MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1754-1758 0270-7306/91/031754-05$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 3

The TISli Primary Response Gene Is a Member of a Gene Family That Encodes Proteins with a Highly Conserved Sequence Containing an Unusual Cys-His Repeat BRIAN C. VARNUM,'t QIUFU MA,' TIANHUAI CHI,' BRADLEY FLETCHER,' AND HARVEY R. HERSCHMANl 2* Department of Biological Chemistry, Molecular Biology Institute,' and Laboratory of Biomedical and Environmental Sciences,2 University of California Los Angeles Center for the Health Sciences, Los Angeles, California 90024 Received 20 August 1990/Accepted 3 December 1990

The TISll primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol13-acetate and growth factors. The predicted TISll protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TISllb and TISlld, that contain the YKTELC sequence. TISll, TISlib, and TISlld proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS1l gene family members are not coordinately expressed: (i) unlike TIS1l, the TISllb and TISlid mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TISllb and TISlld; and (iui) unlike TIS1l, TISllb expression is extinguished in PC12 pheochromocytoma cells. We have isolated cDNAs for a number of primary-re(24) whose mRNA levels are rapidly and transiently elevated in Swiss 3T3 cells in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We term these genes TPA-induced sequences, or TIS genes (14). TIS genes are also induced in both BALB/c 3T3 and Swiss 373 cells by polypeptide mitogens such as epidermal growth factor (EGF) or fibroblast growth factor (FGF) (14, 15) and during nonmitogenic responses such as nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells (12) and neurotransmitter stimulation of cultured glial cells (1). Several of the TIS genes have also been cloned from serum-treated BALB/c 3T3 cells and NGFtreated PC12 cells (reviewed in reference 8). Corrected sequence of TISll. The sequence of the predicted protein encoded by the TIS11 gene (23) includes both an internal repeat, which we term the YKTELC repeat, and a repeated cysteine-histidine motif, CX8CX5CX3H, which begins at the cysteine residues of each YKTELC sequence. During the preparation of the manuscript the tris-tetrapolin (TTP [13]) and Nup475 (5) cDNAs were cloned from insulintreated NIH 3T3 cells and serum-treated BALB/c 3T3 cells, respectively. TIS11 and TTP/Nup475 are identical over a 103-amino-acid region that includes the repeated YKTEL and Cys-His motifs, but diverge at their amino- and carboxyflanking sequences. The C-terminal divergence is the result of an additional nucleotide in TTP/Nup475 that shifts the reading frame. Differences in the amino-terminal portion of TIS11 and TTP/Nup475 cannot be resolved by shifts in the reading frame; their nucleotide sequences differ completely. We carried out polymerase chain reaction (PCR) amplification from mRNA of TPA-treated Swiss 3T3 cells, using a primer common to TTP/Nup475 and TIS11 in their 3' regions and primers unique to the TIS11 and TTP/Nup475 sequences in their 5' (amino-terminal) regions. A PCR product was obtained with the TTPINup475 5' primer, but not with the

TIS11 5' primer. We conclude that the original TIS11 cDNA is likely to be the product of a cloning artifact that created a chimeric cDNA molecule. The PCR product was cloned into M13 and sequenced. The additional C residue reported in TTP (13) was clearly present and shifted the TIS11 reading frame to give a protein whose C-terminal sequence is identical to that of TTP/Nup475 (5, 13). Our sequence of the 5' region of the cloned cDNA from the PCR was identical to that described for TTP/Nup475 (Fig. 1). Antiserum to recombinant Nup475 protein detects transiently elevated nuclear staining in serum-treated cells (5). The repeated Cys-His motif, serine-proline-rich domains, and nuclear localization all suggest that the TIS11/TTPINup475 protein may be a rapidly inducible transcription factor. Cloning and sequence of TISllb cDNA. Arguing by analogy to the importance of conserved amino acid sequence motifs within the fos, jun, and egr families of primary response genes (8), we suspected that the YKTELC and Cys-His repeats in the TIS11 protein might represent an important, conserved domain present in another family of proteins. We used a degenerate oligonucleotide for the YKTELC sequence to screen a mitogen-induced primary response library from BALB/c 3T3 cells (23) for cDNA clones of related sequence. Filter replicas containing 30,000 lambda gtlO phage plaques were prepared (14) and probed either with a random-primed (18) TIS11 cDNA probe or with the kinase-treated (18) degenerate oligonucleotide TCAA(G/A) ACNGA(A/G)CT(C/G)TG(T/G), the reverse translation of the YKTELC sequence. One clone, TISlib, that hybridized with the degenerate oligonucleotide but not the TIS11 cDNA, was picked and plaque purified. The cDNA insert was subcloned and sequenced. A 338-amino-acid open reading frame initiating from an ATG that is consistent with Kozak's rules (11) was identified (Fig. 1). TIS11 and TISlib proteins share a 67-amino-acid region of 72% identity which includes the repeated YKTELC and CX8CX5CX3H motifs. Gomperts et al. (6) recently reported the sequence of cMG1, a cDNA cloned from EGF-treated rat epithelial cells. TISllb and cMG1 differ only at amino acid 78, with a serine in the murine protein and a threonine in the rat protein.

