GENOMICS

13,658-664

(19%)

Characterization and Chromosomal Mapping of the Gene Encoding the Cellular DNA Binding Protein HTLF CHING LI, *, ’ ALDONS J. LUSIS,* ROBERT SwmKEs, t SANH-MOC

TRAN, * AND RICHARD GAYNOR*

*Department of Medicine, Microbiology and Molecular Genetics, and tDivision of Medical Genetics, Department of Medicine, University of California, Los Angeles, California 90024; and *Division of Molecular Virology, Department of Medicine and Microbiology, University of Texas Southwestern Medical School, Dallas, Texas 75235-8594 Received

December

19, 1991;

INTRODUCTION

The regulation of viral gene expression is dependent on the interplay of both viral and cellular proteins (Mitchell and Tjian, 1989). The mechanisms by which these proteins induce gene expression are not known. However, it is possible that viral regulatory proteins may function by interacting either directly or indirectly with cellular factors to stimulate the binding or activity of these factors. Thus an identification study of different cellular factors that bind to viral regulatory regions is critical for understanding the mechanisms governing viral gene expression. The human T-cell leukemia virus (HTLV-I), an oncogenie retrovirus, is the causative agent of adult T-cell leukemia (Poiesz et al., 1980; Yoshida et al., 1982). Numerous studies have investigated &-acting regulatory elements involved in the regulation of HTLV-I LTR

O&38-7543/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

February

26, 1992

(Brady et al., 1987; Shimotohno et al., 1986; Bosselut et al., 1990). Both cellular factors binding to different elements in the long terminal repeat (Altman et al., 1988) and the transactivator protein tax (Chen et al., 1985; Felber et al., 1985; Fujisawa et al., 1985) are involved in regulating HTLV-I gene expression. DNase I footprinting and extensive mutagenesis indicate that three conserved sequences in the LTR known as 21-bp repeats are required for activation by tax (Shimotohno et al., 1986; Brady et al., 1987). In addition, a regulatory element located between the second and third 21-bp repeats is also required for high level viral gene expression (Bosselut et al., 1990). Multiple purine-rich sequences located in this latter element have homology to consensus binding sites for the ets family of DNA binding proteins (Watson et al., 1988; Karim et al., 1990). The Etsl and Ets2 proteins are capable of binding to and activating gene expression via multiple motifs in this latter element (Bosselut et al., 1990; Gitlin et al., 1991). The tax protein does not bind directly to the HTLV-I LTR but rather interacts indirectly with cellular DNA binding proteins (Marriot et al., 1989). Thus, to understand tax activation, an identification of different cellular factors binding to the HTLV-I LTR is critical. In an attempt to characterize other cellular factors that bind to purine-rich regulatory sequences, X gtll expression cloning was performed (Singh et al., 1988). Oligonucleotides corresponding to a region extending between -155 and -117 in the HTLV-I LTR were used to screen X gtll expression libraries. We identified a cellular factor, which we designated human T-cell leukemia virus enhancer factor (HTLF), that bound specifically to this element. HTLF contains a DNA binding domain that contains a region of st,rong homology with the Drosophila fork head protein (Weigel et al., 1989). The fork head region of homology has previously been demonstrated to be required for the DNA binding of other cellular transcription factors including HNF-3 (Lai et al., 1990, 1991) and ILF (Li et al., 1991). HTLF also binds specifically to purine-rich regulatory elements found in the HIV-l LTR and IL2 promoter, which serve as bind-

A region of the human T-cell leukemia virus long terminal repeat (HTLV-I LTR) located between -155 and - 117 is important in the regulation of gene expression by the ets family of transcription factors. In an attempt to identify additional cellular transcription factors that bind to this portion of the HTLV-I LTR, we used X gtl 1 expression cloning with oligonucleotides corresponding to this element. A 1239-bp cDNA was isolated from a Jurkat cDNA library, which encoded a protein capable of binding to this purine-rich region. This protein, which we designated human T-cell leukemia virus enhancer factor (HTLF), contains a domain with homology to the recently described fork head DNA binding domain. Chromosome mapping of the HTLF gene demonstrated that it was localized to human chromosome 2plSp22. HTLF is a unique cellular gene that may function in the transcriptional regulation of HTLV-I LTR. 8 1992 Academic Press. hc.

’ Current address: Institute of Biomedical Science, National Hua University, Hsin Chu 300, Taiwan, Republic of China.

revised

Tsing

658

CELLULAR

DNA

BINDING

ing sites for ILF. Chromosomal mapping of the HTLF gene indicates that it is located on human chromosome 2p16-~22.

