Transcription factors and the control of Drosophila development

WEVIEWS 10 Piret, J., Bernan, V., Harasym, M. and Brandt, M. (1988) in Biology of Actinomycetes '88 (Okami, Y., Beppu, T.

19 Buttner, M.J. Mol. Microbiol. (in press) 20 Chater, K.F. et al. Cell (in press)

and Ogawara, H., eds), pp. 321-323, Japan Scientific Societies Press 11 Champness, W.C. (1988)J. Bacteriol. 170, 1168-1174 12 Lawlor, E.J., Baylis, H.A. and Chater, K.F. (1987) Genes Dev. 1, 1305-1310 13 Cashel, M. and Rudd, K.E. (1987) in Escherichia coli

21 Adams, T.H., Boylan, M.T. and Timberlake. W.E. (1988) Cell 54, 353-362 22 Helmann, J.D. and Chamberlin, M.J. (1988) Annu. Rev. Biochem. 42, 839-872 23 Zuber, P., Healy, J.M and Losick, R. (1987)J, Bacteriol. 169, 461469 24 Schauer, A. et al. (1988) Science 240, 768-772 25 Chater, K.F. and Merrick, M.J. (1979) in Developmental Biology of Prokao,otes (Parish, J.H., ed), pp. 93-114, Blackwell Scientific Publications 26 Sherman, D.H. et al. (1988) in Proceedings of the 8th

and Salmonella typhimurium Cellular and Molecular Biology (Neidhardt, F.C. et al., eds), pp. 1410-1438,

American Society for Microbiology 14 Stein, D. and Cohen, S.N. (1989)J. Bacteriol. 171,

2258-2261

International Symposium of Biotechnology, Paris

15 Hardisson, C., Manzanal, B., Mendez, C. and Brafla, A. (1986) in Biological, Biochemical and Biomedical Aspects ofActinomycetes (Szab6, G., Bir6, S.

(Durand, G., Bobichon, L. and Florent, J., eds), pp. 123-137, Societe Frangaise de Microbiologie 27 Helmann, J.D., Marquez, L.M. and Chamberlin, M.J. (1988).L Bacteriol. 170, 1568-1574 28 McVittie, A. (1974) J. Gen. MicrobioL 81,291-302

and Goodfellow, M., eds), pp. 433~142, Akad~miai Kiad6 16 Wildermuth, H. (1970) J. Gen. Microbiol. 60, 43-50 l Y Williams, S.T., Sharpies, G.P. and Bradshaw, R.M. (1973) in Actinomycetales: Characteristics and Practical Importance (Sykes, G. and Skinner, F.A., eds), pp. 113-130, Academic Press 18 Guijarro, J., Santamaria, R., Schauer, A. and Losick, R. (1988)J. Bacteriol. 170, 1895-1901

K.F. CHATER IS IN THE JOHN INNES INSTITUTE AND A F R C INSTITUTE OF PLANT SCIENCE RESEARCH, COZNEY LANE, NORWICH N R 4 7UH, UK.

G e n e t i c analyses have identified a group of genes controlling development in Drosophila that form a temporally ordered regulatory cascade (reviewed in Ref. 1). Many of these genes act by controlling spatial and temporal patterns of gene expression (for examples, see Fig. 1). The genes controlling development appear to encode a variety of proteins, including membrane-bound receptors, proteases and growth factor-like molecules, but over half are thought to be transcription factors'% This suggests that an important part of the regulatory cascade involves transcriptional control. However, informative as the genetic data are, they do not provide a molecular understanding of how these patterns of gene expression are regulated, nor are they likely to have identified all the transcription factors involved. The elucidation of regulatory mechanisms requires a thorough biochemical and functional analysis of the proteins acting directly on promoters. Such studies, together with the genetic data, will provide a better understanding of the developmental regulation of gene expression than would be possible using either genetic or biochemical analyses separately. In this review we focus on recent molecular studies of transcription factors that regulate expression of genes controlling development along the anteroposterior axis of the embryo. Two different, yet complementary, approaches have been employed to detect transcriptional regulatory proteins. In the first, biochemical methods are used to purify promoter-selective transcription factors from crude embryonic extracts. In the second approach, proteins encoded by developmental regulatory genes are assayed in vivo and in vitro to provide direct evidence that these genes encode transcription factors. Thus far, these

Transcriptionfactors and the control of Drosophila development MARK D. BIGGINAND ROBERTTJIAN

Drosophila is a uniquely advantageous system for carrying out both biochemical and genettc analyses of proteins that regulate spatial and temporal patterns of transcription. Here we discuss what is known about the mechanisms of action and biologicalfunctions of transcription factors that act on genes controlling Drosophila embryogenesis. studies have only identified some of the proteins regulating transcription but, as we discuss below, this work is providing important information about how complex patterns of gene expression are generated.

