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Proc. Natl. Sci. Counc. ROC(B) Vol. 24, No. 2, 2000. pp. 47-55 (Invited Review Paper) Structure and Function of the Groucho Gene Family and Encoded Transcriptional Corepressor Proteins from Human, Mouse, Rat, Xenopus, Drosophila and Nematode STEVEN SHOEI-LUNG LI Institute of Biomedical Sciences National Sun Yat-Sen University Kaohsiung, Taiwan, R.O.C. (Received March 29, 1999; Accepted June 7, 1999) ABSTRACT A gene family of the Groucho, TLE, ESG and AES proteins has been characterized from Drosophila, nematode, Xenopus, mouse, rat and human, and their structural relationships have been analyzed. The genomic organization of nematode ESG, human and mouse AES genes has been determined, and the expression of ESG and AES genes from Xenopus and human has been analyzed. The Groucho, TLE and ESG proteins all share a similar structure, consisting of a conserved amino-terminal domain, a variable middle region, and highly conserved carboxyl-terminal WD-40 repeats. The Drosophila Groucho transcriptional corepressor protein has been shown to interact with the DNA-binding bHLH domain of Enhancer of split, Hairy and Deadpan proteins, which proteins are involved in neurogenesis, segmentation and sex-determination, respectively. Human TLE1 protein has been demonstrated to interact with mammalian AML1 protein, which regulates hematopoiesis and osteoblast differentiation. The AES proteins from human, mouse, rat and Xenopus exhibit strong similarity to the amino-terminal domain of Groucho proteins; however, the biological function remains to be elucidated. Key Words: groucho, Enhancer of split, corepressor, human, mouse, rat, Xenopus, Drosophila, nematode I. Introduction The expression of eukaryotic genes may be regulated by transcriptional activation and repression. Although activation has been studied and is now better understood, recent work has shown that repression is as important as activation in regulating the expression of many eukaryotic genes (Gray and Levine, 1996). The transcriptional repression system utilizes a sequence-specific DNA-binding protein that also contains a protein domain required for repression. While it has been proposed that many repressor domains interact directly with the general transcriptional machinery or with activators, some of the repressors appear to act indirectly by recruiting corepressor proteins that bring about repression. A corepressor is defined as a protein that is required for the repressor activity of a specific transcription factor but does not have the ability to bind DNA alone. A family of transcriptional corepres- sors, known as Groucho proteins, has been reported from Drosophila, nematode, Xenopus, mouse, rat and human. The Groucho gene was identified initially as a viable mutation that affects the development of the Drosophila nervous system, with an allele resulting in thick tufts of sensory bristles over the eyes, resembling the bushy eyebrows of the comedian Groucho Marx. The Drosophila Groucho gene, the founding member of the Groucho gene family, encodes a transcriptional corepressor protein of 719 amino acids. This protein sequence includes the repeat of approximately 40 amino acids demarcated by Trp-Asp (WD-40 repeat) present in a guanine nucleotide binding protein (G-protein) β-subunit (Fong et al., 1986; Hartley et al., 1988). Stifani et al. (1992) reported human homologs, Transducin-Like Enhancer of split (TLE)l, TLE2, TLE3 and TLE4, of the Drosophila Groucho protein. My associates and I described a mouse homolog, Enhancer of split Groucho (ESG), of the Drosophila Abbreviations used: AES, Amino Enhancer of split; AML1, acute myeloid leukemia transcriptional activator 1; bHLH, basic helix-loop-helix; CKII, casein kinase II; cdc2K, cdc2 kinase; ESG, Enhancer of split groucho; G-protein, guanine nucleotide binding protein; Grg, Groucho related gene; NLS, nuclear localization sequence; TLE, transducin-like Enhancer of split; WD-40 repeat, the repeat of approximately 40 amino