Homeodomain proteins and the regulation of gene expression

Homeodomain

proteins and the regulation gene expression

M. Affolter, Biozentrum, Current

A. Schier and W.J. Cehring University Opinion

The homeobox (HB), a 180 bp DNA sequence element, was first identified as a region of sequence similarity between homeotic genes and several other genes involved in the control of Drasc@ih development (McGinnis et al, Nature 1984, 308:428-433; Scott and Weiner, Proc NatlAcudSci CBA 1984, 81:411H119). HB genes have now been found in numerous copies in eukaryotic organisms ranging from yeast to man (Gehring, Science 1987,

Scott et al, Biocbim Bioplys Acta 1989,

989:25-48). The HI3 is part of the coding sequence of these genes and the 60 amino acids it encodes are referred to as the homeodomain (HD). Evidence is now accumulating that I-ID proteins are transcription factors in which the HD is partly or fully responsible for sequencespecific recognition of DNA. In this review, we will describe some of the structural and functional properties of speciiic I-IDS or I-ID proteins. We will then focus on the molecular mechanisms by which well characterized I-ID proteins regulate the expression of their target genes and discuss these examples in the light of the involvement of numerous I-ID proteins in developmental pathways in higher eukaryotes.

Structure

and function

of Basel, Basel, Switzerland

in Cell Biology

Introduction

236:1245-1252;

of the homeodomain

It was suggested, based on sequence similarities of prokaryotic DNA-binding domains and the I-ID, that the structure of the I-ID includes a helix-turn-helix motif designed for sequence-specitic DNA binding (Iaughon and Scott, Nature 1984, 310:25-31; Shepherd et al, Nature 1984, 310:70-71). This hypothesis was directly confirmed by the recent determination of the three-dimensional solution structure of the HD of the Antemu@ diu (Antp) gene product by nuclear magnetic resonance spectroscopy [ 11. The structurally well deLined portion of the Antp I-ID includes four a-helices (Fig.1). Helices I and II are aligned in an almost antiparallel fashion, and helices III and IV are arranged approximately perpendic-

1990, 2A85-495

ular to helices I and II. The conformation of the helixturn-helix motif present at the C-terminus of the Antp I-ID (made up of helices II and III) is virtually identical to the helix-turn-helix motif found in many prokaryotic gene regulatory proteins, except that both helices in the Antp I-ID are longer than those in the prokaryotic DNA-binding proteins (Otting et al, EMBO J 1988, 73430-309; [ 11 and references therein). X-ray crystallographic analysis of the prokaryotic protein-DNA complexes, and functional studies using mutant proteins and mutant binding sites have revealed that amino acids within the second, so-called recognition, helix of the helix-turn-helix motif contact groups exposed in the major groove of the target DNA (Ebright et al, Nature 1984, 311:232-235; Wharton and Ptashne, Nature 1987, 326888-891; Bass

et aA, Science 1988, 242:240-245; Lehming et al, Prcc Nat1 Acud Sci USA 1988, 85:7947-7951; Aggamal et al., Science 1988, 242:89%907; Jordan and Pabo, Science 1988, 242:89%399; Wolberger et aA, Nature 1988, 335:78!+795; Otwinowski et al, Nature 1988, 335321-329). Functional analysis of the Drasqphila HD proteins Bicoid and Paired demonstrated that the corresponding helix in the HD (helix III) is also involved in sequence-specific recognition of DNA, although the most important amino acid positions determining the binding speciikity do not overlap with those determining the specikity of the prokaryotic repressors [ 2,3]. Interestingly, the putative recognition helix of the Antp HD is elongated by an additional fourth helical segment [ 1 I. Be cause the axes of these two helices form an angle with respect to each other, it has been pointed out that this kink might allow tight contacts between the entire length ofhelicesIIIandIVandDNA [l]. Another characteristic structural feature of the Antp HD is its overall globular conformation (excluding helix IV) with a hydrophobic core that includes 10 amino acid side chains [ 11. Two of these, Tip-48 and Phe-49, are found to be strictly conserved among 83 I-IDS analysed. Seven out of the remaining eight positions found in the hydrophobic core of the Antp HD are either highly conserved and/or almost exclusively hydrophobic in all those I-IDS (Scott et al, 1989). This favors the idea that d#er-

Abbreviations cDNA-complementary Prl-prolactin;

of

DNA; W-growth snRNA-small nuclear

@ Current

Biology

hormone; HB-homeobox; HD-homeodomain; RNA; UAS-upstream activation sequence.

Ltd ISSN 0955-0674

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_ RIO

R28

Fig. 1. Structure

of the Antennapedia homeodomain. The Antp HD consists of four a-helices (I-IV). The a-helices are represented as cylinders and the Ca atoms of the connecting backbone segments are linked by straight lines. The first and last amino acid of each helix are indicated by the one letter symbol. The helix-turnhelix motif consists of helices II and Ill. Helix Ill ends at position 52 and helix IV starts at position 53. Data are taken from 111.

ent HDs, defined as such by the conservation of these (and other, see Scott et al, 1989) residues, adopt similar three-dimensional structures and, thus, perform similar funchons. The DNA-binding properties of HD proteins have been investigated in various systems. In a series of elegant experiments, Johnson and Herskowitz (Cerr 1985, 42:237-247) showed that the yeast HB gene 1U47&2 encodes a sequence-specific DNA-binding protein and subsequent studies demonstrated that the HD was required for target site recognition (Hall and Johnson, Science 1987, 237:1007-1012). In vitm DNA-binding studies demonstrate that Drasqhla and vertebrate HD proteins are also capable of sequence-specific DNA recognition (Desplan et al, Nature 1985, 318:630-635; Fainsod et al, Proc Nut1 Acud Sci USA 1986, 83~9532-9536; Hoey and Levine, Nature 1988, 332:858&l; Cho et al, EMBO J 1988, 7~21392149; Miiller et al, EMBO J 1988, 7:4299-4304; Beachy et al, Cell 1988, 55:1069-1081; Iaughon et al, Development 1988,104:75-83; Odenwald et al, Gems Dev 1989, 3:158-172) and that, in certain cases, the isolated HD retains sequence-specific binding activity (Miiuer et al, 1988; Mihara and Kaiser, Science 1988, 242:92>927). Interestingly, the target sequences identified for several HD proteins are very similar to each other. Indeed, it has been shown that several different HD proteins (containing distantly related HDs) can recognize the same sequence elements in vim (Hoey and Levine, 1988; Desplan et al, Cell 1988,54:1081-1090). Equally intriguing is the finding that the same HD protein can bind to remarkably degenerate sequence motifs (Baumruker et al, Genes Dev 1988, 2:1400-1413). Nevertheless, certain HD proteins do not recognize sequences bound by

other HD proteins, and this specificity has been attributed to a single amino acid residue in the recognition helix of these HD proteins [ 2,3] (see also ‘Summary and perspectives’). Although these in vitro results do not demonstrate that HD proteins function as sequence-specific DNA binding proteins in vivo, several observations are consistent with such a function: HD proteins generally accumulate in the nucleus, point mutations in the HB region can greatly tiect wild-type gene activity (Laughon and Scott, 1984; Frasch et al, Genes Dev 1988, 2:1824-1838; Porter and Smith, Nature 1986, 320:766-768) [4] and HD proteins can regulate transcription in vivo and in vitro in a binding site-dependent manner (see below). Recent evidence suggests that the HD portion of a particular HD protein can I in addition, assist in protein complex formation; therefore, HDs are probably also involved in functions other than DNA binding.