MATERIALS

AND

METHODS

Identification of HTLFgene and construction of fusions withglutathione S-transferme. A 266-bp cDNA was identified by screening a Jurkat X gtll cDNA expression library (obtained commercially from Clontech, Inc.) with 32P-labeled double-stranded ligated oligonucleotides corresponding to a sequence located in HTLV-I LTR extending from -155 to -117. The phage were induced with IPTG to produce fusion proteins, which bound to the labeled oligonucleotides as described (Singh et al., 1988). The sequences of the oligonucleotides are 5’.GGAAGCCACCAAGAACCACCCATTTCCTCCCCATGTTTG3’ and its complement. Positive phage were subsequently purified with both wildtype and mutated HTLV-I oligonucleotides to identify clones with specific binding. The 266.bp fragment was labeled by random priming and used to screen a human Jurkat cDNA A ZAP library in an attempt to identify full-length cDNA clones. A 1239-bp cDNA fragment was identified, and DNA sequence analysis of the clone was performed using the Sanger method with the Sequenase system. For expression of HTLF as a glutathione S-transferase (GST) fusion protein in bacteria (Smith and Johnson, 1988), a 0.9 -kbPfmI/ PuuII fragment extending from nucleotides 290 to 1210 in the HTLF cDNA was isolated, treated with mungbean nuclease, and subsequently cloned in frame into the SmaI site of pGEX-3X (Pharmacia) to express a glutathione S-transferase GST/HTLF fusion protein of 60 kDa. Escherichia coli strains and preparation of bacterial expressed proteins. E. coli strains used in these experiments were Y1090, for screening the h gtll expression cDNA library, XL-1 Blue, for screening the A ZAP cDNA library, and TG-1, for transformation of recombinant plasmids and expression of the GST wildtype and GST/HTLF fusion proteins in bacteria. GST and GST/HTLF fusion proteins were prepared by induction of E. coli TG-1 cultures with 0.5 mM IPTG at an OD,,, of 0.6-1.0. E. coli were harvested and lysed, and supernatants were subjected to glutathione Sepharose 4B column chromatography (Pharmacia) for the purification of the wildtype and fusion proteins as described by the manufacturer. DTT and PMSF were each added to all buffers to a final concentration of 0.5 m&f. Gel retardation analysis. Double-stranded oligonucleotides corresponding to the HTLF motif in the HTLV-I LTR between -155 and -117 were end-labeled with [y-s’P]ATP and used in gel retardation analysis with either a 5- or a 20-fold molar excess of (1) HTLF-specific binding site oligonucleotides, motifs in the HIV-I LTR between (2) -283 and -256 (distal) (3) or between -223 and -195 (proximal), (4) the NFAT motif in the IL2 promoter between -285 and -254, (5) an AP-1 binding site in the adenovirus early region 3 promoter between -103 and -83, and (6) an HNF-3A binding site in the mouse transthyretin promoter extending from -111 to -85. The sequence of the oligonucleotides are shown below: (1) HTLV-I LTR 5’-GGAAGCCACCAAGAACCACCATTTCCTCCCCATGTTTG-3’ (2) HIV LTR (distal) B-GAACAGGCCAATGAAGGAGAGAACAACA-3’ (3) HIV LTR (proximal) S-GAGGACGCGGAGAAAGAAGTGTTAGTGTG-3’ (4) IL2 5’.AATTGGAGGAAAAACTGTTTCATACAGAAGGCGT-3’ (5) AP-15’-GAAGTTCAGATGACTAACTCA-3’ (6) HNF-3A 5’-TCGAGTTGACTAAGTCAATAATCAGAATCAG-3’ Chromosomal mapping. Chromosomal assignment was determined by analysis of a panel of mouse-human somatic cell hybrids varying in human chromosome content. A panel of 17 mouse-human

PROTEIN

659

HTLF

somatic cell hybrid clones was constructed and analyzed for chromosome content as described previously (Mohandas et al., 1986). DNA was isolated from nuclei of these clones as well as from the parental mouse cell line (B82, GM 0347A) and the human male fibroblast parental cell line (IMR91). DNA isolation and filter hybridization analysis were performed using a HTLF cDNA labeled with “P to a specific activity of about lo9 cpm/mg, essentially as previously described (Frank et al., 1988). Regional mapping to human lymphocytes was performed by in situ hybridization. The HTLF cDNA probe was labeled by oligonucleotide priming with 3H-labeled deoxyribonucleotides to a specific activity of about 4 X 10’ cpm/mg. The probe was then hybridized to chromosomes from normal human lymphocytes using a method developed by Harper and Saunders (1981) as modified by Cannizzaro and Emanuel (1984). The slides were exposed for 10 days, and all silver grains on or touching chromosomes were scored.