acids demarcated by Trp-Asp (WD); WRPW, Trp-Arg-Pro-Trp. –47– S.S.L. Li Fig. 1. Amino acid sequence comparison of the Groucho, TLE, ESG and AES proteins from Drosophila, nematode, Xenopus, mouse, rat and human. The amino acid sequences were aligned using the Wisconsin GCG package. Human TLE4 is a partial sequence. Identical residues among all the sequences are shown in boldface, and gaps are shown using hyphens. The NLS, CKII and cdc2K sites are indicated, and the WD-sequences demarcating WD-40 repeats are underlined. The translation stop codon is indicated by a star. –48– Groucho Transcriptional Corepressor Proteins Groucho protein (Miyasaka et al., 1993). In addition, we first reported human and mouse Amino Enhancer of split (AES) proteins, which exhibit strong similarity to the amino-terminal domain of Drosophila Groucho, human TLE and mouse ESG proteins, but lack WD-40 repeats (Miyasaka et al., 1993). Mallo et al. (1993) also identified mouse AES (Groucho related gene, Grg) protein. Schmidt and Sladek (1993) described rat homologs, Resp1 and R-esp2 of mouse AES and ESG proteins, respectively. In 1997 my laboratory reported Xenopus AES, ESG1 and ESG2 as well as nematode ESG proteins (Choudhury et al., 1997; Sharief et al., 1997). Pflugrad et al. (1997) independently identified the nematode UNC-37 (ESG) protein. This review will briefly describe the structure and function of the Groucho gene family and encoded transcriptional corepressor proteins. II. Primary Structure of Groucho and Related Proteins The amino acid sequences of the Groucho, TLE, ESG and AES proteins from Drosophila, nematode, Xenopus, mouse, rat and human are compared in Fig. 1, and their structural/evolutionary relationships are indicated in the diagram in Fig. 2 (Sharief et al., 1997). The AES proteins of 197 amino acids from human, mouse, rat and Xenopus are homologous with the amino-terminal domain of the Groucho, TLE and ESG proteins. There is 13% identity at the 221 positions when the amino-terminal domains of Groucho and AES proteins are compared. The middle region of the Groucho proteins exhibits only 5% identity for the 234 positions compared. The carboxyl-terminal WD-40 repeats of the Groucho proteins exhibit 47% identity at the 369 positions compared among the different proteins from human to nematode. The Groucho, TLE and ESG proteins are clustered into a group while the four AES proteins are clustered into a separate group. The nematode ESG (UNC-37) protein of 612 amino acids has been found to be the smallest of the Groucho proteins that are known, and it appears to be the ancestor of both the Groucho and AES proteins. Human TLE2 is structurally Fig. 2. The structural and evolutionary relationships among the Groucho, TLE, ESG and AES proteins. These relationships were constructed using the UPGMA method included in the Wisconsin GCG package. [Reprinted, by permission, from Sharief et al. (1997)] closer to Drosophila Groucho and Xenopus ESG1. The mouse ESG protein appears to be most similar to human TLE3 protein while the rat R-esp2 protein is more similar to the human TLE1 protein. III. Genomic Organization of Groucho Gene Family The nematode ESG (UNC-37) gene is located on chromosome 1, and its protein-coding sequence is interrupted by five introns (Fig. 3). The sizes of exons 1 to 6 are 81, 144, 511, 198, 849 and 156 basepairs, respectively, Fig. 3. The exon organization and protein domains of the nematode ESG (UNC-37) gene. The six exons are indicated by hatched bars. The Groucho protein consists of the conserved NH2 domain, the variable middle region and highly conserved WD-40 repeats. [Redrawed, by permission, from Sharief et al. (1997)] –49– S.S.L. Li Fig. 4. The exon-intron organization of the human AES gene. The deduced amino acids (single letter code) are shown below individual codons. The intron sequence is shown in lower case. The polyadenylation signal AATAAA is underlined. [Summarized, by permission, from Hou and Li (1998)] while introns 1 to 5 contain 52, 252, 87, 53 and 