Homeodomain regulators

proteins

as transcriptional

The interaction between ctiacting DNA regulatory elements and transacting factors regulates transcription in eukaryotes (for reviews, see Ptashne, Nature 1988, 335683-689; Johnson and McKnight, Ann Rev Bib&em 1989, 581799-839; Struhl, Ann Rev Bidem 1989, 58:1051-1077; Mitchell and Tjian, Science 1989, 245:371-378). Recent experiments have demonstrated that HD proteins can act as site-specific transcriptional regulators. The yeast al and a2 HD proteins have been found to have properties of transcriptional repressors, and several well characterized transcription factors have turned out to be HD proteins (Ott-1, Ott-2 and GHF-l/Pit-l, see below). Furthermore, Drosophila HD proteins can regulate transcription in a binding site-dependent manner in transient assays in cultured Draw@du cells (Jaynes and O’Farrell, Nature 1988, 336:744-749; Thali et al, Nature 1988, 336:598&X; Han et al, Cell 1989, 56:573583; Winslow et al, Cell 1989, 57:1017-1030; Krasnow et al, Cell 1989, 57:1031-1043; Dearolf et al, Nature 1989, 341:340-343) [5], in Droscghih embryos [4,6], in yeast (Fitzpatrick and Ingles, Nature 1989, 377666668; Samson et al, Cell 1989, 57:1045-1052; Driever et UL, Nature 1989, 342:149-154) [2,4], and in in vitro transcription systems (Biggin and Tjian, cell 1989, 58:433-443; Ohkuma et al, Cell, in press). The numerous HB genes involved in developmental processes (Gehring, 1987; Akam, Development 1987, lOl:l22; Ingham, Nature 1988, 335:25-34; Scott 1989; Harvey and Melton, Cell 1988, 59687-697; Wright et al, Cell 1989, 59:81-93; Wolgemuth et al, Nature 1989, 337~4-7; Balling et UL, Cell 1989, 58:337-347; Way and Challie, Cell 1988, 545-16) are therefore thought to regulate specihc target genes directly. The complexity of the HB regulatory network in Dros;ophila and the recent demonstration that proteins containing quite distantly re-

Homeodomain

lated I-IDS can bind to the same sequence element in vitro (Hoey and Ievine, 1988) or regulate gene expression in transient assays through identical target sequences (Jaynes and O’Farrell, 1988, Thali et al, 1988; Han et al, 1989) poses multiple problems for the elucidation of defined regulatory circuits. The apparent lack of target specificity of certain I-ID proteins in artilicial situations also seems to be dilhcult to reconcile with the speciiic role of I-ID proteins during development. In the following, we focus on several well characterized cases of regulation of gene activity in which an I-ID protein(s) participates and for which interactions at the molecular level have been characterized in some detail. Afterwards, we discuss how these examples could illustrate the mechanisms by which I-ID proteins regulate transcription during development (see also Levine and Hoey, Cell 1988, 55:536-540). Although it is very likely that certain I-ID proteins directly regulate the expression of HB genes (see for example: Gehring and Hiromi, Ann Rev Genet 1986, 20:147-173; Akam, 1987; Scott and Carroll, Cell 1987, 5689-698; Ingham, 1988) we shall not discuss the regulation of the regulators at length because in most cases, the studies are still preliminary.

Yeast homeodomain

proteins

In the yeast Saccbaromyces cerevisthe, three HB genes have been identilied to date. The MATal and MAa2 genes encode proteins involved in the regulation of celltype-specific gene expression; the PH02 gene product plays a role in the regulation of diverse metabolic pathways. Target operator sequences for these regulatory proteins have been identilied and, particularly in the case of al and a2, the molecular interaction of individual components at the operator sites characterized.

al and ti

proteins

The life cycle of S. cerevisiae is made up of three cell types: the a and a mating types are haploid, and upon fusion give rise to the third cell type, the a/a diploid cell. Different cell-type-specilic proteins are expressed in a, a and a/a cells as a consequence of the information present at the mating type locus MAT(for recent reviews, see Nasmyth and Shore, Science 1987, 237:1162-1170; Herskowitz, Nature 1989, 342:749-757). MA% codes for two proteins directly involved in haploid

a cell-type-specific gene expression; al is a positive regulator required for the activation of a-specific genes (Bender and Sprague, Cell 1987, 50681491) whereas a2, an I-ID-containing repressor, is involved in silencing the aspeci6c genes (Johnson and Herskowitz, 1985; Hall and Johnson, 1987). The a2 target sites, the a2 operators, consist of a 32 bp sequence that is highly conserved between the different operators (Johnson and Herskowitz, 1985; see also Miller et al, Nature 1985, 314:598-603). Not only a2, but also another gene product, Mcml (also called PRTF or GRM), can bind to the a2 operator (Pass-

proteins/regulation

of gene expression

Affolter,

Schier, Gehring

more et al, Genes Dev 1989, 3:921-935) [7]. Mcml, which is present in all yeast cell types, seems to function as a sequence-spectic, weak transcriptional activator enhancing the expression of a-specific genes in the absence of a2 (e.g. in a cells) by binding to the P box in the center of the a2 operator [7]. In a cells, the expression of a-specilic genes is repressed by the interaction of a2 with both ends of the operator (Hall and Johnson, 1987; Sauer et al, Genes Dev 1988,2:807-816), and it has been shown that although the I-ID is required for repression, it is not sufficient (Hall and Johnson, 1987). Mcml and a2 can co-occupy the operator in vitro (therefore making repression through competition for the binding site unlikely), and binding of the two is actually highly cooperative [7]. In the presence of Mcml, the aflinity of a2 for the operator increases approximately 500-fold (Keleher et al, Mol Cell BioLl989,9:52285230). This effect requires the presence of N-terminal sequences of a2, suggesting that Mcml/a2 cooperative binding is mediated through protein-protein interaction [7]. Therefore, the transcriptional activator protein Mcml whose aEmity for its a2 operator target site is approximately 100 times higher than the binding affinity of a2 (see Sauer et al., 1988 and Tan et al, EMBO J 1988, 7:4255-4264), seems to drag its own ‘inactivator’ to the target site. It is not known whether a2, w-hose selectivity for the operator relative to other sequences is fairly poor [7], would be able to fully occupy the operator in vivo in the absence of Mcml. III the scenario just described, the I-ID-containing a2 protein somehow negates or masks the activity of Mcml by binding to sequences adjacent to the P-box. It thereby reduces considerably the transcription-stimulatory effect that the a2 operator confers on downstream promoters in a cells by virtue of its ability to bind Mcml. This mechanism might account for the inactivation (in a cells) of the EAR Igene, whose major upstream activation sequence (UAS) maps to the a2 operator (Kronstad et al, Cell 1987, 50369377). However, in a cells, the a2 operator is also capable of repressing transcription driven by a UAS when placed in a test gene construct between the TATA box and the UAS, or upstream of a UAS (Miller et al, 1985) [7]. This repression requires both functional Mcml- and a2-binding sites and the presence of the corresponding @amacting factors [7]; therefore, Mcml in this situation acts as a corepressor. The ‘locking’ model proposed for repression in these cases implies that a2 does not simply mask the activity of Mcml but that the Mcml-a2 complex locks the transcriptional machinery in place [7]. It is not known how many of the a-speck genes contain a UAS upstream (or downstream) of the a2 operator(s). A UAS upstream of the a2 operator has been identilied in the S7EG gene, but not precisely localized (Wilson and Herskowitz, Proc Nat1 Acud Sci LISA 1986, 83:253&2540). Besides acting as a repressor of a-specifk genes together with Mcml, ~2, in conjunction with the product of the HB gene M4Tu2, is also required for repression of haploid-specilic genes (including al ) in diploid a/a cells. The al/a2 operator is a - 20 bp sequence element that

