Y proteins: flexible regulators of transcription and chromatin structure

Biochimica et Biophysica Acta 1519 (2001) 13^29

www.bba-direct.com

Review

HMGI/Y proteins: £exible regulators of transcription and chromatin structure Raymond Reeves *, Lois Beckerbauer Department of Biochemistry/Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA Received 10 January 2001; accepted 22 March 2001

Abstract The mammalian HMGI/Y (HMGA) non-histone proteins participate in a wide variety of cellular processes including regulation of inducible gene transcription, integration of retroviruses into chromosomes and the induction of neoplastic transformation and promotion of metastatic progression of cancer cells. Recent advances have contributed greatly to our understanding of how the HMGI/Y proteins participate in the molecular mechanisms underlying these biological events. All members of the HMGI/Y family of `high mobility group' proteins are characterized by the presence of multiple copies of a conserved DNA-binding peptide motif called the `AT hook' that preferentially binds to the narrow minor groove of stretches of AT-rich sequence. The mammalian HMGI/Y proteins have little, if any, secondary structure in solution but assume distinct conformations when bound to substrates such as DNA or other proteins. Their intrinsic flexibility allows the HMGI/Y proteins to participate in specific protein-DNA and protein-protein interactions that induce both structural changes in chromatin substrates and the formation of stereospecific complexes called `enhanceosomes' on the promoter/enhancer regions of genes whose transcription they regulate. The formation of such regulatory complexes is characterized by reciprocal inductions of conformational changes in both the HMGI/Y proteins themselves and in their interacting substrates. It may well be that the inherent flexibility of the HMGI/Y proteins, combined with their ability to undergo reversible disordered-to-ordered structural transitions, has been a significant factor in the evolutionary selection of these proteins for their functional role(s) in cells. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : HMGA; HMGI/Y ; Chromatin ; AT-hooks; Transcription; Cancer

1. Introduction In summing up a recent meeting on the high mobility group (HMG) of non-histone chromatin proteins [1], Alan P. Wol¡e observed: `There must be money in proteins that control your fat, your teeth, your sex and your health (besides minor things such as transcription, cell division and DNA recombination and repair).' Although somewhat facetious, this statement aptly illustrates the extent of renewed interest being lavished on these small, nonhistone chromosomal proteins that for many years remained an enigma but are now recognized as founding members of a new class of gene regulatory proteins called `architectural transcription factors' [2]. The structure and function of the three currently recognized families of HMG proteins, i.e., HMG1/2, HMG-14/17 and HMGI/

* Corresponding author. Fax: 509-335-9688 ; E-mail : [email protected]

Y, have been extensively reviewed [1,3^5]. However, given the rapid advances recently made in our understanding of the multiple biological roles played by members of the HMGI/Y family it is timely that we should, in this review, focus exclusively on these proteins. The most extensively studied function of the HMGI/Y proteins is their role in the promotion of gene activation where, by acting as a sort of molecular `glue', they facilitate formation of stereospeci¢c complexes called `enhanceosomes' [6] on the promoter/enhancer regions of inducible genes as a consequence of both speci¢c protein-DNA and protein-protein interactions. Nevertheless, because of their unique combination of structural and biological characteristics, the HMGI/Y proteins are also involved in a diverse range of other cellular processes including the regulation of chromatin structure and active participation in pathologic processes such as neoplastic transformation and metastatic progression. The HMGI/Y proteins also perform important functions as host-supplied factors involved in viral gene regulation and retroviral integration events. Owing to the burgeoning growth of studies on the HMGI/Y pro-

0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 2 1 5 - 9

BBAEXP 93527 29-5-01

Cyaan Magenta Geel Zwart

14

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

Table 1 New nomenclature for the mammalian HMGA families Protein new name

Protein, old (alternative) name

Gene, old name(s)

Gene new name(s)d

Accession No.a

HMGA1a HMGA1b

HMG-I ; HMGI ; HMG I HMG-I/Y; K protein HMG-Y ; HMGY ; HMG Y

HMGIY (H) Hmgiy (M) HMGIY (H)

HMGA1c HMGA2

HMG-I/R HMGI-C ; HMGIC

HMGIY (H) HMGI-C (H) Hmgi-c (M)

HMGA1 (H) Hmga1 (M) HMGA1 (H) Hmga1 (M) HMGA1 (H) HMGA2 (H) Hmga2 (M)

H: L17131b M: AF286367c H: M23618c M: J04179c H: AF176039c H: L46353b M: X99915b

a

The accession number is the unique identi¢er to either the GenBank record containing the gene sequence encoding that protein or to the `reference sequence'. The letters `H' and `M' indicate human and mouse genes, respectively, but the same designations are also given to all other mammalian HMGA genes. At the present time the HMGI/Y gene and protein designations for plants and other non-mammalian species have not been changed. b Full-length gene coding sequences. c cDNA coding for mRNA splice variants. d The human and mouse genomes both contain numerous HMGA `pseudogenes' which should be designated with a `p' su¤x after the appropriate subfamily name (e.g., HMGA1p1, etc). Genomic sequences containing translocations involving either authentic HMGA genes or pseudogenes should be marked with the pre¢x `d' and described.

teins, this review is, of necessity, selective in its coverage and we therefore apologize to any of our colleagues whose work we have inadequately discussed. Wherever possible, we have tried to refer readers to other reviews or papers that will enable them to access most of the original publications in the various areas covered. At the outset it is important to mention that the names of the HMG gene and protein families have recently been changed in order to simplify the old nomenclatures and avoid possible ambiguities among the di¡erent proteins [1]. A Web page containing details of the new HMG nomenclatures can be accessed at: http://www.informatics. jax.org/mgihome/nomen/genefamilies/hmgfamily.shtml. In the new terminology, the HMGI/Y family, which includes the previously designated HMGI/Y and HMGIC proteins, is now referred to as the `HMGA' family. All of the members of the HMGA family are characterized by the presence of highly conserved DNA-binding domains called `AT hooks' that preferentially bind to stretches of DNA with a narrow minor groove. Table 1 lists both the old and new names for the HMGA genes and proteins. In addition to their DNA-binding properties, all members of the HMGA family share similar structural and biochemical characteristics and, where examined, also seem to have many common biological functions. For these reasons, in this review we will collectively refer to them simply as either HMGI/Y or HMGA proteins, unless the discussion of individual proteins is indicated. 2. The HMGA (a.k.a., HMGI/Y) genes and proteins All of the members of the canonical mammalian HMGA protein families share important characteristics that, together, distinguish them from all other classes of eukaryotic nuclear proteins (reviewed in [3]). Circular dichroism (CD) and nuclear magnetic resonance (NMR) studies suggest that the HMGA proteins possess little, if