Transcriptional control in Drosophila Before describing the regulation of developmental control genes in detail, we briefly discuss general features of Drosophila promoters that are transcribed by RNA polymerase II. Like mammalian promoters, many Drosophila promoters contain a combination of enhancer elements distal to the RNA start siteS.6, proximal transcription control elements 5-9, and TATA box consensus sequences 7.to. The transcription factors that act on these promoters can be divided into at least two broad classes: the general transcription factors, and a much larger group of proteins, the promoterselective (or promoter-specific) transcription factors.

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The promoter-selective transcription factors are sequence-specific DNA-binding proteins that each act only on a subset of promoters. In Drosophila, most of the data accumulating on promoter-selective transcription factors have been obtained from the study of developmental control genes (Table 1). These proteins can be divided into two types: one group that is expressed in most cells and appears to regulate transcription of a wide range of genes, and a second group that is expressed in highly regulated spatial patterns and may have a more specific regulatory function.

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One approach that has identified promoter-selective transcription factors / f involves the purification of proteins, from embryonic extracts, that bind to cL;-regulatory DNA elements. Fortunately, extracts ~ t , ) Tissue specific derived from Drosophila embryos have genes proven to be highly active for in vitro trant Wt~t~..~ ddc, adh, white scription, and the proteins isolated from 14hr them by biochemical fractionation have been demonstrated to direct transcription FIGI! in vitro from the Ultrabithorax (Ubx), Expression of representative members of each of the different classes of engrailed (en), Antennapedia P1 (Antp) genes controlling pattern formation along the anteroposterior axis of the and Antp P2 promoters 14-18. These factors embryo. As development proceeds (top to bottom of diagram, the time postfertilization is given) successive classes of genes are switched on in include the ADF-1, DTF-1, GAGA, NTF-1 increasingly more complex patterns of expression. The arrows indicate (or Elf-l) and zeste proteins (the acronyms genetically defined regulation of the members of one gene class by those of for these factors are explained in Table 1). another class of genes (reviewed in Ref. 1). This regulatory cascade places Each of these proteins recognizes distinct different combinations of these proteins in various cells of the embryo, DNA sequences, and binds in different specifying their developmental fates and directing them to go through combinations to the different promoters distinct programs of cell division and differentiation. For this reason, the (Table 1; Fig. 2). For example, DTF-1 binds developmental control genes are assumed to be ultimately responsible for to Antp P2 but not to Antp P1 or Uhx, while controlling expression of tissue specificallyexpressed genes, though it is not zeste protein acts on Ubx but not on An!p known if they do this directly or by acting on intermediary regulators. P2 (Fig. 2). The antp, en and Ubx genes have General factors complicated patterns of expression in the embryo (Fig. The general transcription factors are believed to be 1), and their promoters are correspondingly essential for basal transcription from all RNA complex 6.19. For example, the Ubx promoter is compolymerase II promoters, and many of these proteins, posed of multiple, distinct cis-regulatory regions (Fig. including the polymerase, are conserved throughout 2a), some of which are proximal to the RNA start site eukaryotes. Several general factors have been detected while others lie up to tens of kilobase pairs both in Drosophila by biochemical fractionation ]l, including upstream and downstream of the RNA start site. a factor binding to TATA box elements 10. These However, in vitro transcription reactions, which are proteins are probably equivalent to the group of six generally not responsive to proteins bound to distant general transcription factors identified in mammalian cis elements, have thus far only identified transcription systems (Ref. 12; reviewed in Ref. 13). Although these factors binding close to the RNA start site on Antp, en factors may not be regulatory proteins themselves, and Ubx (for example Fig. 2a). understanding the mechanisms by which promoterspecific transcription factors act will ultimately require Biological functions for these transcription factors an analysis of their interactions with the general Although none of the purified transcription factors factors. However, since little is known about the activates all promoters tested, some proteins, such as action of general factors on genes that regulate GAGA, Adf-1, NTF-1 and zeste protein, can act on /

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more than one gene. These different target genes have very different patterns of expression; some are developmental control genes such as Ubx and en and others are tissue specifically expressed genes such as Adh (Ref. 7), ddc (Ref. 5) or white (Ref. 20) (Fig. 1; Table 1). What could be the biological roles of promoterspecific transcription factors that act on these different genes?