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is similar in sequence to the ends of the &2 operator, although the symmetrical half sites seem to be shorter and closer to each other (see Miller et al, 1985; Siliciano and Tatchell, Proc Nat1 Acud Sci USA 1986, 83:232&2324) [8]. At present, no data on the exact molecular architecture of the operator complex are available. The simplest model accounting for the data obtained in in vitro DNAbinding studies proposes an interaction between the HDcontaining al and a2 proteins, resulting in an al/a2 heterodimer on the operator [8] (see also HaJl and Johnson, 1987). In addition to the I-ID, the al/a2 binding activity seems to require an N-terminal region of a2 different from that required for a2 binding [ 81. This indicates that distinct portions of the same regulatory protein may perform different tasks in various complexes. It is not yet clear whether a2 has, in the presence of al, an increased affinity for the al/a2 operator or whether the interaction of al with a2 induces structural changes in the a2 HD and/or Banking protein sequences enabling it to recognize the operator. How the functional al/a2 operator complex represses transcription has not yet been investigated. Although al plays a key role in the repression of haploid-specific genes in diploid cells and is present in a cells, it is not essential for the a cell type and a-specific genes are expressed without any action by MATa.

ing with these HD proteins require at least two different gene products for function. Although this might, in certain cases, increase the target site speciScity of the HD proteins involved (Mcml/a2; al/a2), studies with PH02 suggest that, at other operators, the second component might be required for the cooperative activation of the transcriptional machinery. The yeast system also provides an example of a single HD protein (a2) acting, at operators of different sequences, with two distinct proteins (Mcml and al) in the same cell (a/a cells). At the a2 operator, this interaction seems to modify the function of a general transcription factor (Mcml), whereas at the al/a2 operator, a new regulatory activity is assembled from two celLspeci6c factors. This emphasizes that the combinatorial assembly of regulatory species (using ubiquitous and specific factors) on different &acting elements can be used to generate cell-specific gene expression. The absence of a component (a2) required for a distinct regulatory species (al/a21 then explains why the expression of a particular HD protein in a specific cell type (al in a cells) does not have any consequences on cell-type-specific gene expression.

Mammalian

PHo2 In contrast to al and a2, the HD protein encoded by the PH02 regulatory gene (also known as LWz Sengstag and Hinnen, Nucleic Acids Res 1987, 15:233246; Burglin, Cell 1988, 53:339-340) is involved in the coordination of phosphate regulation of diverse metabolic pathways. It is required for expression of the acid phosphatase gene PH05 (O&ma, In Molecular Biology of he Yeast Saccbaromyces, edited by Strathem JN, Jones EW, and Broach @ Cold Spring Harbor Laboratory, 1982, pp 15!&180), for the basal level of transcription of the histidine biosynthetic HIS4gene (Amdt et al., Scie?rce 1987, 237:874880), and possibly for the modulation of expression of the XW4 gene which encodes a ttyptophan biosynthetic enzyme (Braus et al, EMBO J 1989,8:939-945). Although putative target sites for PH02 have been identified, experiments addressing the precise mechanisms of action have not been reported yet. However, genetic studies indicate that PH02 cannot act alone: basal transcription of the HIS4 gene requires both functional PH02 and BRSI genes (Tice-Baldwin et al, Science 1989, 246:931-935) and PH05 transcription requires both the PH02 and PHO4 gene products (Oshima, 1982). On the HIS4 promoter, BASl and PH02 interact in vitro with sequences adjacent to each other, but in a preliminary study no cooperativity of binding was observed (Tice-Baldwin et al., 1989). Whether transcriptional activation requires direct interaction between PH02 and BASl remains to be seen. In yeas4 HD proteins can thus act both as negative (al, a2) or positive (PH02) transcriptional regulators. Interestingly, and in contrast to certain other yeast transcriptional regulators (see Struhl, 1989), operators interact-

homeodomain

proteins

Little is known about the function(s) of the numerous mammalian HB genes that have been isolated mostly by virtue of their crosshybridization with Drosophila HB sequences. Although DNA-binding studies have shown that some of the HD proteins encoded by these genes can interact with specific DNA sequence elements in vitro, the search for genes that are regulated by these proteins in vivo is still underway. However, it has been demonstrated recently that three well characterized transcription factors, Ott-1, Ott-2 and GHF-l/Pit-l, for which target sites have been identified, contain HD sequences. Surprisingly, these three transcription factors not only share a similar HB (Ko et al., Cell 1988, 553135-144; Ingraham et al., Cell 1988, 55~519-529; Sturm et al, Genes Dev 1988, 2:1582-1599; Clerc et al, Genes Dev 1988, 2:157O-1581) [9-111, but also an extremely conserved so-called POU-specific box, 5’ to the HB [ 121. The POUspecific box encodes a 75arnino acid ‘domain’ separated from the HD by 20-30 ammo acids. The same structural arrangement is also found in the uric-86 gene of the nematode Caenorbabditti elegans (Finney et al., Cell 1988, 55:757-769), which led to its designation as ‘POU’ domain (for Pit, Ott and Uric) [12]. The function of the POU-specific domain is not well defined at the moment. In Ott-1 and Ott-2, it seems to be required for efficient binding to the target sequences (Stumr and Herr, Nature 1988, 366601604; Muller-Imrnergluck et al, EMBO J 1990, 9~1625-1634; Gerster et al., EMBO J 1990,9:1635-1643) whereas in GHP-l/Pit-l, its presence or absence has only a minor effect on sequence-specific DNA binding in vitro (Theill et al, Nature 1989, 342:945-948). In other organisms, POU-specific boxes have also been found in close association with HBs (Johnson and Hirsh, Nature 1990, 343:467+70).