BBAEXP 93527 29-5-01

any, secondary structure while free in solution but assume induced structural features when bound to other molecules (e.g. to DNA or other proteins). Each HMGA protein possesses a set of three similar, but independent, AT hooks which have an invariant peptide core motif of Arg-Gly-Arg-Pro (i.e., R-G-R-P) £anked on either side by other conserved positively charged amino acid residues. The HMGA proteins bind, via the AT hooks, to the minor groove of stretches of AT-rich DNA but recognize substrate structure, rather than nucleotide sequence, and hence can also preferentially bind to other types of distorted DNA structures. As will be seen, this unique constellation of traits provides the HMGA proteins with an unusually sophisticated repertoire of mechanisms for participating in a variety of cellular functions. As shown in Table 1, the HMGA family consists of four known members: HMGA1a (a.k.a., HMGI ; 107 amino acids (aa); approx. 11.7 kDa), HMGA1b (a.k.a., HMGY; 96 aa; approx. 10.6 kDa) and HMGA1c (a.k.a., HMG-I/R ; 179 aa; approx. 19.7 kDa) and HMGA2 (a.k.a, HMGI-C ; 109 aa; approx. 12 kDa). The HMGA1a, b and c proteins are produced by translation of alternatively spliced transcripts coded for by the HMGA1 (a.k.a, HMGIY) gene located at chromosomal locus 6p21 in humans [7,8] and in mice by the Hmga1 (a.k.a., Hmgiy) gene located in the t-complex region of chromosome 17 [9]. Importantly, in both humans and mice, translation of an alternatively spliced mRNA transcript gives rise to two isoform proteins, HMGA1a (a.k.a., HMGI) and HMGA1b (a.k.a., HMGY). HMGA1a and HMGA1b are identical in sequence except for a deletion of a stretch of 11 amino acid residues between the ¢rst and second AT hook DNA-binding domains in the latter [10,11]. The related HMGA2 (HMGI-C) protein is coded for by a separate gene, HMGA2 (a.k.a., HMGI-C) located at chromosomal locus 12q14-15 in man [12] and Hmga2 (a.k.a., Hmgi-c) locate at the pygmy (or `mini-mouse') locus on chromosome 10 in mice [13]. Mutations in the

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

Fig. 1. Schematic diagram of the structure of the transcribed regions of the mammalian HMGA genes compared to a plant HMGI/Y homologue (intron sequences not shown). hHMG-I/Y (hHMGA1), coding region of the human gene [7]; mHmgiy (mHmga1), coding region of the murine gene [16,17]; aHMG-I/Y, coding region of the plant Arabidopsis gene homologue [32]. The diagram is to scale and both the key and the size marker (in bp) are self-explanatory.

avian HMGI-C gene homologue are also thought to be associated with the chicken dwarf phenotype [14]. 2.1. HMGA genes and their homologues Homologues of the mammalian HMGA proteins have been found in yeast, insects, plants and birds, as well as in all mammalian species examined (reviewed in [3,15]). The HMGA genes from several species have been cloned and characterized. Fig. 1 diagrams the organization of the protein-coding exons (without the intervening intron sequences) of the HMGA genes from man, mouse and the plant Arabidopsis. This ¢gure illustrates the point that all HMGA gene homologues, regardless of the species, are characterized by the presence of multiple, highly conserved, AT hook DNA-binding motifs even though other regions of the genes (and proteins) often vary signi¢cantly. For example, the transcribed regions of the human [7] and mouse [16] HMGA1 genes are approx. 80% identical at the nucleotide sequence level and greater than 90% identical when only the protein-coding exons are considered. Both the human and mouse genes also contain independent exons coding for three separate AT hook DNA-binding domains (Fig. 1). Given this high degree of similarity it is noteworthy that the murine and human HMGA1 genes have di¡erent numbers of transcribed exons and introns. The human homologue [7] has an extra exon and intron located in the 5P untranslated region (5P UTR) that are missing from the mouse gene [16,17]. As in all mammalian HMGA1 genes, each of the three separate AT hook motifs is located on an independent exon in both the mouse [13] and human [12,18] HMGA2 (a.k.a., HMGI-C) genes. The HMGA2 genes are much larger than the HMGA1 genes, in both mice and humans, because of longer 3P and 5P untranslated regions (Fig. 1). In addition, intron 3 of the HMGA2 genes is extremely long ( s 25 kb in the human gene and s 60 kb in the mouse gene [19]) and separates the three exons that contain the AT hook motifs from the remainder of the 3P untranslated tail region of the gene [12,20]. Chromosomal translocations within the exceptionally long third intron of the HMGA2 genes are commonly

BBAEXP 93527 29-5-01

15

observed in benign mesenchymal tumors (see below). The HMGA1 and HMGA2 proteins in both mice and humans share approx. 50% overall amino acid sequence similarity with almost all of the homology being con¢ned to the three AT hook domains and the acidic carboxyl tail regions of the proteins. Aside from these conserved regions, however, there is little amino acid sequence similarity between the mouse and human HMGA1 and HMGA2 proteins [3,20]. Intriguingly, the HMGI protein from the insect Chironomus tentans is the only characterized nonmammalian HMGA-like protein also containing three AT hook domains and an acidic C-terminal tail [21]. The eventual determination of the structure of the gene coding for this insect protein will be of considerable evolutionary interest. Plant homologues of the mammalian HMGA genes exist in maize, Arabidopsis, tobacco, pea, wheat, Japanese Jack bean, soybean, oat, and rice [22^30]. The plant HMGI/Y proteins (whose nomenclature for the moment has not been changed; Table 1) all contain at least three AT hooks but can have as many as seven [27,31] and are almost twice as large as their mammalian counterparts. Unlike mammalian genes, however, the plant HMGI/Y genes contain only one intron and all of the AT hook motifs are coded for by the same exon (cf., Fig. 1). In addition, plant HMGI/Y protein homologues do not contain acidic carboxyl terminal domains but instead have an N-terminal histone H1-like domain (Fig. 1) [31]. Interestingly, both the rice and Arabidopsis proteins contain an alanine-rich, hydrophobic stretch of nine amino acids located between the ¢rst and second AT hook motifs in approximately the same position as the unique, hydrophobic, stretch of 11 amino acids found in mammalian HMGA1 (i.e., HMGI) isoform proteins. The presence of this hydrophobic peptide segment has led to speculation that the Arabidopsis HMGI/Y homologues may then be evolutionarily more closely related to the mammalian HMGA1 proteins than the other plant homologues [32]. 2.2. The AT hook motif: regulation by induction of conformational change As already noted, the unifying feature common to all HMGA proteins is the presence of unique DNA-binding peptide sequences called AT hook motifs which allow the proteins to preferentially bind to the minor groove of stretches of AT-rich B-form DNA [33]. In mammalian HMGA proteins the AT hook motif is a consensus palindromic peptide with the sequence Pro-Arg-Gly-Arg-Pro (P-R-G-R-P) £anked by other positively charged residues [33]. Experiments involving both synthetic AT hook peptides [33] and chimeric recombinant proteins containing a single AT hook consensus sequence [34] have demonstrated that it is this peptide motif that mediates binding of the HMGA proteins to the minor groove of AT-rich regions of B-form DNA. Experiments have likewise shown

