis is shown in Table 2. TFE3 showed significant linkage to proximal longand short-arm markers from DXS7, with maximum lod score, 2, of 5.14 at 11% recombination, to DXS159, with maximum lod score of 7.63 at 9% re-

Lod

AL.

combination. There were no doubly informative meioses with crossovers between DXSl and TFE3 or DXS14 and TFE3, as shown by the maximum lod scores at no recombination (0 = 0) for these markers. However, both DXSl and DXS14 are known to be proximal to TFE3 from the analysis of hybrid 88H5 (see Table 1). The highest lod score of 13.83 was found for TFE3 at only 2% recombination from DXS255, a tandem repeat RFLP for which 93% of the meioses in our data set were informative. We observed only a single crossover in 52 meioses informative for DXS255 and TFE3. There was also only a single crossover between DXS146 and TFE3, producing a maximum lod score between these loci of 7.44 at 3% recombination. Multipoint linkage analysis was carried out with TFE3, moving through the pericentromeric region from DXS7 through DXS159 (Fig. 2). Genetic distances between the marker loci were determined using multipoint ILINK on our own data set, as

12345676

11.4

ET

MM-DXS159.

Because DXS14 is known to be on Xp while DXSl, a relatively uninformative marker, is on Xq (Mandel et aZ., 1989), a genetic distance of 0.001 M was assigned to the interval between these markers. All of the above distances are in agreement with published data (Keats et al., 1989; Mahtani et al., 1989). The maximum lod scores for each interval are indicated on the graph in Fig. 2. TFE3 was found to be 1.95 X 106-fold more likely to be proximal to DXS7 on Xp than distal, and similarly 2.29 X 104-fold more likely to be distal to DXS14 on Xp than distal to DXS159 on Xq. The most likely interval for the location of TFE3 was between DXS146 and DXS255. This position was 229and 22.4-fold more likely than the flanking intervals 2

(0) between

TFE3

and RFLP

Loci

from

Xp21.1

to Xq13.3

(nrobe)

e=o

0.001

0.01

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

2”

tlb

DXS7 (L128) DXS255 (M27b) DXS146 (pTAK8) DXS14 (58.1) Centromere DXSl (~8) DXS159 (cpX73) PGKl (pPGK-RI.9)

-03 --a,

2.41 13.77 7.26 5.19

4.66 13.46 7.36 4.89

5.13 12.44 6.68 4.49

5.03 11.22 6.24 4.05

4.67 9.89 5.50 3.58

4.14 8.43 4.70 3.08

3.47 6.89 3.34 2.54

2.69 5.24 2.92 1.97

1.83 3.51 1.97 1.36

5.14 13.83 7.44 5.26

.ll .02 .03

5.;

-1.48 12.99 6.39 5.25

4.79 -al -03

4.79 1.41 -.92

4.72 5.26 1.02

4.42 7.36 2.14

4.04 7.62 2.38

3.62 7.30 2.35

3.19 6.69 2.19

2.74 5.89 1.96

2.26 4.93 1.67

1.75 3.34 1.32

1.20 2.62 .93

4.79 7.63 2.39

Locus

a Maximum lod score. b Recombination fraction

at which

the maximum

lod score

is observed.

0 0 .09 .12

ASSIGNMENT

377

OF TFE3 TO Xp11.22

high incidence of malignancy (see Blaese, 1989). Xinactivation studies show that the WAS defect affects all cells derived from bone marrow stem cells (Greer et al., 1989; Prchal, et aZ., 1980), and defective glycosylation of lymphocyte membrane proteins has been noted (Shelley et al., 1989). The involvement of TFE3 in any of these disorders can now be addressed.

15.63

11.27

ACKNOWLEDGMENTS

‘I -0.2

-0.1

XP

0.0

0.1

4 Li ::

444 * % SE@ 23 0 0

I . . 0.3

0.2

4 d ii ::

. ( 0.4 M

xq

The authors thank Charles Bailey for assistance in running linkage analysis programs and Ian Craig for providing the M27fi probe. This work was supported hy the Lucille P. Markey Charitable Trust, American Cancer Society Grant IN-135K (to P.S.H.), the Howard Hughes Medical Institute (to T.K.), and Grants MOD1077 and HD23679 (to J.M.P.).

REFERENCES

FIG. 2. Seven-point multipoint linkage analysis for TFE3 using the Xp markers DXS7 through DXS14 and the Xq markers DXSl and DXS159. Lod scores represent the log,, likelihood that TFE3 lies at each position shown relative to an unlinked position at the Xp telomere.

1.

proximal tively.

3.

to DXS146

or distal

to DXS255,

respec-

2.

DISCUSSION

We have used somatic cell hybrid lines and linkage analysis to assign the TFE3 gene locus to Xp11.22, most likely between DXS255 and DXS146. The TFE3 locus contains a highly polymorphic RFLP with the enzyme RsaI, and provides a very useful marker in a region of the X chromosome that seems to show a lack of recombination (Zonana et al, 1988). Even though TFE3 is ubiquitously expressed, defects in the TFE3 gene could cause disease phenotypes limited to specific tissues. Consequently, based on its location, TFE3 could be implicated in a number of clinical and genetic disorders localized to the same region. A rare case of pediatric renal cell carcinoma, with the translocation t(X;17)(p11.2;q25), has been reported (Tomlinson et al., 1990). A Xp11.2 translocation breakpoint has also been seen in one of the few other cases of this tumor (deJong et al., 1986). We are currently investigating the possibility that the TFE3 locus may be near or at the translocation breakpoint in this tumor. In addition, a number of disease gene loci are located near TFE3 (Mandel et al., 1989; Davies, et al., 1990), including eye diseases (retinitis pigmentosa, Aland Island eye disease, and congenital stationary night blindness) and Wiskott-Aldrich syndrome (WAS, Greer et aZ., 1990), an immunodeficiency characterized by recurrent infections and autoimmune disease, thrombocytopenia, eczema, and a

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