518 basepairs, respectively (Sharief et al., 1997; Pflugrad et al., 1997). The UNC-37 gene transcripts have been shown to be transpliced to either SL1 or SL2 leaders at a position five nucleotides upstream of the ATG start. This finding is consistent with the location of a consenses 3’ splice site TTTCAG in the genomic sequence abutting the short 5’ untranslated region TAAAA (Pflugrad et al., 1997). In humans, there are four Groucho (TLE1, TLE2, TLE3 and TLE4) genes and one AES gene. We previously reported that human TLE3 and AES genes are located on human chromosomes 15 and 19, respectively (Miyasaka et al., 1993). Liu et al. (1996) showed that the human TLE3 gene is located at chromosome band 15q22 while both the TLE1 and TLE2 genes have been mapped to chromosome band 19p13.3. It is very interesting that we have also localized the human AES gene to the same chromosome band 19p13.3 (Hou and Li, 1998). As indicated in Fig. 4, the protein-coding sequence of the human AES gene is interrupted by six introns (Hou and Li, 1998), and the exon-intron junction sites have been found to be identical to those of the mouse AES (Grg) gene (Mallo et al., 1994). The first six exons of the human and mouse AES genes are rather small, ranging from 45 to 98 basepairs, while exon 7 contains 219 nucleotides of coding sequence and a 3’ untranslated region. IV. Expression of Groucho and AES Genes The embryogenesis of the African frog Xenopus laevis has been extensively studied, and it is an excellent model for investigating vertebrate development. We have previously described the tissue-specific and developmental expression of the Xenopus ESG1, EGS2, and AES genes (Choudhury et al., 1997). Northern blot analysis using the ESG1 coding-probe revealed two transcripts of 3.6 kb and 2.8 kb in all the adult tissues examined, except in muscle, where the 2.8 kb species was apparently undetectable (Fig. 5(a)). However, only the 3.6 kb band was detected from the same Northern blot using the ESG2-specific 3’ noncoding-probe. These results of Northern blot and sequence analyses suggest that the Xenopus ESG1 and ESG2 transcripts may derive from either two different genes or may be spliced variants using different exons as 3’-terminal sequences of the same gene. The ESG1 –50– Groucho Transcriptional Corepressor Proteins cDNA, presumably derived from the 2.8 kb transcript, is abundantly expressed in the brain, lung, testis and ovary whereas the 3.6 kb ESG2 transcript is abundantly expressed in the spleen and ovary. Differences in mRNA level among these adult tissues suggest that Xenopus ESG mRNAs are subjected to tissue-specific regulation. Northern blot analysis of AES mRNA from adult tissues using the AES specific coding-probe showed that the AES transcript of 2.2 kb is widespread and is predominantly present in the brain, testis and ovary (Fig. 5(a)). The Xenopus ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, have been found to be ubiquitously expressed in developing embryos (Fig. 5(b)). These two transcripts are also expressed in both unfertilized and fertilized eggs, suggesting the ESG1 and AES genes are expressed maternally. Unlike the results obtained from adult tissues, the ESG1 probe detected only a 2.8 kb transcript in all embryonic stages, and the 2.8 kb transcript appeared to disappear after stage 9, fine cell blastula. Sequential hybridization of the same blot with the ESG2 specific probe detected none of the 3.6 kb tran- script, suggesting that the ESG2 gene is not expressed in the early developmental stages. In Drosophila, embryogenesis has also been shown to require maternally deposited Groucho proteins (Paroush et al., 1994) whereas postembryonic development relies on zygotic Groucho expression (Delidakis et al., 1991). In nematode, some of UNC-37 mutations have been shown to be maternal effect lethals (Pflugrad et al., 1997). Northern blot analysis of mouse poly(A)-RNAs indicated that the AES transcripts of 1.5 kb and 1.3 kb are more abundant in the muscle, heart and brain than