Cyaan Magenta Geel Zwart

16

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

Fig. 2. Structure of the second and third DNA-binding domains of the HMGA1a (a.k.a., HMG-I) protein in complex with a synthetic duplex oligonucleotide substrate [48]. (A) Cartoon representation of an unstructured AT hook peptide that is free in solution. (B^E) Various views of the second AT hook peptide bound to the minor groove of a synthetic duplex with a central sequence of 5P-AAATTT-3P. (B) The best-¢t structure of the peptide backbone of the second AT hook as it looks when bound to B-form DNA. (C,D) Co-complex of the second AT hook with B-form DNA showing the ¢nal set of 35 NMR simulated annealing structures for the peptide backbone (frayed `rope' structure) superimposed on the DNA (ribbon structure). (C) Side view looking into the DNA minor groove. (D) Polar view looking down the long axis of the DNA. (E) Polar view of complex showing the side chains of the arginine residues of the AT hook motif projecting into the minor groove and making hydrophobic contacts with adenine bases. (F) Side view of a ball-and-stick model of the third AT hook peptide bound to the minor groove. See text for discussion.

that it is the AT hook peptide that mediates preferential binding of HMGA proteins to DNA substrates with altered structures such as synthetic four-way junctions [35^ 37] (although, see [38]) and regions of DNA distortion found in supercoiled plasmids [39] and on the surface of isolated nucleosome particles [37,40]. The `core' of the AT hook peptide motif (i.e., G-R-P £anked by positively charged residues) has been highly conserved during evolution as a DNA-binding element and is found in numbers ranging from one to more than 15 in various proteins and transcription factors in organisms ranging from bacteria to humans [41]. For example, an HMGA-like protein, carD, has been identi¢ed in the bacterium Myxococcus xanthus [42]. The carD protein, which contains four AT hook-like motifs as well and an acidic peptide domain, is a bacterial transcription factor that is induced in response to either light exposure or starvation [42]. The role of carD in regulating the response of Myxococcus to light exposure is especially intriguing given the recent ¢nding that the HMGI/Y protein is involved with light regulation of the plastocyanin gene in pea plants [43,44]. The involvement of the HMGA proteins with photo-regulated gene activity extends to mammals as recently demonstrated by Ono and his colleagues [45] who demonstrated that mammalian HMGA proteins play an important role in regulating the diurnal transcrip-

BBAEXP 93527 29-5-01

tion of the rhodopsin gene in the mouse retina. Further con¢rming its fundamental importance as a functional peptide motif during eukaryotic evolution, the AT hook has even been incorporated into proteins that are essential components of the chromatin-remodeling machinery of both yeast [46] and mammals [47] (see below). NMR studies of a co-complex of individual AT hooks with a synthetic DNA substrate [48] elucidated the physical basis for recognition of the minor groove of AT-DNA by HMGA proteins. These studies also provided a molecular explanation for the observed directional binding of this peptide motif to DNA substrates [36,49]. They also strongly supported the notion that the intrinsic £exibility of the unstructured HMGA proteins is an important contributing factor for substrate recognition. All of these points are illustrated in Fig. 2. The curved line in Fig. 2A is drawn to illustrate that, when free in solution, the mammalian AT hook peptide motif (as well as the native HMGA1 proteins themselves) has little, if any, detectable secondary structure as detected by either NMR or CD analyses [48,50^52]. For example, the CD spectrum indicates that the HMGI protein has a random-coil content of 73 þ 6% when unbound to DNA [52]. Nevertheless, as shown by the outline of the backbone of the second DNA-binding domain of the HMGI protein in Fig. 2B, following binding of the protein to a synthetic DNA B-

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

form duplex, the AT hook peptide motif (shown in red) assumes a planar, crescent-shaped con¢guration that is dictated by the shape of a narrow minor groove of the DNA substrate. In reality, the apparently rigid structure of the HMGI protein shown in Fig. 2B is somewhat misleading since it is the best ¢t mean structure obtained by averaging the coordinates of the ¢nal set of 35 simulated annealing structures for the peptide backbone bound to DNA [48]. For, as illustrated by the `frayed rope' structures in Fig. 2C (a view of the minor groove perpendicular to the DNA long axis) and D (a view along the DNA long axis), when all 35 of the ¢nal best ¢t annealing structures are superimposed, it is evident that only the region of the peptide backbone that is intimately in contact with the DNA (e.g., the AT hook itself) has assumed an ordered con¢guration. Regions of the peptide backbone not directly associated with DNA (i.e., still free in solution) remain unstructured. The ball-and-stick model of the third DNA-binding domain of the HMGI protein, alone, bound to DNA shown in Fig. 2F further illustrates the ordered con¢guration of the AT hook peptide once it has bound to the minor groove. The biologically important role that intrinsically disordered regions, and transitions from disordered-to-ordered structures, play in the functional activity of many di¡erent proteins is now becoming widely recognized [53^55]. Reversible transitions between disordered and ordered con¢gurations of the HMGA proteins are also likely to play an essential role in the biological activity of these non-histone proteins [37,52,56]. The intrinsic features of the AT hook that allow it to undergo a speci¢c type of disordered-to-ordered conformational change when binding DNA (Fig. 2) are imbedded in the motif itself. Even though the motif peptide has no detectable secondary structure prior to substrate binding, NMR studies have demonstrated that all of the proline residues in the mammalian consensus motif (i.e., P-R-G-R-P; Fig. 2E) exist in a trans con¢guration [48,50]. The trans conformation of the proline residues restricts the £exibility of the peptide backbone on either side of a freely rotating central glycine residue and thereby predisposes the AT hook peptide to adopt dynamic turn-like con¢gurations in solution [50]. Following substrate binding these dynamic peptide structures are `trapped' into a planar convex con¢guration that makes optimal contacts with the narrow minor groove of regions of AT-rich B-form DNA [48]. The crucial importance of both the structure and position of these conserved prolines is demonstrated by the fact that when they are either arti¢cially mutated to alanine residues or when their position in the peptide motif is altered, the resulting mutant peptides will no longer preferentially bind to AT-rich sequences of DNA in vitro [57]. The biological signi¢cance of these prolines is demonstrated by the fact that proteins with speci¢c mutations in these residues act as dominant negative competitors for HMGA function in vivo when introduced into mammalian cells [58].