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In the case of NTF-1 (Elf-l), a factor that binds the ddc and Ubx promoters (M. Biggin, U. Heberlein and R. Tjian, unpublished), several lines of evidence suggest that it is a specific regulatory protein controlling spatial patterns of gene expression. The specific DNAbinding activity of NTF-1 was only detectable in embryos at certain stages of developmenO 4, and the mRNA encoding this protein is spatially restricted in

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HdlB (a) Cis-regulatory elements controlling expression of Ubx. The top of the diagram shows the whole promoter, the scale is in kilobase pairs and +1 indicates the start site of RNA initiation. The c/s-regulatory regions are shown in stippled bars. These were identified either as regions to which genetic mutations map [abx, bx and bxd (Ref. 22)] or by P-element transformation and in vitro transcription studies (abx, vm and p; Refs 6, 14, 15, 33, and M. Bienz, pers. commun.). The intron and exon structure of a Ubx transcript is drawn. Below this are shown the transcription factors binding to the proximal promoter region p, and here the scale is in base pairs. The proteins shown are: Z, zeste; G, GAGA; NT, NTF-1; and HD, homeodomain proteins. Element A is a transcriptionally important sequence, but the factor(s) binding to it have not yet been purified. (b) Transcription factors binding the proximal promoter region of the Antp P2 promote,Iv,Is. The scale is in base pairs and the proreins shown are: DT, DTF-1 and AP, dAP-1. Elements B and C are transcriptionally important, but the factors binding to them have not yet been purified. the e m b r y o (Ref. 21, and B. Dynlacht and R, Tjian, pers. commun.). Furthermore, this protein binds to a DNA element that regulates tissue-specific expression of the ddc gene in neural cells but not in other tissuesS,-'L All these data suggest that NTF-1 directly contributes to the control of patterns of gene expression. A function of zeste protein is suggested by earlier genetic studies. The zeste gene is required for an unusual, minor regulatory process, termed transvection, which involves distant cis control elements of Ubx on one chromosome, directing the expression of a second copy of the Ubx gene on a separate chromosome 22,2-~. These data, together with the demonstration that zeste protein activates Ubx transcription in vitro when b o u n d close to the RNA start site, suggests that zeste protein bound to proximal promoter elements can mediate the control of transcription by interacting with factors bound to distant cis-regulatory elements ls.20. Interestingly, in other organisms promoter-proximal transcription factors, such as

SP1 and CTE appear to enable distant enhancer elements to function (see, for example, Ref. 24), and this may also suggest a similar function for other transcription factors such as GAGA, Adf-1 and DTF-1. But could proteins such as zeste, GAGA and Adf-1 have other functions in controlling transcription? Are they just constitutively required c o m p o n e n t s of some promoters or do they, like NTF-1, regulate spatial patterns of gene expression? In contrast to NTF-1, A d f l , zeste and GAGA proteins have been shown to be evenly expressed in most cells in the embryo, although their levels of expression do vary markedly during d e v e l o p m e n t (Ref. 25, and B. England and W. Soeller, unpublished). Despite this fact, these proteins may still play an important role in the spatial regulation of their target promoters, since different promoters are b o u n d by unique combinations of these factors. Thus they could act by a combinatorial mechanism. For example, different regulatory proteins

TIG NOVEMBER1989 VOL. 5, N(). II

]REVIEWS may only interact productively with promoters to which a particular subset of these widely expressed transcription factors is bound. Another function for these proteins is suggested by the temporal variation in their levels of expression. Thus, they could modulate the general level of transcription of a wide range of genes during development. The genes encoding GAGA, NTF-1 and A d f l have recently been isolated, and the chromosomal locations of these genes do not correspond to those of any genetically identified regulators of their target promoters (Ref. 21, and W. Soeller, B. Dynlacht and B. England, pers. commun.). Thus, genetic screens may not have identified all the loci involved in regulating developmental control genes, possibly because previ()us genetic screens would not detect regulators acting on a broad range of target genes, or because these proteins share redundant functions. It will now be critical to study these biochemically defined regulators by genetic analysis, and to assess how mutating the sequences to which they bind affects the expression of their target genes. For instance, it is already known that the 150 bp region of the Ubx promoter bound by GAGA, zeste and NTF-1 is required for transcription of the Ubx promoter in the embryo~,, suggesting that these proteins are important for the regulation of gene expression.