in the spleen, liver, kidney and testis (Miyasaka et al., 1993). The mouse AES-1 and AES-2 cDNAs, containing different 5’ untranslated regions, and their first 14 and 9 amino acids, respectively, presumably derive from these two different transcripts. Furthermore, the mouse AES-2 cDNA, transcribed from the proximal promoter, was identified by Mallo et al. (1994) while the AES-1 cDNA presumably derives from the distal promoter of the same AES gene. The rat R-esp2 protein have been found to contain a deletion of 25 amino acids in positions 110-144 (Fig. 1) (a) (b) Fig. 5. Northern blot analyses of the Xenopus ESG1, ESG2 and AES genes. (a) Adult tissues. (b) Unfertilized and fertilized eggs, and embryos at different developmental stages: stage 6, late morula (~1,000 cells); stage 9, fine cell blastula (~6,000 cells); stage 11, mid gastrula (~30,000 cells); stage 20, late neurula; stage 24, early tailbud; stage 32, late tailbud. [Reprinted, by permission, from Choudhury et al. (1997)] –51– S.S.L. Li Fig. 6. The structural domains of the Groucho, TLE, ESG and AES proteins. The hatched area indicates similarity among the amino-terminal domains of the Groucho, TLE and ESG, and AES proteins. The variable middle region contains NLS, CKII and cdc2K sites. The C-terminal region of the Groucho, TLE and ESG proteins contains highly conserved WD-40 repeats (dotted area). [Redrawed, by permission, from Choudhury et al. (1997)] (Schmidt and Sladek, 1993). The corresponding amino acids of the mouse AES protein are encoded by exon 6 of the mouse AES gene (Mallo et al., 1994), suggesting that the deletion of 25 amino acids might be due to alternative splicing of rat R-esp2 gene. Northern blot analysis of human poly(A)-RNAs from adult tissues indicated that the AES transcripts of both 1.6 kb and 1.4 kb are predominantly present in the muscle, heart and placenta (Miyasaka et al., 1993). The transcript of 1.4 kb is expressed in the lung, liver, kidney and pancreas while the transcript of 1.6 kb appears to be present in the brain. This size difference of human AES transcripts may be due to alternative splicing in the 5’ region, as is the case for mouse AES transcripts. It should also be noted that three CAG repeats are present at the acceptor site of intron 6 of the human AES gene. Alternative use of the first two AG dinucleotides as acceptor sites may explain our earlier finding of either the presence or absence of G1n at amino acid 125 in two different human AES cDNA clones (Miyasaka et al., 1993). V. Biological Functions of Groucho-Related Proteins The Drosophila Groucho gene was first found to interact genetically with members of the Enhancer of split gene complex. Molecular studies showed that most of the transcripts from this gene complex encode basic helixloop-helix (bHLH) DNA-binding proteins, which play a critical role by repressing target genes during neurogenesis. More recent data has demonstrated that the Drosophila Groucho protein also interacts with Hairy and Deadpan regulatory proteins in segmentation and sex determination, respectively (Delidakis and Artavanis-Tsakonas, 1992; Knust et al., 1992). The role of Groucho proteins in repression by Hairy-related proteins has been studied both biochemically and genetically. Initially, a yeast twohybrid screening performed using the Drosophila Hairy protein identified the Drosophila Groucho protein. Subsequently, both the Drosophila Groucho and human TLE1 proteins were shown to bind several Hairy-related proteins. The Trp-Arg-Pro-Trp (WRPW) motif found at the carboxyl terminus of the Hairy-related proteins has been shown to be both necessary and sufficient for interaction with Groucho proteins. This interaction between the WRPW motif and Groucho protein is required for transcriptional repression by these proteins in Drosophila embryos and cultured cells. Embryos lacking Groucho proteins exhibit mutant phenotypes consistent with functional roles for Groucho proteins as corepressors for the Hairy, Enhancer of split, and Deadpan proteins