SHORT COMMUNICATION

Mapping of the Human Gene for the Human lmmunodeficiency Virus Type 1 Enhancer Binding Protein HIWEP2

to Chromosome 6q23-q24

TATSUHIKO SuDo,**t KAZUO OZAWA,* EI-ICHI SOEDA,* NOBUO NOMURA,$ AND SHUNSUKE ISHII*,’

*Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, lbaraki 305; tlnstitute of Medical Science, University of Tokyo, Minato-ku, Tokyo 708; and *Institute of

Gerontofogy, Nippon Medical School, Nakahara-ku, Yokohama, Kanagawa 211, Japan

Received April 22, 1991; revised September 3, 1991

The human gene encoding the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 has been isolated. Using Southern analysis of human-rodent so- matic cell hybrid DNA with a human HIV-EPS-specific cDNA probe, the HIV-EP2 gene was assigned to chromo- some 6. The gene was further localized to the region 6q23- 24 by fluorescence in situ hybridization. o 1992 Academic

Press, Inc.

Human immunodeficiency virus (HIV), a cyto- pathic retrovirus, is the etiologic agent of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinousi et al., 1983; Gallo et al., 1984). Transcription of HIV-l in latently infected T lymphocytes is induced by com- pounds such as phorbol esters, which induce the bind- ing of a transcriptional activator to the HIV-l en- hancer (Nabel and Baltimore, 1987). The HIV-l en- hancer contains a sequence homologous to the immunoglobulin K gene enhancer (Rosen et al, 1985), and similar sequences are found in the enhancers of genes such as that for 02 microglobulin.

By cDNA cloning, two different types of proteins were found to bind to the HIV-l enhancer, HIV-EPl (Maekawa et al., 1989), and NF-KB (Kieran et al., 1990; Ghosh et al., 1990). HIV-EPl contains the metal finger structure in its DNA-binding domain. PRDII-BFl and MBP-1, which were isolated using the interferon p gene promoter probe and MHC en- hancer probe, respectively, were shown to be identical to HIV-EPl (Fan and Maniatis, 1990; Baldwin et al., 1990). In contrast, NF-KB has homology with the rel oncoprotein in its DNA-binding domain. By cDNA cloning, we have identified a new member of the HIV- EPl family, HIV-EP2, which is expressed at a high level in T cells (Nomura et al., 1991). HIV-EP2 pro- tein is highly homologous with HIV-EPl in three re- gions that contain the DNA-binding domain, the po- tential nuclear localization signal, and a cluster of

1 To whom correspondence should be addressed.

acidic amino acids. The DNA-binding properties of HIV-EP2 protein are similar to those of HIV-EPl. Because the enhancer element recognized by HIV- EP2 is important for the transcriptional regulation of various genes, HIV-EP2 could be implicated in some diseases. In this study, we used hybridization with DNA isolated from a panel of human-rodent somatic cell hybrids and fluorescent in situ hybridization to locate the HIV-EP2 gene on the chromosome.

The 1988-bp BamHI fragment (nucleotides 187- 2175) of human HIV-EP2 cDNA was used for South- ern analysis of human-rodent somatic cell hybrid DNA (purchased from Bios Corp., New Haven, CT). These were genomic DNAs from a panel of 25 hu- man-hamster hybrid cell lines plus human and ham- ster control DNA (Kouri et al, 1989). Hybridization was done at 42°C in a mixture containing 50% form- amide, 5~ SSC, 5X Denhardt’s solution, and 0.1% SDS. The blots were washed at room temperature in 0.1X SSC plus 0.1% SDS. In situ hybridization was done by the procedures of Lichter et al., (1990) fol- lowed by staining (G banding) of chromosomes as de- scribed by Zabel et al. (1983). A biotinylated probe was prepared by nick translation of a plasmid con- taining the full-length cDNA of HIV-EP2 with biotin-14dATP. Human metaphase cells were pre- pared from phytohemagglutinin-stimulated periph- eral blood lymphocytes.