BBAEXP 93527 29-5-01

17

Once the backbone of the AT hook peptide has undergone the transition to a planar, crescent-shaped con¢guration following binding, the side chains of the arginine residues are oriented parallel to the minor groove and extend toward the central axis of the DNA (Fig. 2E) thereby allowing their guanidino groups to make hydrogen bond contacts with the O2 atoms of thymidines [48]. The snug ¢t of the hooked peptide backbone, together with the inward projecting arginine side chains, displaces water molecules from the minor groove, thereby allowing numerous hydrophobic interactions, particularly to adenine bases, to form that further stabilize the overall molecular interactions. Due to hydrophobic interactions between the inward projecting arginine side chains and the adenine bases, the AT hook binds in only one orientation in the minor groove. The side chains of the arginine and lysine residues that £ank either side of the AT hook `core' residues make electrostatic contacts with the phosphates on the surface of the groove, both neutralizing the charge of these residues and providing even more stability to the protein-DNA complex (Fig. 2E). These multiple proteinDNA interactions contribute to the marked selectivity of the AT hook peptide for stretches of AT sequence: guanine residues have a bulky 6-NH2 side group that projects into the minor groove and disrupts numerous molecular contacts required for tight binding. Conformational changes induced by substrate binding are not con¢ned to the HMGA proteins themselves. Rather, in co-complexes a reciprocal interaction occurs with the HMGA proteins likewise inducing signi¢cant structural alterations in the bound DNA substrates [59]. Depending on the sequence, organization and length of the substrate, binding of full-length HMGA proteins can bend, straighten, unwind and induce loop formation in linear DNA molecules in vitro [40,52,60^63]. The HMGA proteins can also change the topological conformation and introduce supercoils in relaxed plasmid DNAs in vitro [39]. It therefore came as something of a surprise when physical studies ¢rst demonstrated that binding of individual AT hook motifs or synthetic AT hook peptides to short duplex oligonucleotides did not signi¢cantly alter the structure of these substrates [48,52]. The solution to what, at ¢rst glance, appears to be a paradox resides in the observation that for signi¢cant distortions in DNA structure to occur the substrate must contain su¤cient numbers of sites to allow for binding of multiple AT hooks [63]. As shown in Fig. 1, each mammalian HMGA protein has three independent AT hook DNA-binding domains (DBDs) that are separated by unstructured linker peptides. Furthermore, a single DBD peptide preferentially binds to stretches of between four and six base pairs of AT-rich sequence, or approximately one-half turn of a B-form double helix [33], and partially neutralizes the negatively charged backbone phosphates on only one face of the DNA helix [48]. This £exible arrangement of the DBDs provides the protein with considerable latitude for inter-

Cyaan Magenta Geel Zwart

18

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

Table 2 Genes regulated by HMGA proteins Positive regulation

Negative regulation

Vascular endothelial tissue related CD44 [151] E-Selectin [152,153] IGFBP-1 [154] iNOS [151,155] COX-2 [132] SM22K [78] Immune system related MGSA/GROK [109] CXCL1 [108,109] IFN-L [157,67,158] GM-CSF [159,160] TNF-L [161] IL-2 [58,159] IL-2RK [49,91,92] HLA-II [162] c-fos [78] IgG heavy chain [79] Viral genes HIV-1 LTR [114] HSV-1 IE-3 [112] HSV-1 EBNA1 [113] BV EBNA1 [111] JV virus early and late genes [110] Other Tyrosinase [163] Rhodopsin [45] Neurogranin IRC3 [103] PKCQ [103] mRANTES [164] Plant related Plastocyanin [32,43] Nodulin N23 gene [165] Ferredoxin [166] Phytochrome A3 [30] Glutamine synthetase [27] Soybean heat shock hsp17.5E [167]

L-Globin [61] IL-4 [102,168,169] IgE [101] GP 91-phox [170] TCRK [171]

local change neutralization e¡ects, cooperative protein binding and the cross-association of protein molecules between di¡erent regions of the same DNA molecule may all play contributing roles. The number and spacing of AT-rich binding sites in DNA in£uences not only the conformation of bound DNA substrates but also the biological e¡ects elicited by HMGA proteins in vivo. For example, the correct helical phasing of HMGA binding sites in the promoter of the human L-interferon gene has been demonstrated to be essential for both enhanceosome formation and maximal levels of gene transcription [6,70]. Likewise, arti¢cially created `multiple AT hook' (MATH) proteins containing 20 AT hook motifs separated by £exible peptide linkers have been shown to both condense chromatin and inhibit chromosome assembly in Xenopus egg extracts in vitro [71] and, when expressed in vivo, to regulate gene expression in transgenic Drosophila by suppression of position e¡ect variegation (PEV) at the white-mottled-4 (wm4 ) locus [72]. 3. HMGA proteins regulate gene transcription in vivo

action with DNA substrates containing several potential binding sites. Thus, through its three AT hooks, an individual HMGA protein is capable of `reading the bar code' of DNA substrates with multiple binding sites that are separated by varying numbers of nucleotides [33,64^66]. Importantly, such multiple DBD contacts also modulate the charge characteristics of DNA molecules in complex and individualistic ways. Simultaneous binding of two or more AT hooks increases the strength of interactions of HMGA proteins with DNA [21,66^68] and, depending on which face of the helix the binding sites are situated, also in£uences the conformation assumed by the substrate. On relatively short fragments containing multiple AT-rich binding sites these conformational alterations are likely the result of asymmetric neutralization of charges on the phosphodiester backbone of the DNA due to binding of the multiple hooks of the HMGA protein [63,69]. In the case of topological changes and loop formation induced by HMGA binding to very long DNA substrates [39,62],

