Cell, Vol. 66, 411-414,

February

7, 1992, Copyright

0 1992 by Cell Press

Minireview

More Is Better: Activators and Repressors from the Same Gene Nicholas S. Foulkes and Paolo Sassone-Corsi Laboratoire de Genetique Moleculaire des Eucaryotes CNRS U184 INSERM Faculte de Medecine 67085 Strasbourg France

The initiation of transcription in eukaryotes is an intricately controlled process. Short sequence motifs in the promoter regions of genes interact in a specific manner with DNAbinding transcription factors. These bound factors interact directly or indirectly with components of the general transcription machinery and thereby recruit RNA polymerases to transcribe the gene. For a given gene, the combination of cis control elements and the availability of transcription factors are major determinants of transcriptional activity. In turn, protein-protein interactions and posttranslational modifications of these factors are also important control points. A multitude of factors that activate and downregulate transcription have been identified and characterized in great detail. The presence of negative control in gene regulation has a number of advantages: for example, the silencing of genes whose expression is inappropriate and the downregulation of induced gene expression (reviewed by Mitchell and Tjian, 1989; Lewin, 1990). Transcriptional downregulators seem to work in different ways. Some factors possess DNA-binding domains but lack functional activation domains; they compete with activators for binding to the same sites and thereby block activation. Alternatively, they can heterodimerize with activators, thereby reducing either their DNA-binding affinity or their ability to activate transcription. In addition, there are reports of repressors that interact with activator factors when they are bound to DNA and block their transactivation function. Another classof down-regulatorscomprises inhibitory proteins that sequester the activator in a complex that is unable to bind DNA (reviewed by Jackson, 1991; Jones, 1991). A recent twist in our understanding of transcription regulation has come with the discovery that activators and repressors can be encoded by the same gene. This finding has important implications for how positive and negative

Examples

of Genes

that Encode

Activators

and Repressors

Gene

Mechanism

erbAn mTFE3 fosB

Splicing modifies C-terminal domain and prevents ligand binding Splicing deletes amphipathic helix (part of activation domain) Splicing out of a coding intron truncates C-terminus and excludes hepta-proline sequence (part of activation domain) Coordinate removal of two glutamine-rich activation domains by splicing Use of internal initiation codon causes truncation of N-terminus and exclusion of activation domain

CREM Lap

for Activator

effects upon transcription are integrated to define the final transcriptional response. In this review, we focus upon five examples that represent paradigms of this phenomenon (see table). Switches by Alternative Splicing There are many examples of the generation of proteins with diverse function from a single gene” by alternative splicing (Smith et al., 1989; Montmayeur and Borrelli, 1991). Amongst these are a number of genes that encode transcription factors, but in many of these cases the functional significance of alternative splicing is unclear. In some, however, alternative splicing constitutes a switch that dramatically reverses function (Figure 1A). One of the first documented cases is the proto-oncogene erbAa, which encodes the a subtype of the nuclear thyroid hormone receptor(Koenigetal., 1989; Rentoumiset al., 1990, and references therein). Binding of thyroid hormone to its receptor can result in both transcriptional activation and repression from different thyroid hormone-responsive elements. The N-terminal segment of the receptor contains the DNA-binding domain, and the ligand-binding domain is at the C-terminus. Alternative splicing of this gene generates three different transcripts designated a 7, a2, and a3. Their encoded proteins are identical throughout the N-terminal 370 amino acids but diverge thereafter: the a7 transcript encodes a C-terminal sequence of 40 amino acids, the a2 transcript encodes a distinct C-terminal sequence of 120 amino acids, and the a3 protein is identical to a2 except for a deletion of 39 amino acids at the boundary of the C-terminal domain. Functional analysis has revealed that neither a2 nor a3 is able to bind thyroid hormone, although both are still able to bind to target DNA sequences. The a2 receptor alone neither activates nor represses transcription from thyroid hormone response elements in the presence of the hormone. However, coexpression of a2 with al blocks activation induced by the hormone. At least three possible mechanisms could explain this observed down-regulation: competition by a2 homodimers for binding to the response element; the formation of inactive, DNA-binding heterodimers between al and a2; or competition by a2 for some accessory transcription factor necessary to mediate activation. In all these mechanisms, however, the alternative splicing affecting the C-terminal

to Repressor

Switch

Reference Koenig et al., 1989; Rentoumis et al., 1990 Roman et al., 1991 Dobrzanski et al., 1991; Yen et al., 1991; Nakabeppu and Nathans, 1991; Mumberg et al.,1991 Foulkes

et al., 1991

Descombes

and Schibler,

1991

Cdl 412

A. Alternative splicing. DNA-Binding Dime&&on

Activation

I Alternative Splicing /

Activator @L

B. Alternative Translation Initiation.

uali

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1

Repressor

Activator

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C. Model for transcriptional j

regulation.