involved in segmentation, neurogenesis, and sex-determination, respectively. Studies using in vitro biochemistry, transcriptional repression assays in cells, and Drosophila genetics found that the Groucho proteins are essential corepressors for the Hairy-related proteins (Paroush et al., 1994; Fisher et al., 1996; Grbavec and Stifani, 1996; Jimenez et al., 1997; Fisher and Caudy, 1998). Together, these results have led to the prevailing view that the Hairy protein functions as a promoter-bound repressor; specifically, an intact bHLH region is required for the Hairy protein to bind to specific DNA sites, where it then uses its WRPW motif to recruit the Groucho corepressor protein. The Groucho protein, once recruited as a non-DNA binding corepressor, can repress both basal and activated transcription for specific subsets of DNA-binding transcription factors. Using a yeast two-hybrid assay, in vitro association and pull-down experiments, human TLE1 protein was demonstrated to interact specifically with mammalian AML1 protein, which regulates hematopoiesis and osteoblast differentiation. The TLE1 protein was also shown to inhibit AML1-dependent transactivation of the T cell receptor in transfected Jurkat T cells (Levanon et al., 1998). The biological function of AES proteins resembling the amino-terminal domain of the Groucho proteins is not yet understood. Although they may act as corepressors, these AES proteins could also act as antagonists of the Groucho proteins by titrating an effector or by binding to the targets of repression and acting as dominant-negative –52– Groucho Transcriptional Corepressor Proteins inhibitors of the Groucho proteins (Fisher and Caudy, 1998). VI. Structure-Function Relationship of Groucho-related Proteins The overall structural domains of the Groucho, TLE, ESG and AES proteins are illustrated in Fig. 6 (Choudhury et al., 1997). The Groucho, TLE and ESG proteins all share a similar structure, consisting of a conserved amino-terminal domain, a variable middle region, and a series of highly conserved carboxyl-terminal WD-40 repeats. It should be noted that the amino-terminal domain of the nematode Groucho protein is encoded by exon 1, exon 2 and a portion of exon 3. The middle region is encoded by a portion of exon 3 and a portion of exon 4. The carboxyl-terminal WD-40 repeats are encoded by portions of exon 4, exon 5 and exon 6 (Fig. 3). The conserved amino-terminal domain of the Groucho proteins has been shown to be able to repress both basal and activated transcription, and to act as a dimerization domain (Fisher et al., 1996). We previously noted that the amino-terminal domain of the Groucho and AES proteins contains a potential leucine zipper, which may provide a basis for direct interaction between proteins (Miyasaka et al., 1993). The variable middle region contains potential phosphorylation sites for cdc2 kinase (cdc2K) and casein kinase II (CKII) in close proximity with a nuclear localization sequence (NLS). Moreover, the phosphorylated forms of Drosophila Groucho and human TLE proteins have been shown to be present in the nucleus (Hartley et al., 1988; Stifani et al., 1992). Based on the results of studies on other WD-40 repeat proteins (Neer et al., 1994; Sondek et al., 1996), the WD-40 repeats found at the carboxyl terminus of the Groucho proteins most likely are involved in protein-protein interaction. The WD-40 repeats have been found to form a beta-propeller structure, in which each repeat projects outwards radially and is available for interaction with other proteins. Recently, the WD-40 repeats of the nematode Groucho (UNC-37) protein have been found to interact genetically with the homeodomain of the UNC-4 protein in such a way as to control neuronal development (Pflugrad et al., 1997). Interestingly, a chimera containing the amino terminus of the nematode UNC-37 protein and the WD-40 repeats of human TLE1 is able to rescue the UNC-37 mutant phenotype, which suggests that the structure and function of the WD-40 repeats