sponse genes

Corresponding author. t Present address: AMGEN Inc., Amgen Center, Thousand Oaks, CA 91320. *

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PTAP T T A T SA-- - - - FIG. 1. The TISll, TISllb, and TISlld predicted proteins, aligned to identify maximal sequence similarity. Identical amino acids and conservative substitutions are boxed. The 67-amino-acid conserved core region is within the heavy box. liD

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cMG1 and TISlib are very highly conserved; there are substantially greater sequence differences between other primary response gene products cloned from rat and mouse cells. The murine TIS7 and rat PC4 proteins, for example, differ by 13 of 449 amino acids (20, 21). The egrl/TIS8 and NGFI-A proteins differ in size and have substantially more sequence differences than are found for TISlib and cMG1 (16, 19). The nur77/TIS1 and NGFI-A proteins also differ in size and are more divergent in amino acid sequence than are TISlib and cMG1 (7, 17). Cloning and sequence of TISlld cDNA. We thought it likely that there might be still other members of the TIS11 and TIS11b/cMG1 gene family. Because primary response gene expression is often restricted (8, 12, 23), screening selected cDNA libraries with the degenerate oligonucleotides might not identify all TIS11 family members. We adopted an alternative approach and cloned related sequences for the conserved motif of TIS11 and TISlib directly from genomic DNA. The degenerate oligonucleotides 5'-CCGGAATTCTA(T/C)AA(A/G)ACNGA(G/A)CT NTG(T/C)CG-3', which encodes the amino acid sequence YKTELC, and 5'-CCAAGCTTCNGT(C/T)TT(A/G)TA(C/ T)TTNGG(G/A)TG-3', the reverse translation of the amino acid sequence HPKYKTE, were used for PCR amplification with DNA from murine C3H1OT½2 cells. The oligonucleotides also contain terminal EcoRI or HindlIl sites. PCR was performed in buffer containing 18 mM (NH4)2SO4, 6.7 mM MgCl2, 67 mM Tris HCI (pH 8.8), 6.2 mM EDTA, 10%

dimethyl sulfoxide, 4 mM deoxynucleoside triphosphates, 10 puM each degenerate primer, 4 ,ug of genomic DNA, and 2.5 U of Taq polymerase. The first extension reaction was performed at 94°C for 7 min. After being at 55°C for 2.5 min, 40 cycles of polymerization were carried out at 94°C for 1 min, followed by 45°C for 1 min and 55°C for 2 min. The PCR product was purified, digested, and cloned into m13 vectors. Plaques that hybridized with the TIS11 and TISlib cDNA probes at high stringency were eliminated, and the remaining m13 clones that hybridized with the degenerate oligonucleotide probes were sequenced. Two additional genomic clones, TISlic and TISlid, are closely related to TIS11 and TISlib. The inserts from replicative forms of TISlic and TISlid bacteriophages were excised, purified, random primed, and used to screen several cDNA libraries. The TISild probe detected clones in a BALB/MK lambda gtlO cDNA library. The sequence of a 1.2-kb TISlld cDNA contains an open reading frame that satisfies the consensus sequence rules of Kozak (11) for initiation sites and codes for a predicted protein of 367 amino acids (Fig. 1). We have not isolated and sequenced the complete cDNA for TIS1ld. Primer extension analysis suggests that the predicted translation start site is approximately 300 nucleotides from the 5' end of the TISild mRNA (data not shown). It is possible that the TISild translation start site lies 5' of this ATG. The 67-amino-acid region conserved between TIS1l and TISlib is also present in TISlid. TISllb and TISlid share a 94% identity over this region; only four amino acid

