SHORT COMMUNICATION The TCF8 Gene Encoding a Zinc Finger Protein (Nil-2-a) Resides on Human Chromosome 10pl 1.2 THOMAS M. WILLIAMS,* GEORGEMONTOYA,* YING WU,* ROGERL. EDDY,t MARY G. BYERS,t AND THOMAS B. SHOWSt *Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131; and tDepartment of Human Genetics, Roswell Park Memorial Institute, Buffalo, New York Received March 17, 1992; revised June 12, 1992

The TCF8 gene encodes a zinc finger protein (Nil-2a). N i l - 2 - a i n h i b i t s T - l y m p h o c y t e - s p e c i f i c i n t e r l e u k i n 2 (IL2) g e n e e x p r e s s i o n b y b i n d i n g to a n e g a t i v e r e g u l a t o r y d o m a i n 1 0 0 n u c l e o t i d e s 5' o f t h e I L 2 t r a n s c r i p t i o n s t a r t site. S o u t h e r n h y b r i d i z a t i o n a n d s o m a t i c c e l l h y b r i d s a r e u s e d to d e m o n s t r a t e t h a t t h e m u r i n e a n d h u m a n g e n o m e s c o n t a i n r e l a t e d g e n e s for N i l - 2 - a . T C F 8 r e s i d e s o n h u m a n c h r o m o s o m e 1 0 . F l u o r e s c e n t in s i t u h y b r i d i z a t i o n is e m p l o y e d to l o c a l i z e T C F 8 to l O p l 1 . 2 . © 1992 AcademicPress, Inc.

TCF8 encodes a human zinc finger transcription factor (Nil-2-a, negative regulator of IL-2) that represses T-lymphocyte-specific interleukin 2 (IL2) gene expression by interacting with a negative regulatory domain within the IL2 gene promoter/enhancer (15). Transient activation of the IL2 gene is crucial for the immune response (2, 12). Following T cell activation, IL2 m R N A levels rise, peak at about 24 h, and then decline (7, 16). Nil-2-a may play a role in the IL2 m R N A decline by inhibiting transcription of the activated IL2 gene (15). Nil-2-a-related proteins may also suppress immunoglobulin heavy-chain enhancer activity through interaction

A ~

m

m

X

B

8.7 kb

-

+

M

H

-8.7

6.0kb

5.5

4.0 3.8

2.3

2.3

~---

kb

1.8

-2.3 Mouse

Human

1

2

3

4

FIG. 1. Somatic cell hybrid mapping of TCF8. (A) Southern hybridization demonstrates that mouse and humah genomie DNA contain related genes for Nil-2-a. The DNA was digested with the restriction enzymes indicated. (B) Nil-2-a hybridization to mouse/human somatic cell hybrid genomic DNA digested with EcoRI. Hybridizing bands for mouse (lane 3) and human (lane 4) genomie DNA controls are shown with results from representative negative (lane 1) and positive (lane 2) somatic cell hybrid lines. Digested murine and human genomie and somatic cell hybrid DNA (10-12 ~g) isolated by standard methods was eleetrophoresed on 0.7% agarose gels. The DNA was transferred to nylon membranes, which were prehybridized for 4 h at 37°C in a solution containing 25 m M KPO4, pH 7.4, 5× SSC, 5× Denhardt's solution, 50/~g/ml salmon sperm DNA, 50% formamide, and 1% SDS (14). Hybridization to the nick-translated, 32P-labeled Nil-2-a eDNA probe for 14 h at 37°C was in an identical solution except for the addition of 10% dextran sulfate. Membranes were washed for 15 rain at 21°C with solutions containing 2× SSC, 0.1% SDS; 0.5× SSC, 0.1% SDS, and 0.1× SSC, 0.1% SDS. A final wash with 0.1× SSC, 1% SDS was for 30 rain at 42°C.

GENOMICS 14, 194-196 (1992) 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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SHORT COMMUNICATION with a sequence domain similar to its binding site on the IL2 gene promoter (6). The Human Gene Nomenclature Committee has assigned TCF8 as the gene symbol for Nil-2-a. We use DNA gel blot hybridization, somatic cell hybrids, and fluorescent in situ hybridization to show that TCF8 has a related murine counterpart and maps to human chromosome 10p11.2. We performed Southern hybridization of genomic DNA with a human Nil-2-a cDNA probe. These assays suggest that homologous genes encoding Nil-2-a are present in humans and mice (Fig. 1A). EcoRI-digested murine and human DNA yielded 8.7 and 2.3 kb hybridizing bands, respectively, when probed with a gel-purified 1727-bp Nil-2-a cDNA (~8321) (15). We used this probe to screen EcoRI-digested genomic DNA from mouse/ human somatic cell hybrids prepared from 18 unrelated human and 4 murine cell lines. The Southern hybridization results indicate that TCF8 resides on human chromosome 10 (Table 1, Fig. 1B). Each of the 33 mouse/human somatic cell hybrid lines used was characterized by karyotypic and mapped enzyme analysis (9-11). With the use of in situ hybridization (1, 3), we confirmed the presence of TCF8 on chromosome 10 and localized it to band 10p11.2 (Fig. 2). Human metaphase chromosomes were prepared from 5-bromodeoxyuridine