BBAEXP 93527 29-5-01

Table 2 presents a partial list of speci¢c genes whose transcription has been reported to be regulated, in either a positive or negative fashion, by the HMGA proteins in vivo. Previous reviews [3,5] have discussed in detail the most widely accepted model for how the HMGA proteins function in such regulation namely, through either the facilitation, or inhibition, of the formation of `enhanceosomes' [6] on the promoter regions of the genes they regulate. Enhanceosome formation, most extensively studied on the virus-inducible human L-interferon (IFN-L) enhancer, involves the creation of a multi-protein-DNA complex that serves as a stereospeci¢c platform that is apparently involved in recruiting RNA polymerase II and other co-factors necessary for transcription initiation [70,73^76]. In the case of the IFN-L promoter, the ¢rst step of enhanceosome assembly has been demonstrated to involve HMGA recruitment of the transcription factors NF-UB and ATF-2/c-Jun to the enhancer and is mediated by allosteric changes induced in the DNA by HMGA and not by protein-protein interaction between HMGA and these proteins [77]. Although in many instances direct protein interaction with promoter/enhancer DNA seems to be required for transcriptional activation of HMGA-responsive genes, such an association is not universally required. Chin and his colleagues [78] demonstrated, for example, that HMGA induces transcriptional activation of both the cfos and the smooth muscle-speci¢c SM22K genes in vivo by potentiating binding of serum response factor (SRF) to CArG box recognition elements in their promoters but without the HMGA protein itself making contact with DNA [78]. These experiments were the ¢rst to show that HMGA proteins also play an important role in regulating

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

gene transcription through protein-protein interactions rather than strictly functioning through protein-DNAmediated mechanisms. Subsequent studies further support this assertion. When plasmids expressing dominant negative forms of HMGA proteins (which cannot bind to ATrich DNA because of mutations in AT hook proline residues) were transfected into stimulated human lymphocytes both the production of IL-2 (an HMGA-responsive gene) and cell proliferation were inhibited [58]. But perhaps the most direct demonstration of this mode of action by the HMGA protein comes from recent studies of the ETS transcription factor PU.1 which is an important regulator of the immunoglobulin heavy chain gene mu enhancer in B-lymphocytes [79]. The PU.1 protein is a component of a large multi-protein enhanceosome complex that is required for B-cell-speci¢c mu enhancer activation. Experiments by Lewis et al. [79] demonstrate that PU.1 physically interacts with HMGA and that such interaction increases PU.1 a¤nity for the mu enhancer element. Nevertheless, the increased PU.1 a¤nity is not mediated by HMGA-induced changes in DNA structure. Employing protease digestion mapping techniques these workers demonstrated that interaction between PU.1 and HMGA in solution induces a structural change in the PU.1 protein. Subsequent mapping experiments in the presence of both wild-type mu enhancer DNA and HMGA indicated that an additional change in PU.1 structure occurred upon HMGA-induced PU.1/DNA binding. From these results the authors concluded that increased DNA a¤nity under limiting PU.1 concentrations is mediated by an HMGAinduced structural change in the PU.1 protein. Considered together, all of these results support a new mechanism for HMGA-mediated co-activation: HMGA forms proteinprotein interactions with other transcription factors, which alters the three-dimensional structure of the factors resulting in enhanced DNA binding and transcriptional activation. Thus, the importance of HMGA-induced changes in DNA and/or protein substrates during enhanceosome formation may well be both gene- and transcription factorspeci¢c and will, therefore, necessitate analysis of individual promoters to assess the relative contribution of each of these mechanisms to gene activation. The limited available data, summarized in Table 2, infer that HMGA proteins regulate expression of complex constellations of genes in di¡erent types of cells and tissues, often in speci¢c and individualistic ways. For example, HMGA proteins are involved in controlling both the positive and negative expression of numerous inducible genes in the immune system. Some of these, such as the c-fos gene, are commonly expressed in all immune cells while others, such as IL-2, IL-4, IgG, RANTES and GP91PHOX, are restricted to certain cell subtypes such as Tor B-lymphocytes or macrophages. A similar situation apparently exists for the various HMGA-regulated genes expressed in endothelial and smooth muscle cells of vascular tissues. Experimental support for the notion that HMGA

BBAEXP 93527 29-5-01

19

proteins regulate cell type-speci¢c constellations of genes in vivo comes from several sources. For example, microinjection of puri¢ed HMGA protein into one-cell mouse embryos induces the premature onset of transcription of the constellation of genes normally produced in the zygote at the end of the ¢rst cell cycle [80]. Overexpression of HMGA proteins in mammalian cells has also been demonstrated to not only induce aberrant expression of large numbers of genes but to also promote tumor progression and metastasis in human breast epithelial cells (see below). On a more restricted level, it has also been shown that MATH proteins produced from transgenes introduced into Drosophila regulate gene expression by suppression of PEV at the wm4 locus in a cell type-speci¢c manner in the insect eye [72]. In more recent experiments Laemmli's laboratory has demonstrated that synthetic polyamide drugs consisting of oligo-N-methylpyrrole residues that speci¢cally target AT-rich satellite DNAs in Drosophila will likewise suppress eye PEV at the wm4 locus when feed to larvae [81,82]. These are remarkable biological e¡ects and represent a milestone on the way to the therapeutic use of drugs that bind speci¢c sequences of DNA in the minor groove to regulate gene transcription in vivo [83]. These pharmacological studies also point to the importance of chromatin structure in the regulation of gene expression by minor groove-binding ligands. The authors interpret the results of their experiments in mechanistic terms and attribute the biological e¡ects observed to a drug-induced `opening up' of the chromatin structure of satellite DNA sequences which, in turn, leads to transcriptional activation of individual genes [81,82]. 3.1. HMGA proteins, chromatin structure and transcriptional regulation Alterations in chromatin structure, mediated by both biochemical modi¢cation of proteins and `remodeling' of nucleosome core particles, play a major role in regulating the transcription of eukaryotic genes (reviewed in [84^87]). In most instances chromatin exerts a repressive e¡ect and if either nucleosomes or inhibitory chromatin proteins (such as histone H1) are associated with important regulatory regions gene transcription is severely attenuated or shut o¡. Laemmli's laboratory advanced one of the earliest proposals to explain how the HMGA proteins might be involved with transcriptional activation suggesting that HMGA proteins act as anti-repressor molecules that outcompete (or displace) the binding of inhibitory proteins (such as H1) to AT-rich sequences called `sca¡old attachment regions' (SARs) [88]. The binding to SARs was postulated to promote the establishment of an `open' or accessible chromatin domain structure that is permissive for transcription to occur. As already mentioned, subsequent in vivo experiments performed by the Laemmli laboratory involving both MATH proteins and drugs that mimic AT hook motifs lend considerable support to the

Cyaan Magenta Geel Zwart

20

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

Fig. 3. Recombinant HMGA1a (a.k.a., HMG-I) protein binds in vitro to speci¢c regions of DNA on the surface of isolated nucleosome core particles. (A) Sites of protein binding (dashed circles) on the surface of random nucleotide sequence nucleosome core particles (viewed from the front) isolated from chicken erythrocytes [40,90]. (B) Schematic diagram of the site of preferential binding of the HMGA1a protein (dashed oval) to a stretch of ATrich DNA positioned on the surface of an in vitro reconstituted nucleosome core particle of de¢ned nucleotide sequence [40]. The X-ray structure of the nucleosome core particle is from Luger et al. [156].