Homeodomain Octamer

binding

factors

The octamer motif ATGCAAAT has been identiiied in promoters and/or enhancers of a substantial number of genes: it seems to act as a cell-cycle regulated element in histone H2B genes [ 131; it behaves as an enhancer element in certain ubiquitousIy expressed, RNA poIymerase II-transcribed U smalI nuclear RNA (snRNA) genes; and it par@ mediates the virus-induced activation of herpes simplex virus immediate-eariy genes. In addition, its presence in immunogIobuIi.n gene regulatory regions seems to contribute to high-level, tissue-speciIic expression. Two major mammalian proteins that speciIicaIIy recognize the octamer motif have been purified and characterized: the ubiquitousIy expressed Ott-1 protein (also referred to as OTF-1, NF-Al, OBPlOO or NFIII) and the lymphocyte-speciIic Ott-2 protein (also referred to as OTF-2 or NFA2). Both factors can act as sequence-speciIic transcriptional activators in in vitro transcription assays (Fletcher et al, Cell 1987, 51:773-781; Scheidereit et al, Cell 1987, 51:783793). Although encoded by different genes, both proteins recognize the octamer motif in identical fashion as judged from methyiation interference studies (Staudt et al, Nature 1986, 323640-643). Therefore, a major question that must be addressed in order to understand the function(s) of the octamer motif and the factors interacting with it is: how is differential regulation mediated by speciIic octamer sites? An interesting description of how regulation via an octamer motif can be achieved comes from studies on viral gene expression. Immediate-eady genes of the herpes simplex virus contain one or several copies of a conserved TAATGARAT (where R is purine) DNA element (see h4ackem and Roizman, J W-01 1982,44:939-949; Gelman and Silverstein, J Kirol 1987, 61:3167-3172). This motif mediates transcriptional stimuIation through a virai transinducing factor that was identi6ed as ~~16, the major protein of the viral tegument, also referred to as aTLF, Vmw65 or ICP25 (Campbell et al, J Mol Biol1984, 180:1-19). ~~16 does not bind directly to the consensus site (Marsden et al, J Viral 1987, 61:24282437), but interacts (directly or indirectIy) with a cellular factor(s) that recognizes the element (Preston et al, Ceu 1988, 52:425-434; O’Hare and Goding, cell 1988, 52:435-445). In most cases the TAATGARAT motif is preceded by the sequence ATGC, generating a seven out of eight match with the octamer motif (ATGCTAATGARAT). It can therefore be described as OCTMGARAT [ 151 which suggests that the cellular factor that recognizes the motif in non-infected cells could be the ubiquitousiy expressed Ott-1 protein (O’Hare et al, EMBO J 1988,7:42314238; Kemp and Iatchman, EMBO J 1988, 7:423W244). Indeed, Gerster and Roeder [ 151 demonstrated that, in combination with ~~16, purified Ott-1 protein can form high-molecular-weight complexes that include one, or possibIy several, additional cellular protein(s) (see also Kristie et aA, EMBO J 1989, 8:4229-4238). This interaction requires both functional OCTA and GARAT sequences [ 151. The transcription activation potential of the multiprotein complex is most likeIy provided by the

proteins/regulation

of gene expression

Affolter, Schier, Gehring

transactivator ~~16 (Triezenberg et aA, Gme.s Dev 1988, 2:718-729). InterestingIy, although Ott-2 binds to the OCTWGARAT element, it does not efficientIy form a complex with ~~16 (Kristie et al, 1989) [ 15,161. This feature was used in a series of elegant deletion and ‘swapping’ experiments, where different portions of Ott-1 were either deleted or replaced by the equivalent portion of Ott-2, and the altered proteins were subsequentIy tested for complex formation [ 161. Although Ott-1 and Ott-2 contain very similar POU domains and completely divergent N- and Cterminal flanking sequences, it was convincingIy demonstrated that the major determinants that allow complex formation are coniined to the I-ID region of Ott-1. Three amino acid differences (out of a total of seven) between the Ott-1 and Ott-2 I-ID are found in helix II. Substituting these residues in Ott-1 with those present in Ott-2 inactivates complex formation. However, the reciprocal ‘swap’ does not confer complex-forming capacity on Ott-2 [ 161, indicating that additional residues in the Ott-1 I-ID are required. Taking the three-dimensional structure of the ~ntp I-ID as model of the Ott-1 I-ID, the three residues in helix II lie on the surface of the protein [1,16] and could therefore be directly invoIved in protein-protein interactions required for complex formation. Although it is difficult to draw a clear picture of the resuking complex (because it is not known whether ~‘16 itself or a ceIIular factor(s) interacts with the Ott-1 HD), the study by Stem et al [ 161 provides an interesting example of how a non-DNA-binding protein can differentiaIIy iniluence the transcription potential of two I-ID proteins (Ott-1 and Ott-2) that bind indistinguishably to the same sequence. It wiII be interesting to hd out whether the I-ID of Ott-1 (or other I-ID proteins) also directs protein-protein interactions to regulate cellular transcription, and whether this requires cellular factors similar in sequence to ~‘16. In most histone H2B genes, the octamer motif is akio flanked by conserved sequences and is embedded in a 1315 bp-long H2B subtype-speciiic consensus element (the so-called Harvey box; Harvey et al, Nuckic Acid Re.s 1982, 10:7851-7863), present approximately 20 bp upstream of the TATA box. Upon introduction of point mutations in the ATGCAAAT motif of a human H2B promoter, transcriptional induction at the G2/S boundary of the cell cycle is abolished [ 131. This suggests that a cell-cycle-dependent signal is transmitted (directIy or indirectIy) via Ott-1 to control transcription of the H2B gene. The strong conseivation of sequences Ranking the octamer suggests that they may be involved in this reguIation, possibIy in a manner comparable with the octarnerIlanking sequences present in herpes simplex immediate early genes. The Ott-1 protein is also invohred in the high-level expression of certain U snRNA genes in HeIa ceIIs. In most U2 snRNA gene enhancers, the ATGCMAT sequence is adjacent to a so-called GC-box in an upstreamactivating region located near position -200 (Mattaj et al, Nature 1985, 316:163167; Ares et al, Mol Cdl Biol 1985, 5:1560-1570). Both elements are required for ef-