RESULTS

Identification

of HTLF

To characterize cellular factors that bind to a critical purine-rich regulatory element extending between -155 and -117 in the HTLV-I LTR, oligonucleotides corresponding to this region were used to screen a Jurkat Tcell X gtll expression library (Singh et al., 1988). Several identical cDNAs expressed fusion proteins that bound specifically to these oligonucleotides. DNA sequence analysis of each of these cDNAs indicated that they extended from nucleotides 222 to 487 (Fig. 1). One of these cDNAs was used to screen additional Jurkat and HeLa libraries. A number of cDNAs of different sizes but with identical sequences were identified from both types of libraries. The largest cDNA identified, which we designated HTLF, contained a 1239-bp cDNA insert, the sequence of which could potentially encode a protein of 320 amino acids (Fig. 1). Multiple stop codons upstream of a potential initiating methionine (data not shown) were noted. However, no stop codons were identified in the carboxyl-terminus of this cDNA, indicating that the full-length protein coding sequence for the HTLF cDNA had not been identified. HTLF Contains a Fork Head DNA Binding Domain A striking feature of HTLF was the fact that a portion of this protein contained strong homology with the fork head DNA binding domain (Figs. 1 and 2). This domain was originally described in the Drosophila protein fork head, which regulates terminal segment development (Weigel et al., 1989). We have recently described another cellular transcription factor known as ILF (Li et aZ., 1991), which binds to purine-rich motifs in the HIV-l LTR and IL2 promoters. A region of the ILF protein also has strong homology with the fork head DNA binding domain. Fork head DNA binding domains have also been identified in a group of hepatocyte-specific DNA binding proteins known as HNF-3 (Lai et al., 1990, 1991), which regulate the expression of the transthyretin and albumin genes (Costa et al., 1989). A schematic

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ATG 282 (1)

ATT 1239 (320)

FIG. 1. Structure of HTLF cDNA. (A) An open reading frame of 320 amino acids in the HTLF cDNA extending from nucleotides 282 to 1239 is shown. The homology to the fork head DNA binding domain within this cDNA is shaded. (B) Schematic of the HTLF cDNA clone with the position of the original X gtll clone (hl), the region of homology with the jerk head DNA binding domain, and the position of the shaded 320-amino-acid open reading frame.

indicating the degree of homology between these various fork head DNA binding domains is indicated in Fig. 2. HTLF

Binds Specifically

to Purine-Rich

Motifs

The purine-rich elements in the HTLV-I LTR, which bind HTLF, have a high degree of homology with elements in the HIV-l LTR and IL2 promoter, which bind ILF (Fig. 3). To determine the binding specificity of HTLF, we cloned a portion of the HTLF cDNA downstream of the glutathione S-transferase gene in the prokaryotic expression vector pGEX (Smith and Johnson, 1988). This GST/HTLF fusion protein was purified using glutathione sepharose allinity chromatography. Gel retardation analysis was performed using oligonucleotides extending from -155 to -117 in the HTLV-I LTR.

There was no binding of glutathione S-transferase protein alone to this probe (Fig. 4, lane 0). The GST/HTLF protein bound strongly to these same oligonucleotides (Fig. 4, lane 1). Competition analysis was performed to determine the specificity of this binding. Both a 5- and a 20-fold molar excess of those oligonucleotides corresponding to a region of the HTLV-I LTR resulted in competition of the HTLF gel-retarded species (Fig. 4, lanes 2 and 3). Similar purine-rich sequences present in distal and proximal regions of the HIV-l LTR (Fig. 4, lanes 4-7) and the NFAT motif in the IL2 promoter (Fig. 4, lanes 8 and 9) resulted in marked competition of the HTLF gel-retarded species. Two other oligonucleotides corresponding to either HNF-3 or AP-1 binding sites, which markedly differ in their sequences from the HTLV-I oligonucleotides, did not result in significant 50

59

HNF-3Y HNF-3a HNF-3p FORK HEAD ILF HTLF

HNF-3y HNF-3a

ILF HTLF

FIG. 2. Homology of HTLF with other with homologous regions found in the DNA portion of the Drosophila fork head protein

fork head binding proteins. A IlO-amino-acid portion of the HTLF protein (13 to 113) is aligned binding domains of the hepatocyte nuclear factors HNF-3 ~(777136), cu(160-269), P(149-258), a (200-309), and the interleukin enhancer binding factor (ILF) (135 to 244).