`chromatin opening' model of action of the HMGA proteins. Other experiments, however, suggest that the HMGA proteins are not limited to this mode of action but may

BBAEXP 93527 29-5-01

also participate in other mechanisms of activation that involve direct interaction with nucleosomes. HMGA proteins are found associated with monomer nucleosomes isolated from mammalian cells [89] and can be cross-linked

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

21

Fig. 4. Proposed model for participation of HMGA proteins in two steps of transcriptional activation of the human IL-2RK gene promoter: chromatin remodeling and enhanceosome formation. Based on data from John et al. [91,92] and Reeves et al. [49]. See text for discussion.

by chemical reagents to nucleosomal histones in vivo [90]. As illustrated in Fig. 3A, HMGA proteins bind in vitro to four locations on the surface of monomer nucleosome core particles isolated from chicken erythrocytes: at the entrance and exit of the DNA from the nucleosome and at the distorted regions caused by the junction of over- and under-wound DNA located about 1^1.5 bp on either side of the dyad axis [40,90]. On the other hand, a somewhat di¡erent mode of binding is observed if HMGA proteins are mixed with monomer nucleosomes that have been reconstituted into core particles in vitro using cloned DNA fragments of de¢ned sequence that contain multiple ATrich binding sites. In this case, depending on the length, number and sequence of the AT stretches and where they are situated on the reconstituted core particle, the HMGA proteins preferentially bind, as illustrated in Fig. 3B, to these AT stretches even when they are positioned on the surface of the nucleosome away from the edges of the particle [40,49]. Importantly, binding of the HMGA protein to these AT stretches is both direction speci¢c and induces localized changes in the rotational setting of the DNA on the nucleosome surface. The HMGA proteins have been proposed to be involved in the chromatin remodeling that occurs on the promoter region of the gene coding for the K subunit of the human interleukin-2 receptor (IL-2RK) following activation of Tlymphocytes [49]. Transcriptional expression of the human IL-2RK gene is regulated, in part, by interaction of the HMGA protein and other transcription factors with positive regulatory regions (e.g., PRRII and PRRIII) on the gene's promoter during enhanceosome formation [91,92]. Additional in vitro and in vivo experiments have also demonstrated that an inhibitory nucleosome is positioned on the critical PRRII regulatory element in unstimulated lymphoid cells but is `remodeled' during the process of transcriptional activation following lymphocyte stimulation [49]. These ¢ndings led to the model of transcriptional activation of the human IL-2RK gene illustrated in Fig. 4. The most signi¢cant feature of the model is the proposal that HMGA proteins are involved in both the initial re-

BBAEXP 93527 29-5-01

modeling of the inhibitory nucleosome positioned on the PRRII enhancer and the subsequent formation of an enhanceosome on this element. The dual ability of HMGA proteins to bind to AT-rich DNA sequences on the surface of nucleosome particles and to participate in enhanceosome formation makes it likely that a similar scenario may also apply to other inducible genes controlled by these proteins. The precise details of how HMGA proteins might participate in the initial chromatin-remodeling step remain to be determined. Nevertheless, in this connection, it has been demonstrated that drugs that bind to the minor groove of AT tracts can disrupt nucleosome structure [93] and, more intriguingly, that proteins containing AT hook peptides are essential components of the multiprotein, ATP-dependent chromatin-remodeling complexes found in both yeast and mammalian cells. For example, RSC is an essential 15 protein nucleosome-remodeling complex in Saccharomyces cerevisiae that contains one or the other of two closely related proteins, Rsc1 or Rsc2 [46]. Both Rsc1 and Rsc2 contain an AT hook peptide motif that, when mutated, destroys the chromatin-remodeling function of the RSC complex. Similarly, the mammalian SWI/SNF chromatin-remodeling complex relies on two closely related ATPases, known as brm/SNF2K and BRG-1/ SNF2L, for its remodeling activity [47]. Both brm/ SNF2K and BRG-1/SNF2L have an AT hook-like DNAbinding motif in their C-terminal regions and experiments demonstrate that when this region is deleted from the brm/ SNF2K protein the SWI/SNF complex looses its in vivo functional activity and can no longer tether to chromatin. The cumulative evidence thus supports a role for AT hook-containing proteins being important participants in the process of chromatin structural alterations in vivo. 3.2. Biochemical modi¢cations regulate HMGA protein function The HMGA proteins are among the most highly phosphorylated proteins in the mammalian nucleus and the extent of this dynamic secondary biochemical modi¢cation

Cyaan Magenta Geel Zwart

22

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

is dependent on both the phase of the cell cycle and on exposure to various environmental stimuli (reviewed in [3,94]). The HMGA proteins undergo cell cycle-dependent phosphorylation as a result of cdc2 kinase activity in the G2 /M phase of the cycle [95]. The sites of cdc2 phosphorylation of the human HMGA1 proteins are Thr53 and Thr78 and modi¢cation of these residues, which are situated at the amino terminal ends of the second and third DNA-binding domains, signi¢cantly reduces ( s 20-fold) the binding a¤nity of the proteins for AT-rich substrates in vitro [96]. Studies in which amino acids Thr53 , Thr78 and Thr21 (at the amino terminal end of the ¢rst DNAbinding domain ; cf., Fig. 5) were arti¢cially mutated to alanines indicated that these were regulatory residues that controlled the substrate-binding a¤nity of HMGA proteins via reversible in vivo phosphorylations [97]. As summarized in Fig. 5, more recent studies have demonstrated that HMGA proteins are the downstream targets of a number of signal transduction pathways that lead to phosphorylation of other speci¢c residues distributed throughout the length of the proteins. Phosphorylation by casein kinase 2 (CK 2) is an example of the intimate connection between events occurring at the cell surface and the state of phosphorylation of HMGA proteins in the cell nucleus. The human HMGA1 protein has phosphorylation sites for CK 2 at Ser102 and Ser103 , both of which are modi¢ed in vivo [98^100]. Within 15 min of exposure of B-lymphocytes to the cytokine interleukin-4 (IL-4) phosphorylation of Ser102 and Ser103 is detected in the nucleus [102]. Boothby's laboratory has presented persuasive evidence that binding of IL-4 to the K protein subunit of its surface receptor initiates a signaling pathway involving PI-3 kinase, pp70 S6 kinase and CK 2 kinase leading to nuclear phosphorylation of the HMGA1 protein [101,102]. Similarly, within 30 min of treatment of mammary epithelial cells with phorbol esters, chemicals that directly activate