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Ecient transcription in transient expression experiments and are thought to interact with the nuclear factors Ott-1 and Spl, respectively (Ares et al, Genes Dev 1987, 1808-817). The U2 snRNA enhancer, in contrast to other enhancers, only functions in combination with a U2 snRNA proximal element [14] which is notable for the lack of a TATA box. It is the presence of this proximal element that seems to render the minimal U2 snRNA promoter responsive to a strong activation of transcription by the Ott-1 protein in HeIa cells [ 141. It is not known if Ott-1 can speciEcally interact with proteins bound to the proximal element. Recent studies on the function of the Ott-2 protein suggest that the presence of Ott-2 in B-lymphocytes most likely contributes to the high level of transcription of immunoglobulin genes in these cells. Tmnsfection studies have shown that the octamer is the most prominent cell-type-speciEc transcriptional control element in immunoglobulin enhancers and promoters. In addition, cell-type-specific promoters (being highly active in B-cells and only weakly or not at all active in HeIa cells) can be artiEciaUy generated by fusing the octamer site to heterologous, minimal promoters (Mason et al, Cell 1985, 4188-97; Wirth et al, Nature 1987, 329:174-178; Gerster et al, EA4BO J 1987, 6:13231330; Dreyfus et al, EM..0 J 1987, 6:16851690). The activity of these natural or artificial promoters in lymphoid and non-lymphoid cells therefore correlates with the presence of Ott-2, indicating that Ott-2 is the trarzs-acting factor that associates with the octamer in these constructs and that Ott-1 (at the level present in non-lymphoid cells) is incapable of stimulating transcription of these templates to a sign& cant extent. Recently, it has been shown that introduction of an Ott-2 complementary DNA (cDNA) under the control of a constitutive enhancer/promoter into HeLa cells induces tmnscription of a cotransfected lymphocyte-spe ciEc promoter [ 91, demonstrating that at least one aspect of cell-type speciEcity can be transplanted from one cell type to the other by the overexpression of a single tram acting factor. Functional dissection of Ott-2 revealed that efficient activation requires two interdependent nonacidic domains, located N- and C-terminal of the POUdomain (Tanaka and Herr, Cell 1990, 60:37%386; Gerster et al, 1990 see also Mtiller-Immergluck et al, 1990). However, several observations suggest that Ott-2 alone does not determine B-cell speciEcity of immunoglobulin genes in vivo. Irnmunoglobulin heavy chain enhancers, in which the octamer sequence has been deleted, still display B-cell speciEcity (Perez-Mu&r1 et al, Nucleic Acids Res 1988,16:6085-6096). Also, pre-B-cell fines containing extremely low levels of Ott-2, yet expressing high levels of immunoglobulin heavy chain ~RNA, have been found (Johnson et al, Mol Cell Bioll990, 10:982-990). Furthermore, both Ott-1 and Ott-2 can activate transcription of immunoglobulin gene promoters in vitro (LeBowitz et al, Genes Dev 1988, 2:1227-1237; Poellinger et aA, Nature 1989,337:573-576; Johnson et aA, 1990). These Endings underline questions regarding the role(s) of Ott-1 and Ott-2 in the transcription of immunoglobulin genes

in vivo. Definitive answers to these questions may require gene disruption experiments. Pituitary-specific

transcription

factor(s)

Studies aimed at a better understanding of the mechanisms involved in the control of tissue-speciEc gene expression in mammals recently led to the identitication and isolation of an additional HD-containing transcription factor, the pituitary-specific GHF-l/Pit-l protein. Anterior pituitary gland development results in the generation of ftve cell types that are distinguished on the basis of the secreted hormone. The last two phenotypitally distinct cell types to appear are the somatotrophs and the lactotrophs, producing growth hormone (GH) and prolactin (Prl), respectively. Several &acting control elements are required for the tissue-speciEc expression of the GH and Prl genes. Some of these elements overlap with binding sites for GHF-l/Pit-l (see Lefevre et aA, EhfBO J 1987, 6:971-981; Nelson et al, Science 1988, 239:1400-1405, and references therein). In vitro and in cell culture, this HD protein(s) seems to act as a transcriptional activator when bound to sites upstream of the transcriptional start sites of the GH and Prl genes (Nelson et al, 1988; Mangalam et al, Genes Dev 1989, 3946958) and in analogy to the Ott-2 factor, it has been shown that ‘ectopic’ expression of GHF-l/Pit-l in HeLa cells can activate co-transfected pituitary-specific promoters (Ingraham et al., 1988; Mangalam et al, 1989; Theill et al, 1989). Interestingly, GHF-l/Pit-l appears to be expressed in both somatotrophs and lactotrophs in adult rats (Ingraham et aA, 1988) [ll]. Expression of GH and Prl would then have to be under restrictive control in the non-producing cell type. This model is supported by the demonstration that specific sequence arrangements (a tissue-speciEc promoter and a tissue-speciEc enhancer including negatively acting &inking sequences) are necessary to restrict expression of the Prl gene to lactotrophs in transgenic mice (Crenshaw et al, Genes Dev 1989, 3:959-972). Therefore, it has been proposed that GHF-l/Pit-l primarily restricts target gene expression to the anterior pituitary, but that additional restrictive mechanisms have to be involved that suppress GHFl/Pit-1 in certain pituitary cells leading to the observed differential cell-type-speciEc expression. However, conflicting results have been obtained by other investigators: GHF-l/Pit-l seems to have only low afEnity for the Prl control region whereas another factor, IFS-l, speciEcally binds with high alfinity to elements in the Prl but not the GH promoter (Castrillo et al, Science 1989, 243:814-817; see also Karin et al., Trends Genet 1990,6:92-96). This led to the alternative hypothesis that GHF-l/Pit-l activates the GH gene whereas the Prl gene is activated by IFS-l. The precise correlation, both temporally and spatially, of the expression of GHF-1 and GH proteins during mouse embryogenesis strongly supports the suggestion that GHF-1 plays a major role in the specialization of somatotrophic cells (Do@ et al, Cell 1990, 6:809-820). As in the case of Ott-2, however, only gene

Homeodomain

proteins/regulation

of gene expression

Affolter,

Schier, Cehring

The most studied of these examples is bicoid @cd). Embryos derived from bed females develop lacking head and thoracic structures implicating the bcdgene product in the formation of anterior structures. Ecd transcripts are synthesized in the nurse cells during oogenesis and subsequently transported into the egg cell where they are localized at the anterior pole of the egg (Frigerio et aA, Cell 1986, 47~735746; Berleth et al, EFfBO J 1988, 7:174!+-1756). Upon fertilization, these transcripts are translated; a relatively stable Bicoid protein gradient is established over the anterior two-thirds of the early embryo possibly through diffusion of RNA and/or protein and dispersed degradation [Frigerio et al, 1986; Driever and Nusslein-Volhard, Cell 1988a, 54:83-93; Johnston et al, Development 1989, 107(suppl):1319]. Using genetic means to manipulate the density and distribution of Bicoid in embryos and correlating the fate map of the respective embryos with the altered distribution of the protein, Driever and Niisslein-Volhard (CeLl 1988b, 54:95-104) concluded that Bicoid has all the properties of a morphogen that determines position in the anterior half of the embryo in a concentration-dependent manner. The presence of an HB in the L&gene (Frigerio et al, 1986) suggested that the morphogenic effects of Bicoid could be brought about by its direct regulation of zygotic target genes.