of TCF8

with Human

Concordant number of hybrids

Chromosomes

a

Discordant n u m b e r of hybrids

+/+

-/-

+/-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

8 11 18 12 17 17 18 18 2 28 15 19 14 17 17 14 20 19 7 19 21 11 13

3 2 4 3 3 4 2 3 4 5 2 3 2 2 5 3 1 5 4 3 2 2 3

16 16 9 15 11 11 9 10 22 0 10 9 14 11 10 14 6 9 21 9 7 17 12

000000000000 00000000

q

F I G . 2. F l u o r e s c e n t in situ h y b r i d i z a t i o n localization of T C F 8 on h u m a n c h r o m o s o m e 10. T h e idiogram displays positive signals det e c t e d on b o t h c h r o m a t i d s in 20/44 (45%) of t h e m e t a p h a s e s p r e a d s

hybridizedto biotinylatedNil-2-a genomicDNA. Percentage

Chromosome

P

10

TABLE 1 Segregation

195

/+

discordance

1 3 1 2 2 1 3 2 1 0 2 2 3 3 0 2 3 0 1 2 3 3 2

61 59 31 53 39 36 38 36 79 0 41 33 52 42 31 48 30 27 67 33 3O 61 47

T h e N i l - 2 - a c D N A p r o b e w a s h y b r i d i z e d to b l o t s c o n t a i n i n g

EcoRI-digested D N A f r o m h u m a n / m o u s e hybrids. Scoring was determ i n e d by t h e p r e s e n c e or a b s e n c e of t h e hybridizing 2.3-kb h u m a n b a n d a n d c o m p a r e d to t h e p r e s e n c e or a b s e n c e of h u m a n c h r o m o s o m e s in each hybrid. A 0% d i s c o r d a n c y i n d i c a t e s a m a t c h e d segregation of t h e Nil-2-a probe w i t h a h u m a n c h r o m o s o m e .

synchronized lymphocytes. A 12-kb genomic DNA for Nil-2-a was isolated by hybridization screening of an EMBL-3 human genomic library with the Nil-2-a cDNA probe described above. Southern hybridization experiments demonstrated that a 5.5-kb XhoI/BamHI fragment of this clone specifically hybridized to the Nil-2-a cDNA probe (Williams, unpublished data). The Nil-2-a genomic probe, biotinylated with the use of the random primer method, was hybridized to the chromosomes and detected with fluorescein-conjugated avidin. DAPI and propidium counterstains generated Q and R bands, which were evaluated with a Nikon fluorescence microscope to identify chromosomes. Of 44 metaphases examined, a fluorescent signal was detected at 10p11.2 on both chromatids of a single chromosome 10 in 20 of the metaphases. One of the 20 had signals on both chromatids of both chromosomes 10 at 10p11.2. One other of the 20 had an additional single signal on one chromatid of the other chromosome 10 at 10p11.2. Of the remaining 24 cells examined, 16 had no fluorescent signals detected, 2 had a signal on one chromatid of both chromosomes 10 at p11.2, and 6 had a signal on one chromosome 10 at p11.2. Figure 2 demonstrates the localization of the Nil-2-a DNA probe to 10p11.2. A number of genes that encode transcription factors have been cloned at the breakpoints of chromosome translocations associated with neoplasms (13). Several

196

SHORT COMMUNICATION

cases of a d u l t T cell l e u k e m i a h a v e b e e n r e p o r t e d w i t h karyotypes demonstrating translocations involving 1 0 p l l i n c l u d i n g t ( 1 0 ; 1 4 ) ( p l l ; q 3 2 ) (4, 8). T h e s e o b s e r v a t i o n s are i n t e r e s t i n g g i v e n t h e fact t h a t N i l - 2 - a r e g u l a t e s IL2 gene e x p r e s s i o n , a c y t o k i n e c r u c i a l for T cell p r o l i f e r ation. Translocations involving 10pll [t(10;ll)(pllp 1 5 ; q 2 3 ) a n d i n s ( 1 0 ; l l ) ( p l l ; q 2 3 q 2 4 ) ] h a v e b e e n reported by several groups in acute monocytic leukemia [cited i n (5)]. T h e b r e a k p o i n t s of t h e s e t r a n s l o c a t i o n s have not been molecularly cloned, a n d the genes inv o l v e d o n c h r o m o s o m e 10 are n o t k n o w n . S t u d i e s are u n d e r w a y to d e t e r m i n e w h e t h e r t h e e x p r e s s i o n a n d s t r u c t u r e of t h e T C F 8 g e n e is a l t e r e d i n n e o p l a s m s w i t h 10pll translocations.

5.

6.

7.

8.

9. ACKNOWLEDGMENTS We thank C. L. Willman, B. Hjelle, and T. S. McConnell for gifts of genomic DNA and for helpful discussions. This work was supported by the National Institutes of Health (CA-54428, T.M.W., and HG00333 and HD-05196, T.B.S.) and dedicated funds from the University of New Mexico. T.M.W. is the recipient of a Junior Faculty Research Award from the American Cancer Society.

10.

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