DNA-Bindin$K$erization @

Activation Domain

+ Figure Single

1. Generation Gene

of Activation

and Repression

Functions

amino acids is thought to modify the ligand-binding properties of the receptor and, thereby, its activation potential. Thedifferent isoformsexhibit atissue-specificdistribution, which may explain the differential responsiveness to thyroid hormone of various tissues. For example, a2 is the dominant isoform in brain, and it has been postulated that the relative abundance of a2 and al in this organ may account for its lack of response to thyroid hormone. The activity of mTFE3 is also regulated by alternative splicing (Roman et al., 1991). This factor binds to sites in immunoglobulin promoters and enhancers (for example, thepE3siteoftheimmunoglobulin heavychainenhancer). Analysis of cDNA clones has revealed that alternative splicing generates two forms of this factor-one of 326 amino acids (mTFE3-L) and a shorter form of 291 amino acids (mTFE3-S). Both forms include helix-loop-helix and leucine-zipper motifs adjacent to a region of basic amino acids that mediates protein-protein interactions and DNA binding. The 35 amino acids that are absent in mTFE3-S are encoded by a single exon and are located within the activation domain. More specifically, the deletion removes a region carrying a net negative charge predicted to form an amphipathic helix. Both forms of the factor stimulate transcription, but mTFE3-L is a more powerful activator than mTFE3-S. However, cotransfection of mTFE3-S with mTFE3L down-regulates transcriptional activation, even with substoichiometric amounts of mTFE3-S. Two mechanisms have been proposed to explain this negative interaction. One supposes that mTFE3-S protein is more stable than mTFE3L, so the ratio of proteins in the transfected cells does not correspond with the ratio of mRNAs. A more likely model takes into account the observation that mTFE3-S and mTFE3-L can heterodimerize. In this model, mTFE3-S reduces the transcriptional activation of the heterodimer relative to the mTFE3-L homodimer. This model accounts for the substoichiometric down-regulation of transcription by the mTFE3-S form. In all tissues examined to date, the mTFE3-S and mTFE3-L forms are coexpressed with a tissue-specific ratio. Interestingly, the ratios found in different tissues fall within an experimentally determined range over which small changes in the mTFE3-L: mTFE3-S ratio result in large differences in transactivation. This suggests a physiological relevance for the coexpression of the alternatively spliced forms in the modulation of mTFE3 activity. fos6 belongs to the immediate early class of genes and is closely related to the proto-oncogene c-fos. By heterodimerizing with the different Jun family members, FosB constitutes part of the AP-1 transcription factor complex, which activates transcription from elements responsive to

from a

(A) Generation of activator and repressor factors by alternative splicing. Exons are represented by boxes, introns by lines. Hatched and shaded regions denote the sequences encoding the activation and DNA-binding domains, respectively. Inclusion or exclusion of the activation domain exons during splicing generates mRNAs encoding activators or repressors. (6) Generation of activator and repressor factors by alternative translational start sites. An equivalent but intronless gene encodes just one transcript. Translation initiated at the internal AUG excludes the up

stream activation domain-coding sequence and so generates a repressor product. (C) Schematic illustration of postulated mechanisms for downregulation of the activator by the repressor. Factors lacking a functional activation domain can bind to a target sequence, block binding of the activator, and so repress transcription. Alternatively, they can heterodimerize with activator molecules thereby reducing their activation function relative to the activator homodimer.