sion gene constructs in which these Bicoid-binding sites were either successively deleted or which contained different numbers of high- and/or 1owaRinity binding sites was studied in transgenic flies. The experiments revealed several important features of Bicoid-dependent expression patterns [4-6]. First, the quality of binding sites determines the position of the posterior border of expression, i.e. high-afhnity sites allow a transcriptional response to lower levels of Bicoid protein than low-athnity sites. Second, the number of low- or high-affinity binding sites determines the level of expression and the sharpness of the posterior border, i.e. the more high-affinity binding sites present, the higher the level of expression and the sharper the posterior border. Furthermore, it was shown that by varying the number of bed gene copies, the posterior border of expression of the reporter gene could be shifted, i.e. with increasing amounts of Bicoid protein, the expression domain expand4 more posteriorly. These studies strongly suggest that the function of multiple Bicoid-binding sites in the bb promoter is to confer high levels of b6 expression in the anterior half of the embryo and to generate a sharp border at the posterior end of the expression domain [4,5]. This allows relatively small differences in Bicoid concentmtion (approximately two-fold or even smaller) to discriminate between the on or off states of hb expression. At present, it is not known whether the strong cooperativity of 6& function observed with multiple binding sites (as demonstrated by artificial promoter constructs) is a result of cooperative binding or cooperative activation (see also Berk and Schmitt, Genes Dev 1990,4:151-155). Although oligomerized Bicoid-binding sites can confer expression patterns to reporter genes that are similar to the expression pattern of the endogenous hb gene, recent results [4] suggest that Bicoiddependent cooperativity on transcription from the hb promoter might be assisted by additional factors that interact with c&acting elements flanking the Bicoid-binding sites. Therefore, it is possible that the number of Bicoid molecules required to activate transcription depends on the presence of other associated factors which might facilitate or interfere with activation [4]. The highly concentration-dependent gene activation by Bicoid shows how the relatively smoothly graded information of a spectic gene product can be translated into discrete domains of target expression, implying that relatively small differences in the level of Bicoid can have profound effects on gene expression.

Among the first zygotically expressed genes are the socalled gap genes (Gaul and Jackie, Trench Genet 1987, 3:127-131). One of them, the hunchback, @bIgene, is required for anterior development (Lehmann and NiissleinVolhard, Dev Biol 1987, 119402417). Zygotic expression of bb in the anterior half of the embryo is bed dependent (Tautz et al, Nature 1987, 327:383389; Tautz, Nature 1988, 332:281-284). The regulatory sequences of the b6 promoter required for bcddependent expression are within a 300 bp region upstream of the transcript start site (Schroder et al, EMBO J 1988, 7:2881-2888). This region contains three high- and three low-afhnity binding sites for Bicoid [5]. Expression of fu-

A second maternally expressed HB gene product for which putative target sites have been identiJied is the Caudal (Cad) protein. Cad accumulates in a concentration gradient across the anterior-posterior axis of the developing embryo, resulting in high levels at the posterior end of the embryos (Mlodzik et al, EMTBO J 1985, 4:2961-2969; Macdonald and Struhl, Nature 1986, 324~537-545; Mlodzik and Gehring, Cell 1987, 48:46%78). A potential target gene for Cad is the zygotically active segmentation gene fi.~& turazu (jk). A

disruption experiments will provide more definitive information concerning the function(s) of GHF-l/Pit-l and LFS-1.

Drosophila

homeodomain

proteins

Over 30 HB genes have been isolated so far in Drascpbih melunoguster. HD proteins seem to play a role during most stages of fly development ranging from the determination of anterior-posterior polarity to the differentiation of the nervous system. Therefore, considerable effort has been invested in the study of the regulation of HB gene expression and in the search for genes that are directly regulated by HB gene products in vivo. At the moment, there are only a few good examples of direct transcriptional regulation exerted by a HD protein via defined DNA regulatory elements in a target gene. Bicoid

Caudal

491

492

Nucleus

and gene expression

700 bp fragment upstream of the )z transcription start site, called the Zebra element, is sufficient to generate the spatially restricted early expression pattern of& (Hiromi et al, Cell 1985, 43:6OS13; Hiromi and Gehring, Cell 1987,50:%3974), and contains a 350 bp region that confers an expression pattern to a heterologous promoter that is similar to the distribution of the Cad protein during early embryogenesis (Dearolf et al, Genes Dev 1989, 33384-398). Two binding sites were identifkl in that region, to which bacterially expressed Cad protein or Cad protein produced in D?-oxp%h Schneider cells binds. Point mutations that abolish binding to either of the two sites abolished posterior expression of the reporter gene in embryos (Dearolf et al, Nature 1989, 341:340-343). Although these data are consistent with a direct interaction of Cad with the J?Z promoter in viuo, it cannot be ruled out that another, as yet unidentified HD protein, recognizes the Cad-binding site and interacts in vivowith one or both of the elements.

Summary

Regulatory

activity

Regulatory effect

(a)

+

-

and perspectives

Present evidence clearly supports the view that HD proteins act as sequence-q&k DNA-binding proteins that can directly regulate, positively or negatively, the expression of specifk target genes. The initial proposal that the HB encodes a DNA-binding domain containing a helixturn-helix motif similar to that in prokaryotic regulatory proteins has been confirmed by functional and structural anatyses. The examples presented in this review (see also Fig.2) demonstrate that HD proteins regulate gene expression via a variety of mechanisms and that, in certain cases, the spedicity of action with respect to target gene regulation can be attributed to different molecular strategies. Amino acid sequences within the HD can determine the DNA-binding speci6city (see below) or inlluence the ability of a given protein to form specific DNA-protein complexes (e.g. Ott-l/WIG). Ammo acid sequences flanking the HD can tiuence interactions with the transcripFig. 2. Homeodomain

proteins as regulators of gene activity. (aI In yeast, Mcml bound to the a2 operator weakly activates transcription of a-specific genes in a cells. Binding of a2 results in transcriptional repression of these genes in a and a/a cells. al and ~2 bound to the al/a2 operator repress transcription of haploid specific genes in a/a cells. fb) In mammalian cells, Ott-1 can bind to OCTAKARAT motifs but does not efficiently stimulate transcription. The Ott-1 protein forms a complex with the viral VP16 protein and one for more) cellular proteins on a functional OCTAKARAT motif; this complex promotes efficient transcription. Ott-2 stimulates transcription from an OCTA motif but does not form a complex with VP16. fc) In Drosophila, Bicoid protein can stimulate transcription from promoters containing Bicoidbinding sites. Transcription stimulation depends on the level of Bicoid protein and the number and affinity of Bicoid binding sites. For simplicity, only the DNA segments that are the targets for the assembly of HD proteins are indicated. +, activating regulatory effect; -, repressing regulatory effect. 0 indicates that the binding of a factor does not detectably affect the transcription initiation rate from the nearby transcription start site. For details, see text.

-

(b)