CELLULAR HTLV-I LTR

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(p) -229

5 ’ - GACGCGGAGAAAGAA-3’

-291

BINDING

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IL-2

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PROTEIN

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-2n

- AGAAAGGAGGAAAAA-3’

FIG. 3. Homology of purine-rich regulatory elements. Related purine-rich regulatory sequences found in the HTLV-I LTR, two elements in the HIV-l LTR, and the IL2 promoter are shown. The positions of these regions in their respective promoter elements are also indicated. -2.OKb

competition of the HTLF gel-retarded species (Fig. 4, lanes 10-13). These results suggest that HTLF bound specifically to related purine-rich sequences in the HTLV-I, HIV-l, and IL2 promoters. Chromosomal Localization of HTLF The chromosomal localization of the gene for HTLF was determined by Southern blotting analysis of a panel of mouse-human genetic cell hybrids derived by fusion of normal male fibroblast (IMR 91) with thymidine kinase-deficient mouse B82 cells (Mohandas et al., 1986). The hybrids contained various human chromosomes as defined by karyotyping, and they have now been used for the chromosomal assignment of a large number of human genes. After digestion of human genomic DNA with HindIII, a major band of 2.0 kb, as well as intense bands at about 7.0 and 1.0 kb, hybridized to the HLTF cDNA upon Southern blotting analysis (Fig. 5). Despite the relatively stringent hybridization and washing conditions, mouse genomic DNA (GM 0349A) yielded hybridizing HTLV

0

1

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HIV-d

HIV-p

NFAT

HNF3A

AP-1

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133

analysis of HTLF. Oligonucleotides FIG. 4. Gel retardation corresponding to the region of the HTLV-I LTR extending from -155 to -117 were used in gel retardation analysis with either glutathione S-transferase alone (lane 0) or with the glutathione S-transferase/ HTLF fusion protein (lanes l-13). Gel retardation was performed either without competition (lane 1) or with either a 5 or 20-fold molar excess of unlabeled oligonucleotides for competition with each set of binding elements corresponding to the HTLV-I LTR purine-rich regu latory element (lanes 2 and 3), HIV-I LTR distal NFAT element (lanes 4 and 51, HIV-I LTR proximal NFAT element (lanes 6 and 71, IL2 NFAT element (lanes 8 and 9), HNF-3 element (lanes 10 and II), or AP-1 element (lanes 12 and 13).

FIG. 5. Southern hybridization analysis of HTLF gene sequences in DNA from a panel of mouse-human somatic cell hybrid clones. DNA isolated from hybrid cells (84-2 through 84-391, a mouse cell line (GM 0349A), or human leukocytes (Hl was digested with Hind111 and subjected to gel electrophoresis, and Southern analysis was performed using a portion of the HTLF cDNA. The position of the strongly hybridizing 2.0-kb band present in human genomic DNA is indicated.

fragments of about 7.0 and 1.0 kb (Fig. 5). Blotting analysis of HindIII-digested DNA isolated from the panel of 14 mouse-human genetic cell hybrids revealed that the 2.0-kb fragment cosegregated with human chromosome 2 (Fig. 5). Thus, hybrid clones 84-2, 84-4, 84-20, and 84-27 all contained chromosome 2 and upon Southern analysis showed the 2.0-kb species hybridizing to the HTLF cDNA, while the remaining clones that did not contain this species also lacked chromosome 2. The relatively light signal corresponding to the human fragments observed with some of the hybrid clones reflected the relatively low content of human chromosome 2 in these clones. In particular, hybrid clone 84-20 contained human chromosome 2 in only 10-30s of the metaphase chromosomes analyzed. There were three or more discordancies between the human HTLF gene sequences and each of the remaining chromosomes, indicating that the HTLF gene resided on chromosome 2. The regional localization of the HTLF was examined by in situ hybridization of HTLF cDNA to normal metaphase chromosomes. Figure 6A shows the distribution of silver grains in histogram form over the full set of chromosomes observed after exposure to photographic emulsion for 1 week. A significant accumulation of grains was observed on the short arm of chromosome 2 with peak accumulation in the 2p16-p22 region as portrayed in the schematic representation of chromosome 2 (Fig. 6B). These results confirm the somatic cell hybrid analysis studies and further localized HTLF to human chromosome 2p16-~22. DISCUSSION