In a Southern blot experiment, the labeled HIV- EP2 probe recognized both human and rodent HIV- EP2 genes. The HIV-EP2 probe detected two Hind111 fragments (3.1 and 1.9 kb) in human DNA, and two fragments (7.5 and 1.4 kb) in hamster (data reviewed, but not shown). No cross-reaction with any sequence derived from the other related genes was detected under the hybridizing and washing conditions used (data not shown). The results of Southern blot analy- sis of genomic DNA from 18 hamster X human hy- brids probed with HIV-EP2 are shown in Table 1. Two diagnostic bands of 3.1 and 1.9 kb were highly discordant with all chromosomes except chromosome 6, which showed no discordance. From these data we

167 GENOMICS 12,167-170(1992)

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TABL

E 1

Segr

egat

ion

of

Hum

an

HIV

-EPP

in

H

uman

-Ham

ster

H

ybrid

s

Hum

an

chro

mos

ome

Hybr

id HI

V-EP

2 1

2 3

4 5

6 7

8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

X Y

937

654

507

983

1079

1006

756

904

909

967

862

860

1049

683

867

750

212

734

- +

- - -

+ -

+ -

+ -

+ -

- -

- -

- (+

I -

-

- -

- + - - - - - - - - - - - - -

- - + - + - - - - - - (+) - - - - - -

- - - - - (+I - - - - - - - - - - -

+ + + + + + + + + + + + + + + + + +

- - - - - - + + + - - + - - - - - -

- - - - - + + - - - - - - - - - -

- - - - + - - + + - - - - - - -

- - - - - - - - - - + - - - - - - -

- - - + (+) - - - - - - (+I - - - - -

- - - - - - - - - - - - + + - - - -

- - + - - - + + - - - - - (+) - - - -

- - - - - + + - - - - - - - + + - -

+ - + - - - (+I - + - - - - + + + -

+ - - - (+I + - - - - - - - - + - -

- - - - + - - (+) - + - - - - - - - -

+ - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - + - - +

- - - + + - - - - (+I - + + + -

- - (+) - - - + - - - - - - - - - - -

+ - - - - + + + - - - + - + - - - -

- - (+) - - - - - - - - - - + - - - -

- - - - - - - - f - - - - - - - + -

HIV-

EPB/

chro

mso

me

++

0 0

1 0

4 4

1 1

0 1

0 2

1 2

0 1

0 0

2 1

3 0

1 2

-- 12

13

12

13

0

14

13

12

13

12

12

12

11

9 10

12

13

12

10

13

11

12

14

12

+-

4 4

3 4

0 0

3 3

4 3

4 2

3 2

4 3

4 4

2 3

1 4

3 2

-+

2 1

2 1

14

0 1

2 1

2 2

2 3

5 4

2 1

2 4

1 3

2 0

1

Note

. +

and

-, de

finite

sc

orin

g of

th

e pr

esen

ce

or

abse

nce

of

hum

an

chro

mos

ome;

(+

), on

ly pa

rt of

ch

rom

osom

e pr

esen

t (d

ue

to

dele

tion)

or

pr

esen

ce

of

chro

mos

ome

unde

r 55

%

(from

5

to

55%

).

SHORT COMMUNICATION 169

FIG. 1. Localization of HIV-EP2 gene by fluorescence in situ hybridization. (Left) Biotinylated HIV-EP2 cDNA was FITC. Arrow denotes cDNA signal. (Right) G-banding pattern of metaphase chromosomes is indicated.

detected with

concluded that human HIV-EP2 was on chromo- some 6.

To locate the human HIV-EP2 gene more closely, we used fluorescence in situ hybridization. Figure 1 displays the localization of the hybridization signal (FITC) on chromosome 6q23-q24, corroborating the results of the Southern blot analysis of the somatic cell hybrids. This result was confirmed by analysis of 15 cells (data not shown). Under the condition used here, the fluorescent signals were observed on both chromosomes in all 15 cells, and there was no signal on other positions except for chromosome 6q23-q24. Thus, our data demonstrate that the human HIV- EP2 gene is at position 6q23-q24.

Recently, Gaynor et al. (1991) assigned the human HIV-EPl gene to 6p22.3-~24. Thus, the locus of the HIV-EP2 is not closely linked to that of the HIV-EPl gene. Recently we have isolated cDNAs encoding the third member, HIV-EP3, of the HIV-EP gene family (data not shown). All of three members have similar sequence specificity for DNA binding. It will be of particular interest to know whether their chromo- somal position has a role in the regulation of expres- sion of these genes.

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170 SHORT COMMUNICATION

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