o/+

+++

+

+

++++

Homeodomain

tional machinery (e.g. Ott-1, Ott-2) or with well defined factors required for specific action (Mcml/ct2 and al/a2). Potentially, these interactions can also be influenced by differential regulation at the post-transcriptional level (i.e. alternative splicing; Mann and Hogness, Cell 1990,60:597-610) or at the post-translational level (several HD proteins are phosphotylated; see Krause et al, Genes Dev 1988, 2:1021-1036; Gay et al, Nucleic Acids Res 1988, 166637-6646; Odenwald et al, 1989; Krause and Gehring, EMBO J 1989, 8:1197-1204; Tanaka and Herr 1990) [5]. Combined with the spatially and temporally restricted expression of I-W genes, the different levels of expression (e.g. Bicoid) and the availability of other transacting factors in specific cells, these mechanisms might contribute to the unique specificity (with respect to target genes controlled in a speciiic manner) of most HD proteins. Important questions concerning the target site specilicity of HD proteins are presently being addressed. The mechanism by which I-ID proteins bind to DNA is not known, and its elucidation could shed some light on the contribution of the DNA-binding site to selective regulation by various I-ID proteins. Despite the conservation in the Anp HD of a nearly identical helix-turn-helix motif, prokaryotic repressor DNA-binding domains and Anp HD seem to interact with DNA in a substantially different manner. The aEinity of a single Anp I-ID molecule for its recognition site is considerably higher than the atfinity of several prokaryotic DNA-binding domains for their monomer binding site. This is also reflected by the fact that a more extended DNA segment interacts with a single Anp HJI molecule, as shown by methylation and ethylation interference studies (Affolter et al, Proc Nat1 Acud Sci in press). In addition, it has been suggested that the putative recognition helix of I-ID proteins (helix III) may be oriented differently in the major groove than the recognition helix of prokaryotic repressor proteins [ 2,3]. Structural analysis of I-IDDNA complexes will provide a detailed picture of how I-IDS bind to DNA and will reveal which amino acids are potential candidates for providing the specificity of the interaction with respect to the protein component. The function of individual amino acids in site-specilic DNA recognition has already been investigated to a certain extent. The amino acid at position 9 of the putative recognition helix III of certain I-ID proteins is critical for DNA-binding specilicity whereas other surface-exposed residues in helix III do not seem to be very important for target site selection in the cases examined [2,3]. Recent studies suggest, however, that certain proteins with an identical amino acid at position 9 can differentially recognize target sites in vitro and possibly in viva, for example, the target site specificity of the Cad protein seems to ditfer from that of other I-ID proteins with identical amino acids at position 9 (see Dearolf et al, 1989). Therefore, although position 9 seems to be an important deten-ninant of sequence specihcity, other residues, possibly even outside the helix-turn-helix motif, may play equally important roles. Interestingly, a I-ID substitution in the Deformed protein changes the regulatory specilicity of the

proteins/regulation

of gene expression

Affolter, Schier, Gehring

resulting chimeric protein in Dnasqhila embryos (as evidenced by a change in specific gene expression patterns), although the Deformed I-ID and the substituted Ultrabithorax I-ID share 63% identity and no changes occur in the putative recognition helix III (Kuziora and McGinnis, Cell 1989, 50:563571). It is not known, however, whether altered target specificity is due to changes in DNA binding properties, or to changes in the ability to interact with other proteins, or both. The function of conserved protein sequences adjacent to the I-ID (e.g. POU-specilic or paired-box-encoded sequences) also remains to be elucidated. In Ott-1 and Ott-2, the POU-specilic domain is required for highalfinity DNA binding (Strum and Herr, 1988; Miiller-Immergliick et al, EMBO J 1990; Gerster et al, 1990). Studies on the interaction between Ott-1 (called NPIII in those studies) and its binding site in the adenovirus type 2 origin of DNA replication revealed that the Oct1 HD has an intrinsic binding capacity, but the binding affinity of the intact POU domain (I-ID plus POUspecilic domain) is higher. This appears to be because the intact POU-domain makes additional DNA contacts on one side of the recognition sequence, indicating that it interacts with a more extended DNA segment (Verrijzer and Van der Vliet, personal communication). In contrast to Ott-1 and Ott-2, the POU-speciiic domain of GHF- 1 does not interact directly with DNA but somehow potentiates the binding of the I-ID (Theill et al, 1989). The role of POU-specific sequences and the function of the paired box in protein-protein interactions remain to be examined. Biochemical studies in yeast and mammalian cells have shown that I-ID proteins can interact with other regulatory proteins (that may or may not bind DNA themselves) and thereby generate new regulatory activities. It has been shown in tissue-culture experiments that the coexpression, in various combinations, of several Dro.sc@ih HD proteins can synergistically activate or repress transcription of reporter genes that contain multiple HD protein-binding sites (Han et al, 1989; Krasnow et al, 1989; Jaynes and O’FarreR, 1988). Although genetic studies in Drosqddu suggest that crossregulatory interactions among HB genes themselves and/or other regulatory genes occur (see Ingham, 1988), no biochemical evidence for direct interactions between Draw@& I-ID and/or non-HD proteins has yet been obtained. As in most cases the combinatorial assembly of regulatory activities requires a precise organization of c&acting elements (e.g. a2 operator, OCTWGARAT motif), the identilication of functional promoter and enhancer elements and their careful dissection in transgenic animals will be a prerequisite For the characterization of functionally significant interactions among regulatory proteins. Because HB genes are part of a multigene family encoding transacting factors with overlapping target site specificities, it is possible that related, coexpressed I-ID proteins compete in vivo for a speciiic target site(s). It has been shown in transient expression assays (Jaynes and O’Farrell, 1988; Han et al, 1989) as well as in in vitro transcription systems (Ohkuma et al, cell 1990, in press) that certain proteins containing distantly related I-IDS can

493

494

Nucleus

and gene expression

indeed effectively compete for target sites. However, this is not necessarily the case in vivo. At present, there is no evidence that Ott-1 and Ott-2 interfere with each other’s function in lymphocytes. It will be interesting to see whether the products encoded by the homeotic genes of D. mekznoguster among which a possible functional hierarchy exists (Lewis, Nature 1978,276:565-570; GonzalesReyes et al, Nature 1990, 344:78-80), compete for target sites that regulate ‘downstream genes’, or whether the epistatic effects of certain homeotic genes on others are the result of othe’r, non-competitive interactions. The major challenge for the future will be the establishment of direct interactions of HB gene products (and other transcriptional regulators) with defined c&acting elements in the multicellular organisms where HD proteins play major roles in the determination of cell fate. The presence of various HD proteins (probably only a subset of which are known at present) in most cells of a developing embryo makes it extremely difficult to establish definitively which gene product interacts with a delined target site in vivo. Although much information about a possible regulator can be gained by the study of gene expression in a developing transgenic organism carrying a non-functional version of the regulator (see Drasc@Mz homeodomain proteins), new methods will have to be established to answer questions related to this issue. Information gained from genetic and other studies could then be used to supplement homologous, reconstituted in vitro transcription systems with delined transregulators in purified form (see Biggin and Tjian, Trends Cemt 1989, 5:377-383). Using natural c&acting elements, it should then be possible at least to a certain extent, to reproduce cellular transcription phenomena in vitro. Relevant interactions between purified protein and DNA components could then shed more light on how genes are regulated during development. As it stands today, the discovery of the HB has provided a molecular link between the development of complex organisms and the regulation of gene expression. It is a challenge for the future to determine the detailed molecular mechanisms by which HD proteins regulate gene expression to control development.

Acknowledgements

homeodomain determined by NMR spectroscopy in solution: comparison with procaryotic repressors. Cell 1989, 59:573580. The structure of the An41 HD from Drosophih me&znog&erwa.s found to contain, at its C-terminal end, a helix-turn-helix motif virtually identical to those observed in various prokaryotic repressors. This confirms earlier predictions that were based on sequence similarities and supports the view that the third helix of the HD may function as the recognition helix. A structural feature unique to the An@HD is the elongation of the third helix by a more tlexible fourth helix. 2.