the signaling enzyme Ca2‡ /phospholipid-dependent protein kinase C (PKC), results in nuclear phosphorylation of HMGA1 proteins [37]. The major sites of phosphorylation of HMGA1 by PKCK in vitro are Ser44 and Ser64 [37,103] with Thr21 also being heavily phosphorylated. Analysis of the sites of phosphorylation in cells overexpressing the PKCK enzyme using MALDI/TOF mass spectrometry show that Ser44 , Ser64 and Thr21 are also the residues that are most heavily modi¢ed in vivo, con¢rming that both in vivo and in vitro the HMGA proteins are substrates of the PKC enzymes (Fig. 5). Interestingly, the HMGA1 protein of the insect Chironomus has also been demonstrated to be phosphorylated in vivo by both PKC and CK 2 and, in addition, by the mitogen-activated protein (MAP) kinase [104]. Whether or not the mammalian HMGA proteins are also substrates for MAP kinase is unknown. In any event, what is unambiguous is that in vivo the HMGA proteins are the direct downstream targets for phosphorylation by a number of signal transduction pathways. In addition to the elucidation of complex phosphorylation patterns, mass spectrometric analyses of in vivo modi¢ed HMGA proteins have also revealed several other important ¢ndings [37]. They con¢rmed earlier metabolic labeling studies [105] that indicated that the HMGA proteins are acetylated in vivo at a number of sites and they also demonstrated that the HMG-Y (a.k.a., HMGA1b) isoform protein is more extensively modi¢ed than the HMG-I (a.k.a., HMGA1a) isoform protein. And, unexpectedly, they demonstrated that the HMG-Y protein, but apparently not HMG-I, is modi¢ed by methylation in vivo. But, perhaps just as importantly, these analyses also con¢rmed that the HMGA proteins are simultaneously subject to multiple di¡erent secondary modi¢cations (e.g., phosphorylations, acetylations and methylations) in vivo. Indeed, the complex constellation of modi¢cations

Fig. 5. Major sites of in vivo biochemical modi¢cation of the mammalian HMGA1 (a.k.a., HMG-I and HMG-Y) proteins. The line diagram in the center illustrates the full-length HMG-I (HMGA1a) protein. The boxes indicate the three AT hook DNA-binding domains (I, II and III) and the position is indicated where an 11 amino acid deletion occurs (i.e., the `Y splice site') that gives rise to the HMG-Y (HMGA1b) isoform protein as a result of alternative splicing of a precursor mRNA in vivo. The amino acid sequence of the HMG-I protein is given at the bottom with the underlined residues in bold indicating sites of in vivo biochemical modi¢cations. The bold, underlined numbers in between the diagram and the sequence indicate the number of the amino acid residues modi¢ed. The references for the sites of biochemical modi¢cation are: casein kinase 2 (CK 2) [98,100^102]; cdc2 kinase [95]; protein kinase C [37,103]; CBP and P/CAF [74]; methylation sites found only on the HMG-Y isoform protein (M*), as well as additional phosphorylation sites [37].

BBAEXP 93527 29-5-01

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

found on individual proteins has been proposed to represent a biochemical code that determines the way HMGA proteins bind to substrates such as DNA, chromatin and other proteins [37]. Support for this idea comes from experiments that show that isolated native HMGA proteins modi¢ed in vivo in di¡erent ways exhibit di¡erent a¤nities and speci¢cities for binding to various DNA and nucleosomal substrates in vitro [37]. Likewise, puri¢ed HMGA proteins that have been phosphorylated in vitro by either one, or by a combination of di¡erent kinase enzymes exhibit di¡erent DNA-binding a¤nities and patterns in vitro [103,104, 106]. Of particular interest is the ¢nding that simultaneous phosphorylation of HMGA proteins by di¡erent kinase enzymes at sites £anking both sides of an AT hook DNA-binding motif (cf., Fig. 5) has a much greater impact on substrate-binding a¤nity and speci¢city than does modi¢cation of the protein at only one site by a single enzyme [103,104,106]. For example, Xiao et al. [103] have demonstrated that in vitro phosphorylation of recombinant human HMGA1 protein by cdc2 kinase alone decreases binding a¤nity of the protein for an AT-rich DNA substrate by about 12-fold and phosphorylation by PKC alone reduces binding a¤nity by about 28-fold. On the other hand, the simultaneous phosphorylation of the HMGA1 protein by both cdc2 and PKC reduces binding a¤nity of the HMGA1 protein by greater than 120fold. Synergistic e¡ects of multiple modi¢cations on the substrate-binding properties of HMGA proteins is likely to be a very general phenomenon applying to other types of biochemical adducts as well. Evidence that the biochemical modi¢cations, other than phosphorylation, found on HMGA proteins also serve speci¢c regulatory functions comes from experiments by Munshi et al. [74]. These workers demonstrated that acetylation of the HMGA1 protein at residue Lys65 by the CBP acetyltransferase enzyme (but not acetylation of Lys71 by the P/CAF enzyme) results in enhanceosome destabilization and disassembly leading to transcriptional turno¡ of the human IFN-L gene in virus-infected cells. As noted earlier, the HMGA1b (HMG-Y) protein appears to be very highly methylated in vivo [37]. Also, in vitro experiments have shown that the HMGA1 proteins are susceptible to poly(ADP)-ribosylation in vitro [105,107]. The functions of these additional modi¢cations are currently unknown. Nevertheless, it recently has been reported [108] that the enzyme poly(ADP)-ribose polymerase (PARP) is associated with the promoter region of the gene coding for the melanoma growth stimulatory protein CXCL1. The importance of this observation is that the in vivo transcription of this gene has been suggested to be regulated by a complex of HMGI/Y (HMGA1), NF-UB, SP1 and other factors [109].