Homeodomain proteins as transcription factors Homologies to known transcription factors >~ suggest that over half the genes that control development, including the gap genes and a sex determination gene, may encode transcriptional regulators. However, here we limit our discussion to the large group of genes that share a homologous amino acid sequence termed the homeodomain, since currently these are the only developmental control genes that have been experimentally demonstrated to act as transcription factors. Homeodomain proteins have been expressed from cloned cDNAs, and analysed for DNA binding and transcriptional activities both in vivo and in vitro. A large group of proteins, including the products of the en, even-skipped (eve), fushi tarazu (,fiz), paired (prd) and gfbx genes, have been found to recognize at least some of the same DNA sequences with similar affinities (reviewed in Refs 2, 3), but other homeodomain proteins, such as the bicoid (bcd) gene product, appear to have quite distinct DNA binding specificities 2<27. In transient transfection assays, which introduce cDNAs expressing homeodomain proteins along with target promoter template DNAs into tissue culture cells, various homeodomain proteins have been found to act as sequence-specific proximal promoter- and/or enhancer-binding transcription factors 26,28-33 (Table 1). The transcriptional properties of homeodomain proteins have also been tested using purified proteins in reconstituted in vitro transcription reactions33. While these proteins are probably acting directly in both transcription assays, the in vitro assay provides the most unambiguous support for direct regulation, since in a cell-free system a purified factor cannot act indirectly, by influencing the expression of intermediary genes.

How do homeodomain proteins regulate spatially resl*~cted gene expression? The different spatial and temporal patterns of homeodomain gene expression result in different combinations of these proteins in specific cells of the embwo (Fig. 1) I. Genetic data predict that, in a given cell, many homeodomain proteins regulate overlapping sets of genes. Thus, in some cases they regulate the same promoter, while their other target genes are different. For example. Ubx expression appears to be activated by.fiz and repressed by Abdominal B (AbdB) and en. Yet in the same cells, ,/'tz expression is activated by ftz but is not regulated by AbdB or en. Particularly for those homeodomain proteins with similar DNA-binding specificity, how can we explain these types of genetic observations? The cases where several homeodomain proteins simultaneously regulate the same promoter may be explained by the finding that many of their target prorooters contain clusters of multiple homeodomain recognition elements. This allows several proteins with related DNA-binding specificity to bind simultaneously to the same promoter. In cotransfection assays, some combinations of homeodomain genes, such as prd and .fiZ, c a n synergistically activate the same promoter .~°. Bv contrast, other homeodomain genes, such as eve and en, do not activate transcription, but they competitively inhibit activation by other homeodomain genes-'O.YL These interactions between homeodomain proteins could explain an important mechanism by which striped patterns of gene expression are generated. For example, if a promoter is only activated when two particular homeodomain proteins are both bound to it, then it will only be switched on in cells where their expression overlaps (Fig. 3). However, it should be stressed that homeodomain proteins do not necessarily act in combinations, and can regulate transcription in the absence of other homeodomain proteins (see, for example, Ref. 33). Can we also explain the cases where homeodomain proteins with similar DNA-binding specificities show differences in promoter specificities? This paradox is undoubtedly explained, in part, by the finding that even homeodomain proteins with similar binding specificities do show" significantly altered affinities for distinct DNA-binding sites-'. However, this is not a sufficient explanation, since in tissue culture cells some of these proteins can bind and regulate promoters that they do not normally control in the embryo (see, for example, Ref. 31). Perhaps promoter specificity also results from the differential abilities of homeodomain proteins to interact with other proteins bound at target promoters. Such proteins could include the previously described unique combinations of non-homeodomain transcription factors that bind to the Ubx, Antp and en promoters. Indeed, the idea that homeodomain proteins can act together with other promoter-bound proteins is supported by the synergy and repression discussed previously, and by the observation that a cis element bound by homeodomain proteins on Uhx acts in concert with a second element to regulate Ubx expression in the visceral mesoderm<'. Another example of how- promoter specificity of Drosophila homeodomain proteins may be altered is suggested by

TIG NOVEMBER1989 VOL.5, XO. 11 38f

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;

are expressed in the same cells, making it difficult to determine which proteins are acting on a particular element in the embryo. To resolve these uncertainties, it will be necessary to develop more sophisticated in vitro transcription and cotransfection assays that include all the various regulatory factors, and more accurately reflect events occurring in the embryo. It will also be crucial to map additional bona fide target elements for these proteins in the embryo using Pelement transformation assays, if we are to understand fully how homeodomain proteins act during embryogenesis.