Ices SD, BREW R DNA specificity of the Bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell 1989, 57:127%1283. Using a gene activation assay in yeast, it was found that a single amino acid replacement at position 9 of helix Ul is sufficient to switch the DNA-binding specificity of the Bicoid protein. This observation strongly supports the view that the helix-turn-helix motif of the Bicoid protein is involved in the sequence-specilic recognition of DNA l e

TREISMAN J, ~NCZJ’ P, VASHISHTHA M, HARRlS E, DESP~AN C: A single amino acid can determine the DNA binding specificity of homeodomain proteins. Cell 1989, 59:553562. Using crude bacterial extracts containing overexpressed HD proteins and a DNAase I footprint assay, it is shown that a single amino acid replacement at position 9 of the putative recognition helix of the Paired protein switches its DNA-binding specificity to that of Fushi tarazu or Bicoid. 3.

l

4. e.

STRUHI. G, STRUHL K, MACWNAID PM: Tbe gradient morphogen bicoid is a concentration-dependent transcriptional activator. Cell 1989, 57312591273. Multiple regulatory elements in the hb promoter are shown to mediate transcriptional activation in response to Bicoid protein in Dmophih and yeast. The panem of expression depends critically on both the level of Bicoid and the number and quality of the hb elements. 5.

DRJEVTR W, NO~~II~N-VOU+~IUI C: The Bicoid protein is a positive regulator of hunchback transcription in the early Dmsophfla embryo. Nature 1989, 337:138-143. Transient expression assays in Dr@ila embryos as well as in Dras@ila tissue culture cells reveal that a 250 bp hb promoter region is necessary and sufficient for Bicoid-dependent activation of zygotic hb expression. This region contains several binding sites for bacterially ex. pressed Bicoid protein. These results suggest that the effect of Bicoid on hb is direct. l e

6.

DIUEXR W, THOMA G, NIISSIEIN-VO~HARD C: Determination of spatial domains of zygotic gene expression in the D?mophila embryo by the affinity of binding sites for the Bicoid morphogen. Nafure 1989, 340:363367. Bicoid binding sites are shown to be able to confer Bicoid-dependent gene expression in the early embryo. High-alEn@ binding sites for Bicoid protein allow expression further down the Bicoid gradient (towards lower levels of Bicoid) than sites of low affinity. Such a mechanism can translate smoothly graded information into discrete domains of gene expression. l e

7.

KELEHER CA, Gourr~ C, JOHNSCIN AD: The yeast cell-typespecific repressor a2 acts cooperatively with a non-celltype-specific protein. Cell 1988, 53:927-936. The a2 operator consists of binding sites for a2 and for an additional, non-ceil-type-specik protein. The binding of the two proteins is cooperative and the cooperativity is most likety mediated through protein-protein interactions. l e

We thank M. Miiller-Immergliick, W. Schaffner and M. Hall for discussion and A Percival-Smith and K Cadigan for comments on the manuscript Special thanks go to E. Wenger-Marquardt for typing the manuscript and M. J&@ for preparing the figures.

8.

Annotated reading l

em

references

Of interest Of outstanding

and recommended

interest

GOUITE C, JOHNSON AD: al protein alters the DNA binding specificity of a2 repressor. Cell 1988, 52:875-882. The presence of the yeast regulatory protein al is shown to alter the binding specilicity of a2. An a/a cell makes both al and a2, in a ratio that ensures that a2 is distributed between the a2 and the al/a2 binding mode. l e

9. l

1. 00

QIAN YQ, B-R v, Wt?THlUCH K:

M, The

OIITNG G, structure

of

MOUER M, GEHR~NG the Antennupediu

MOUR MM, RUPERT S, SCHAFFNER W, MAIIHLU P: A cloned octamer transcription factor stimulates transcription from lymphoid-specific promoters in non-B cells. Nature 1988, 336544-551.

Homeodomain

proteins/regulation

of gene expression

Affolter,

In uiuo analysis of a human histone H2B promoter demonstrates the activity of the subtype-specitic consensus element containing

A cDNA coding for a lymphocyte-specific octamer-binding transcription factor contains an HB. Expression of this cDNA in HeLa cells is sufficient to activate transcription of a cotransfected B-cell-specific promoter,

GCMAT core is specific for Sphase.

10.

14.

TANAKAM, GRO~N~AUS

l

of the U2 snRNA promoter by the octamer new class of RNA polymerase II enhancer Deu 1988, 231764-1778.

l

%XEIDEREIT BALMACEDA

C, CROMUSH CG, CURIUE R4

specific transcription genes is a homeobox

JA, GERVER T, KAWAKAMI K, ROEDER RG: A human lymphoid-

factor that activates immunoglobulin protein. Nature 1988, 336551-557.

cDNA clones coding for a lymphocyte-specific octamer-binding protein contain an HB. The partial amino acid sequences obtained from purified Oct.2 protein coniirm that the clones represent Oct.2.

11. l

cDNA

BODNER

M, CASTRIUO

JL

THEILL

LE. DEEIUNCK

T, ELLISMAN M,

KAREN M: The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein Cell 1988, 55:50%518. clones are described that encode GHF-1, as conlirmed by partial

GHF-1 peptide sequencing. 12.

HERRW, STURMRA

l

SHARP PA G, HORVIIZ

in the elegant

C~ERC RG, CORCORAN LM, BALTIMORE D, INGRAHAM H4 ROSENFEU) MG. FINNEY M, RWKUN HR: The POU domain: a large conserved region

mammalian pit-l. Octl, uric-86 gene product.

Ott-2,

and

13.

LABELLA F, SIVE HL,

ROEDER

l

lation of a human H2b subtype-specific 2:32-%X

histone H2b consensus

RG, HEINIZ

N: CelLcycle

regu-

gene is mediated by the element. Genes Deo 1988,

that

an AT-

N: Activation motif defines a element. Genes

U, HERR W, HERNANDEZ

in transient expression assaysit is demonstrated that oligomerized octamer sequences can, in HeIa cells. act as an enhancer element for the U2 snRNA promoter, but not for the g-glob&r promoter. This suggests that it is the particular proximal element of the U2 snRN4 promoter that responds to proteins bound to the cctarner sequence (presumably Oct.11 in Heia cells. 15. l

GE-R T, ROEDER RG: A herpes virus wartsactivating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc Natf Acud Sci USA 1988,85:63474351.

ln band-shift gel assays,it is demonstrated that purified Oct.1 but not Oct.2 can form high molecular weight complexes in the presence of VP16 and a functional OCTA/GAgAT sequence element. 16.

Cuenorhabdlrfs

Genes Dev 1988, 2:1513-1516. First description of the POU domain, which contains an HD and a POUspecilic subdomain.

Schier, Cehring

l e

STERN S, TANAKA M, HERR W: The Ott-1 homeodomain rects formation of a multiprotein-DNA complex with HSV transactivator vpl6. N&we 1989, 341:624430.

dithe

Using band-shift gel assays,it is demonstrated that the formation of a multiprotein-DNA complex (in the presence of VP16 and Ott-1) is dependent on specitic ammo acids in the Oct.1 HD. Ammo acid sequence differences within the Oct.1 and Oct.2 HDs are shown to be responsible for the different behavior with respect to VPl6-induced complex formation.

495