1756

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differences are present (Fig. 1). When the TIS11, TISlib, and TISlid proteins are compared, it is clear that the sequence similarities of the core region of TISlib and TISild are greater than the similarities of either of these two proteins with TIS11. Moreover, the 67-amino-acid core region is extended another 8 amino acids for TISlib and TIS1ld. Several regions of strongly conserved sequence also exist in the amino acids flanking the conserved core of these three proteins (Fig. 1). Like TISli, TISlib and TISlid are rich in proline-serine regions. The TISlid protein also contains a run of four glutamine residues. Glutamine strings, like serine-proline-rich regions, are often associated with transactivation regions of transcription factors (4, 9). It seems likely that the conserved 67-amino-acid sequence of the TISli, TISlib, and TISlid proteins, which contains the YKTEL and Cys-His repeats as well as 11 to 13 basic amino acids (Fig. 1), is a DNA-binding domain. Southern analysis of murine genomic DNA demonstrates a unique restriction fragment that hybridizes to the TISlic probe. However, no cDNA corresponding to the TISlic sequence was found in the several cDNA libraries screened, and no mRNA hybridizing to this probe has been found in a number of cell lines that express the other members of the TIS11 gene family. The TISlic sequence may be a portion of another member of this gene family that is expressed in a highly conserved fashion. Alternatively, it may be an unexpressed murine pseudogene. Basal and TPA-induced expression of TISli, TISlib, and TISlld in 3T3 cells. The TIS1l mRNA level is low in quiescent 3T3 cells (14, 15) (Fig. 2A). In contrast, TISlib and TISlid mRNAs are easily detectable (Fig. 2A). The TIS11 mRNA level peaks between 30 and 60 min after TPA addition. In contrast, there is relatively little TISllb or TISlld induction. The original TIS genes are all "superinducible"; their induction is substantially elevated in the presence of cycloheximide (CHX) when compared with induction in the absence of CHX (14). The high basal levels of TISlib and TISlid mRNAs and their relatively low degree of TPA induction (Fig. 2A) suggest that these genes might not be superinducible. CHX is able to elevate the mRNA levels for all three genes (Fig. 2B). TIS1l is superinducible; mRNA accumulation at 3 h is far greater in the presence of both TPA and CHX than in the presence of either agent alone. In contrast, the presence of both TPA and CHX did not substantially elevate TISllb or TISlid mRNA levels beyond that observed with CHX alone; unlike TIS11, TISlib and TISlid are not subject to CHX-mediated superinduction in 3T3 cells. Since our cloning procedure to isolate TIS genes (14) depends on differential screening with cDNAs prepared from 3T3 cells treated with (i) TPA plus CHX and (ii) CHX alone, TISlib and TISlid would not be cloned by our analysis. Induction of TISll, TISllb, and TISlld by EGF, FGF, and NGF. Since the TIS genes can be induced by multiple, independent pathways (2, 3, 15) and are induced to qualitatively different levels by TPA, EGF, and FGF in some cell types (2), we investigated the induction of TISllb by the growth factors. Although the induction of TISll mRNA was rapid and transient, there was only modest induction of TISlib by EGF or FGF in 3T3 cells (Fig. 3). However, cMG1, the rat homolog of TISllb, was cloned by differential screening of a cDNA library prepared from EGF-treated rat epithelial cells (6). These data raise the exciting possibility that cell-type-specific differences in the response of the TISllb/cMG1 gene occur for the same ligand. TIS11 can also be induced in rat PC12 cells by EGF, FGF,

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FIG. 2. TPA induction of TIS11, TIS11b, and TIS11d. (A.) Time Density-arrested 3T3 cells were treated with TPA (50 ng/ml) for the times (in minutes) shown. Total RNA was isolated, and 10 p.g was subjected to electrophoresis. Transfer, blotting, preparation of probes, and hybridization conditions were as described previously (14). CHOB probe was used to provide a constitutive mRNA level for comparison. Each probe was hybridized to a separate filter. The illustration is a composite; band positions do not indicate the relative sizes of the mRNAs. (B) TISlib and TISlid are not CHX superinducible. Density-arrested 3T3 cells were treated for 3 h with TPA (50 nglml) (lanes 1), TPA plus CHX (10 p.g/ml) (lanes 2), or CHX alone (lanes 3). Cells were harvested, and TIS11, TIS11b, and TISl;d mRNA was measured by Northern (RNA) analysis. Each lane contained 10 pLg of RNA. course.

TPA, NGF, and K'-induced depolarization (12). However, TIS gene expression can be restricted; individual cell types

specific TIS genes in response to any inducer. Thus, TIS10 mRNA cannot be detected in PC12 cells despite robust induction of other TIS genes (12), and TISi cannot be expressed in neutrophils, despite induction of TIS8 and TIS11 by TPA or granulocyte-macrophage colony-stimulating factor (22). Although TIS11 could be induced in PC12 cells by NGF or TPA, no TISlib mRNA is detectable in PC12 cells in response to either inducer (Fig. 3). T1S11b/cMG1 joins TISl0 (12) and egr2 (10) as genes that cannot be induced in PC12 cells in response to any ligand tested. The TIS11, TIS11b, and TISild genes are present in human DNA. TPA and GM-CSF can induce TIS11 expression in human neutrophils (22). To search for human homologs of TISlib and TISild genes, we used human DNA as a template for PCR amplification. The identical primers, reaction conditions, cloning procedures, and sequencing methods described for murine DNA were used to characterize genomic sequences for human TIS11 genes. Sequences that identified TIS11, TIS11b, and TISlid human homologs were present among the m13 clones. No human homolog of the TISlic sequence was found. One conservative amino acid sequence change occurred in the 31 amino acids encoded by For lb and TISild the the human TISl1 PCR product. TIST are often unable to express

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