\

Functional domains and post-translational modifications

C]__) HGRI How synergistic activation of a promoter by two proteins can generate new patterns of expression. Two transcription factors (A and B) are expressed in overlapping patterns (top of diagram). They are both required for activation of a target promoter (center of diagram). Consequently the target promoter is only switched on in cells where both transcription factors are expressed (bottom of diagram). In the case shown (center of diagram), both proteins recognize the same DNA sequences and the promoter contains two such DNA elements. the yeast homeodomain protein 0c2, which appears to form a complex in solution with two different proteins, one of which alters its DNA-binding specificity (reviewed in Ref. 3). Informative as the above studies have been, little evidence is available concerning the cis-regulatory elements through which homeodomain proteins control gene expression in the embryo. Probably the best understood case is the activation of the hunchback (hb) promoter by bicoid gene product during early embryogenesis, where it appears that the bicoid protein directly binds and regulates the hb promoter in the embryo 26,34. By contrast, many other transcription studies have used artificial promoters, in which homeodomain recognition elements have been placed in heterologous promoter constructs (Table 1). Where natural target promoters have been used, it is not clear which homeodomain proteins act directly on these DNA sequences in the embryo (see, for example, Refs 6, 33). One reason for this uncertainty is that in cotransfection experiments some proteins will regulate genes in tissue culture cells that they do not appear o act on during embryogenesis (see, for example, Ref. 31). Also, at later stages of development, numerous homeodomain proteins with similar binding specificity

It seems clear that regulating transcription during embryogenesis requires the interplay of numerous proteins, but what are the molecular mechanisms underlying these interactions? Analyses of yeast and mammalian transcription factors have identified distinct functional peptide domains, and have begun to reveal how' different proteins interact on a promoter. For example, transcriptional activators recognize promoters via a DNA-binding domain, and then the transcriptional activation domain directly or indirectly contacts one or more of the general transcription factors to increase the rate of transcription ~2,13.3~-3". Although the dissection of domains within Drosophila transcription factors is in its infancy, it already appears that they are composed of functional units similar to those of mammalian proteins. For example, the homeodomain has been shown to be a DNA-binding domain (reviewed in Ref. 3) and there are sequences outside the homeodomain that are required for transcriptional activation30..~2. Also, Drosophila proteins are expected to contain the same activation motifs as some mammalian and yeast proteins, since transcription factors employ motifs such as negatively charged, glutamine-rich and proline-rich domains in Drosophila cells -~s3s. Drosophila transcription factors are also expected to have domains involved in homo- or heteromeric complex formation :~. For example, zeste protein binds DNA cooperatively e°, indicating that this protein can form multimers with itself. Obtaining an understanding of how proteins interact with each other structurally should reveal the mechanisms underlying the synergistic, competitive and other interactions we have discussed. Another structural aspect of Drosophila transcription factors that may be relevant to the mechanisms by which they act is their post-translational modification by phosphorylation and O-linked glycosylation2<39,4° (Table 1). In other organisms these post-translational modifications may regulate the activities of transcription factors 39,41. Thus it is possible that Drosophila proteins could be regulated in a similar manner, allowing a further level of control, in addition to regulating their patterns of expression.

Concluding remarks The generation of complex spatial and temporal patterns of expression requires the coordinated interplay of both widely expressed and spatially restricted promoter-selective factors. Even proteins that are

T1GNOVEMBER1989 VOL.5, NO. 11 38_~

[k~EVIEWS expressed in most Drosophila cells could, by combinatorial mechanisms, play a crucial role in the differential regulation of gene expression. The number of transcription factors identified in Drosophila is likely to increase rapidly over the next few years as more and more genetically defined trans-regulators are shown to be transcription factors, and as new proteins are isolated that bind to DNA elements regulating spatial and temporal patterns of transcription. Until recently our perception of the regulatory network controlling gene expression was based largely on genetic data. The biochemical analysis of the various classes of proteins involved in this process is beginning to provide a more mechanistic description of developmental gene regulation. This combination of genetic and biochemical data should provide a detailed understanding of these complex events in Drosophila.

Acknowledgements We are grateful to Mariann Bienz, David Hogness, Tom Kornberg, Mark Krasnow, Vincent Pirrotta, Matt Scott, Walter Soeller, Gary Struhl and their co-workers for communicating their unpublished data. We are indebted to Steve Bell, Mariann Bienz, Tim Hoey, Jim Kadonaga, Betsy O'Neill and Trew)r Williams for helpful comments on the manuscript.

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M.D.

BIGGIN IS IN THE DEPARTMENT OF MOLECULAR

810PHYSICS AND BIOCHEMISTRY, YALE UNIVERSITY, 2 6 0 WHITNEY AVENUE, P O B o x 6 6 6 6 NEW HAVEN, CT 06511,

USA," R. TJIAN IS IN THE HOWARD HUGHES MEDICAL INSTITUTE, DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF CALIFORNIA, BERKELEY, CA 94 720, USA.

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