A variety of cellular factors are involved in regulating HTLV-I gene expression. Previous studies have indi-

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2 FIG. 6. In situ hybridization of HTLF cDNA to human metaphase chromosomes. depicting grains observed in a total of 503 cells, 80 of which contained grains. Of a total (B) The grain density over the individual bands of chromosome 2 are shown.

cated that three conserved 21-bp repeat sequences are critical for activation by the transactivator protein tax (Chen et al., 1985; Felber et al., 1985; Fujisawa et al., 1985). The 21-bp repeat sequences serve as the binding site for a variety of different cellular transcription factors (Altman et al., 1988; Shimotohno et al., 1986; Brady et al., 1987). In addition, a purine-rich region located between the second and third 21-bp repeats has been shown to be involved in the activation of HTLV-I (Bosselut et al., 1990). Recently it was demonstrated that members of the ets family of transcription factors are capable of binding to this purine-rich element (Watson et al., 1988, Klemsz et al., 1990; Karim et al., 1990; Bosselut et al., 1990; Gitlin et al., 1991). Both the Etsl and Ets2 are capable of activating gene expression via this sequence, but it is not known whether Etsl or Ets2 are able to interact with tax (Bosselut et al., 1990).

(A) A schematic representation of 103 grains, 20 grains occurred

of human chromosomes in the region 2p16-~21.

In an attempt to identify additional cellular factors that bind to this purine-rich sequence in the HTLV-I LTR, X gtll expression cloning was performed with oligonucleotides corresponding to this region (Singh et al., 1988). Several cDNAs that bound specifically to these oligonucleotides were isolated. One cDNA designated HTLF contained an open reading frame of 320 amino acids, although no stop codons were present in the 3’ portion of the clone, indicating that a full-length protein had not been identified. The coding sequence of HTLF exhibited a high degree of homology in its DNA binding domain with several other DNA binding proteins that contain the recently described fork head DNA binding domain (Lai et al., 1990, 1991; Li et al., 1991). Northern analysis revealed that HTLF mRNA was ubiquitously expressed with a transcript size of approximately 3.0 kb by Northern analysis.

CELLULAR

DNA

BINDING

We recently identified another DNA binding protein known as ILF, which binds to purine-rich sequences in the HIV-l LTR and IL2 promoter (Li et al., 1991). ILF also contains a fork head DNA binding domain, which is required for its binding to related purine-rich sequences (Li et al., 1991). In addition, we have recently demonstrated that the ILF protein is capable of binding to the HTLV-I LTR between -155 and -117 (unpublished observation). These results suggest that the fork head DNA binding domain may be capable of binding to a number of related purine-rich motifs in both viral and cellular promoters. Interestingly, several members of the ets family of proteins share a common DNA binding domain, which is important in binding to purine-rich sequences (Karim et al., 1990). The ets proteins have been demonstrated to be transcriptional activators (Klemsz et al., 1990; Bosselut et al., 1990). Thus it is possible that transcription factors containing either ets or fork head DNA binding domain may compete for binding to similar motifs with different effects on either viral or cellular gene expression. Studies are underway to address whether ets or HTLF proteins may be targets of the transactivator protein tax. The chromosomal localization of HTLF indicates that it is localized to human chromosome 2p16-~22. This region is the site of several known genes, including carbamoyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase, fl-spectrin, and a fibronectinlike protein (Povey and Falk, 1989). The HTLF gene is distinct from another fork head domain DNA binding protein, ILF, which is localized to chromosome 17q25. Interestingly, translocations of chromosome 2p16-p22 with chromosome llq23 have been reported in some cases of human leukemia (Bloomfield and de la Chappelle, 1987). Since the ets-1 oncogene is localized to this latter region (Watson et al., 1988), it is possible that translocations in human leukemia could potentially result in fusions between different portions of the ets-1 and HTLF genes. Further studies will be necessary to determine whether translocations of these two DNA binding proteins occur in human leukemia. ACKNOWLEDGMENTS This work was supported by NIH Grants HL28481 and HL42488 to A.J.L. and by grants from the NIH, Veterans Administration, and American Cancer Society to R.G. We are grateful for the technical assistance of Ivana Klisak, Anh Diep, Yurong Xia, and Ping-Xi Wen.

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PROTEIN

663

HTLF

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Smith, D. B., and Johnson, K. S. (1988). Single-step polypeptides expressed in E. coli as fusions with transferase. Gene 67: 31-40.

purification glutathione

of S-

Duesberg, ets-I and Acad. Sci. H. (1989). and is exCell 57:

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