are highly conserved among different Groucho proteins from human to nematode. The conserved carboxyl-terminal WD-40 repeats of Groucho proteins have also been found in an expanding group of proteins, including TUP1 (a mediator of glucose repression), SLF2 (suppressor gene for floculation), AAR1 (al-a2 repression and cell control) and AER2 (control of heme-regulated and catabolite-repressed gene). The TUP1, SLF2, AAR1 and AER2 genes have been identified on the basis of different phenotypes, but all the genes have been found to be identical (Yochem and Byers, 1987; Fujita et al., 1990; Williams and Trumbly, 1990; Mukai et al., 1991; Zhang et al., 1991). It is interesting that both the Groucho and yeast TUP1 proteins containing the carboxyl-terminal WD-40 repeats have common a function as transcriptional corepressors; however, there are major structural and mechanistic differences between them (Fisher and Caudy, 1998). VII. Conclusion A gene family of the Groucho, TLE, ESG and AES proteins has been characterized from Drosophila, nematode, Xenopus, mouse, rat and human. In humans, there are four TLE genes, TLE1, TLE2, TLE3, and TLE4, and one AES gene. Human TLE2 is structurally closer to Drosophila Groucho and Xenopus ESG1. The mouse ESG protein appears to be most similar to the human TLE3 protein, and the rat R-esp2 protein is similar to the human TLE1 protein. The Groucho transcriptional corepressor protein has been found to interact with the DNAbinding bHLH domain of the Enhancer of split, Hairy and Deadpan proteins, which are involved in neurogenesis, segmentation and sex-determination, respectively. The human TLE1 protein has been found to interact with the mammalian AML1 protein, which regulates hematopoiesis and osteoblast differentiation. These genetic and biochemical studies have provided new insight into mechanisms of transcriptional repression as well as greater understanding of the significance of transcriptional repression during development. The AES proteins, which exhibit a strong similarity to the amino-terminal domain of the Groucho proteins, have been identified in Xenopus, mouse, rat and human, but their biological functions are not yet understood. Acknowledgment This investigation was supported in part by grants NSC 86-2313B-110-002 and NSC 87-2313-B-110-002 from the National Science Council, R.O.C., as well as the Intramural Research Program of NIEHS, NIH, U.S.A. References Choudhury, B.K., Kim, J., Kung, H.F. and Li, S.S.L. (1997) Cloning and developmental expression of Xenopus cDNAs encoding the Enhancer of split Groucho and related proteins. Gene, 195:41-48. Delidakis, C. and Artavanis-Tsakonas, S. (1992) The Enhancer of split (E(spl)) locus of Drosophila encodes seven independent helix-loophelix proteins. Proc. Natl. Acad. Sci., U.S.A., 89:8731-8735. Delidakis, C., Preiss, A., Hartley, D.A. and Artavanis-Tsakonas, S. (1991) Two genetically and molecularly distinct functions involved –53– S.S.L. Li in early neurogenesis reside within the Enhancer of split locus of Drosophila melanogaster. Genetics, 129:803-823. Fisher, A.L. and Caudy, M. 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Stifani, S., Blaumueller, C.M., Redhead, N.J., Hill, R.E. and ArtavanisTsakonas, S. (1992) Human homologs of Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nature Genet., 2:119-127. Williams, F.E. and Trumbly, R.J. (1990) Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae. Mol. Cell. Biol., 10:6500-6511. Yochem, J. and Byers, B. (1987) Structural comparison of the yeast cell division cycle gene CDC4 and a related pseudogene. J. Mol. Biol., 195:233-245. Zhang, M., Rosenblum-Vos, L.S., Lowry, C.V., Boakye, K.A. and Zitomer, R.S. (1991) A yeast protein with homology to the beta-subunit of G proteins is involved in control of heme-regulated and catabolite-repressed genes. Gene, 91:153- 161. –54– Groucho Transcriptional Corepressor Proteins Groucho ESG ESG Groucho AES TLE TLE ESG AES ESG WD-40 DNA Deadpan AML1 bHLH TLE1 AES AES –55– AES Groucho Hairy Groucho