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the phorbol ester, TPA. An alternative-spliced form of fosfl encodes AFosB, which lacks the C-terminal 101 amino acidsof FosB (Nakabeppu and Nathans, 1991; Mumberg etal., 1991; Yenetal., 1991; Dobrzanskiet al., 1991). This is generated by a deletion of 140 bp 3’to the leucine zipper motif that shifts the reading frame and so introduces a stop codon immediately 3’to the site of the deletion. The deleted sequence contains consensus donor and acceptor splice sites and so can be regarded as a coding intron. AFosB down-regulates activation by c-Fos-c-Jun or FosB-c-Jun heterodimers (Nakabeppu and Nathans, 1991; Yen et al., 1991). The AFosB protein shares a leutine zipper and basic domain region with FosB and so is predicted to retain the dimerization and DNA-binding function. However, it lacks a hepta-proline repeat that has been postulated to constitute part of the activation domain. Since AFosB, like c-Fos and FosB, is unable to form homodimers, the most plausible model to explain downregulation is that AFosB engages in the formation of inactive, DNA-binding AFosB-c-Jun heterodimers. One notable property of AFosB is that it is able to suppress transformation by c-Fos, v-Fos, or FosB in a focus assay (Mumberg et al., 1991; Yen et al., 1991). This finding suggests that AFosB might play a role as a transformation suppressor by antagonizing Fos function. Some controversy remains, however, about the precise functional significance of AFosB, since it still seems to function as a transcription activator (Dobrzanski et al., 1991; Mumberg et al., 1991). The first cDNA clones characterized from the CREM gene (CAMP response element modulator) encode antagonists of CAMP-induced transcription (Foulkes et al., 1991). The central role of splicing in the regulation of this gene was suggested by the presence of two alternative DNA-binding domains that are used differentially in a cellspecific fashion. The CREM antagonists are able to bind to CAMP response elements as homodimers and also as heterodimers with the transcriptional activator CREB (CAMP response element-binding protein). It is postulated that CREM may function either by blocking the CAMP response element for CREB binding or by forming nonfunctional heterodimers with CREB. In cotransfection experiments, substoichiometric amounts of CREM cause significant down-regulation of activation through CAMP response elements(Foulkes et al., 1991), and thus, similarly to mTFE3-S, heterodimerization may be a more plausible model. The CREM antagonists share extensive homology with CREB, but they lack two glutamine-rich domains that are necessary for transcriptional activation. In addition, the CREM gene also encodes an activator of transcription (Foulkes et al., 1992). In the adult testis, an isoform, CREMr, has been identified that resembles one of the antagonist forms (CREMf3) but includes two exons that encode two glutamine-rich domains. This form has been demonstrated to transactivate transcription from a CAMP response element. The CREMr isoform is expressed alone in adult testis and it constitutes an abundant species in late spermatocytes and spermatids. The CREM mRNA isoforms illustrate how alternative splicing can modulate

the developmental function of a transcription factor in a tissue-specific manner. Alternative Translation Initiation: Diversity without lntrons In all the cases described above, the genes encoding these factors are multiexonic in structure. The exons of these genes define functional domains, and the splicing pattern of the transcript can be used to regulate the function of the final product. However, even from an intronless gene there is the possibility to generate functionally different factors (Figure 1 B). Descombes and Schibler (1991) have described such acase, which concerns the LAP transcriptional activator. The lap gene is transcribed in many tissues, but LAP protein is most abundant in the liver, in terminally differentiated parenchymal hepatocytes. A repressor form, termed LIP, is generated by the use of an alternative translation start site within the coding sequence, which gives rise to a protein with a truncated N-terminus (see Figure 1 B). It is postulated that this is achieved by a leaky ribosomal scanning mechanism (Kozak, 1989). Both LAP and LIP share a region of basic amino acids and a leucine zipper motif, but LIP lacks the N-terminal activation domain identified in LAP. LIP and LAP can form heterodimers that bind DNA with an efficiency equivalent to the homodimers. Cotransfection experiments demonstrate that LIP represses transcriptional activation by LAP in stoichiometric quantities when the factors are encoded by two separate plasmids. However, curiously, when the two forms are produced from the same plasmid via the alternative usage of translation initiation codons, then LIP down regulates even at substoichiometric amounts. It is postulated that the LIP repression occurs through the formation of heterodimers: since these complexes only contain one activation domain, they are postulated to function as less eff icient transactivators. In addition, it is possible that more efficient heterodimerization occurs when LIP and LAP are being cotranslated from the same template. Thus the coexpression of the LIP form with LAP is predicted to be a major determinant of the activation function of LAP. During postnatal development in the liver, the LAP:LIP protein ratio increases significantly from 3 at birth to 15 in the adult. Thus, the production of LIP is a dynamic and developmentally regulated process. Activators and Repressors: Offspring of a One-Parent Family A number of common features emerge from these examples. First, the modularity of transcription factor function allows the production of both activators and repressors from the same gene. In all cases, there is evidence for a distinct activationdomain; deletionof specificcomponents of this domain can abolish or reduce the transcriptional activation function, but not dramatically affect other functions such as dimerization or DNA binding. Second, the activator and repressor are consistently coexpressed in vivo, and in the cases of erbAa, CREM, mTFE3, and lap the ratio of the different forms is modulated in a temporal and tissue-specific fashion. These observations imply that the coexpression of positive and negative regulator forms

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is central to the modulation of transcriptional activation by these factors. Splicing and translation initiation can thus be regarded as important mechanisms in transcriptional control. It will be fascinating to determine whether, for example, these splicing events constitute targets for intracellular signal transduction pathways. The potential to obtain factors with opposite functions from the same gene represents an economical solution to the problem of just how many genes are required to execute transcriptional control. Another advantage is the provision of an added level of complexity and versatility in the regulation of gene expression: the functional repertoire of one gene can be expanded. It is tempting to speculate that in terms of evolution this may be a more ancient strategy, and that by successive duplication events genes have become dedicated to encoding exclusively single factors. For the future, it will clearly be interesting to define how widespread this phenomenon is and also to deduce the physiological relevance of individual cases. This new facet to transcription factor biology seems set to teach us much more about the evolution and control of gene regulatory mechanisms. References Descombes,

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