BBAEXP 93527 29-5-01

23

4. HMGA proteins and viral function HMGA proteins have been implicated as host cell-supplied factors involved in controlling transcriptional expression of a number of viral genes (Table 2). Examples include: regulation of expression of the early and late genes of the human papovavirus JC virus [110]; regulation of the EBNA1 gene of Epstein-Bar virus (EBV) involved in controlling viral latency [111]; regulation of the IE-3 gene of herpes simplex virus-1 (HSV-1) that codes for the immediate-early protein ICP4 [112]; regulation of the latencyactive promoter 2 (LAP-2) of HSV-1 [113] ; and regulation of the 5P long terminal repeat (LTR) promoter/enhancer of the human HIV-1 virus [114]. In all of these cases, the HMGA proteins bind to AT-rich regions of viral promoter/enhancer regulatory regions and, in conjunction with other proteins contributed by both the virus and host, control virus gene transcription in apparently much the same manner as they regulate transcription of the nuclear genes of the host cell. The commandeering of host-supplied HMGA proteins for viral function is not con¢ned to involvement in viral gene regulation. The HMGA proteins are also employed as co-factors by HIV-1 [115], Moloney murine leukemia virus (MoMuLv) [116] and avian sarcoma virus (ASV) [117] to facilitate integration of double-stranded linear cDNA copies of their genomes into host cell chromosomes (reviewed in [118]). The HMGA1 protein has been demonstrated to be associated with competent viral preintegration complexes (PICs) of both HIV-1 [115] and MoMuLv [63] that have been isolated from freshly infected cells. PIC reconstitution experiments suggest that HMGA proteins are involved with the early processing and joining reactions of the viral integrase-mediated chromosome integration process, rather than with the later stages of the reaction [118,119]. Furthermore, Li et al. [63] used an in vitro viral cDNA integration assay system and wild-type or mutant recombinant HMGA proteins to demonstrate that each HMGA monomer protein must contain multiple AT hooks, and the viral substrate multiple binding sites, in order for the HMGA protein to stimulate integration. The same was also found to be true for HMGA-induced bending and condensation of viral cDNA substrates in vitro. These data led the authors to propose that binding of multivalent HMGA monomer proteins to multiple ATrich sites on retroviral cDNAs in PICs leads to DNA compaction and the formation of active integrase-cDNA complexes [63]. Again, as with viral gene transcription, it seems that the role played by HMGA proteins in the retroviral integration process mechanistically mimics the normal function(s) of these proteins in uninfected eukaryotic cells.

Cyaan Magenta Geel Zwart

24

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

5. HMGA proteins and cancer

Table 3 Cancers associated with aberrant expression of HMGA proteins

5.1. HMGA genes and cell proliferation

Overexpression of full-length proteins

The level of expression of HMGA genes is maximal during embryonic development and in rapidly proliferating cells but is low, or undetectable, in fully di¡erentiated or non-dividing adult cells and tissues (reviewed in [3]). Nevertheless, HMGA gene products are rapidly induced in quiescent normal cells exposed to factors that stimulate metabolic activation and growth and are thought to be involved in the control of cell proliferation [7,120^122]. Lanahan et al. [123] detected synthesis of HMGA mRNAs within 1^2 h of exposure of quiescent NIH3T3 cells to growth stimulatory factors and placed the HMGA genes in the category of `delayed-early response' genes whose transcription is required for subsequent DNA synthesis to occur. Consistent with such a growth regulatory role, homozygous mutations in the Hmg2a (a.k.a., Hmgi-c) gene result in the pygmy (pg) or `mini-mouse' phenotype in mice [124] and the dwarf phenotype in chickens [14]. Intriguingly, careful analysis of the phenotype of homozygous Hmga2 (3/3) mice implicates the Hmga2 gene in the control of fat cell proliferation and the regulation of obesity [125]. Available data thus directly implicate HMGA gene products in the regulation of cell proliferation, embryonic growth and mesenchymal cell function. 5.2. Factors that regulate HMGA gene expression The structure of the human HMGA1 gene is complex and consists of eight transcribed exons that produce multiple forms of transcripts as a result of a complicated pattern of alternative mRNA splicing [7]. Additionally, the gene has four di¡erent promoter/enhancer regions that are capable of independently initiating transcription depending on the cell type and the nature of the stimulatory signal [7,121,122]. Among the agents that induce HMGA1 gene transcription are: serum [120,126]; transforming growth factor K (TGF-K) [127]; epidermal growth factor (EGF) [122]; platelet-derived growth factor (PDGF) and ¢broblast growth factor (FGF) [123]; phorbol esters and calcium ionophores [7,121,128]; interferon-L1 (IFN-L1) and endotoxin [129]; retinoic acid [130]; morphine [131] and hypoxia [132]; as well as the transcription factors AP1 [121], c-Myc [133] and the human papillomavirus E6 protein [134]. Many of these same agents induce expression of the HMGA2 (a.k.a., HMGIC) genes with both FGF-1 and PDGF being particularly strong inducers acting via both the PI-3 kinase and MAP kinase signaling pathways [135]. Even from this partial list of inducing agents it is obvious that many di¡erent signal transduction pathways, and a variety of transcription factors, participate in the in vivo control of expression of HMGA genes thus paving the way for a plethora of possible mechanisms

BBAEXP 93527 29-5-01

Colorectal [172,173] Prostrate [139,140,174] Thyroid neoplasias [141,172,175] Breast [127,176,177] Squamous carcinoma of uterine cervix [178] Non-small cell lung carcinoma [179] Lipomas [13,180] Lewis lung carcinoma [100,137] Pancreatic duct cell carcinoma [181] Neuroblastomas [130,182] Burkitt's lymphoma [133] Chromosomal translocations/AT hook rearrangements Thyroid neoplasias [136] Pulmonary chondroid hamartomas [183^185] Lipomas [13,186,187] Endometrial polyps [188,189] Breast hamartoma [180] Myeloid leukemias [190] Uterine leiomyomas [191^193]

that might lead to their aberrant regulation and pathological expression. 5.3. HMGA genes are oncogenic In neoplastically transformed cells, as opposed to normal adult somatic cells, the constitutive level of HMGA gene products is often exceptionally high with increasing concentrations being correlated with increasing degrees of malignancy or metastatic potential (recently reviewed in [3,136]). This correlation is so consistent and widespread that it has been suggested that elevated concentrations of HMGA proteins are diagnostic markers of both neoplastic transformation [137,138] and increased metastatic potential of cancers [139,140]. In remarkable con¢rmation of these predictions, as illustrated in Table 3, there is an almost perfect correlation between HMGA overexpression, neoplastic transformation and tumor progression in every type of cancer that has been carefully and critically examined! These include colorectal, prostate, breast, cervical and lung carcinomas, amongst others (reviewed in [136]). It is noteworthy that in all of these malignant cancers the overexpressed proteins are full-length, native HMGA proteins rather than mutant or truncated forms of these proteins. Of potential clinical importance, in this connection, are reports indicating that antisense mediated inhibition of synthesis of full-length HMGA proteins suppresses neoplastic transformation of rat thyroid cells that have been infected with retroviruses [141]. Likewise, antisense retroviral expression vectors induce apoptotic death in anaplastic human thyroid carcinoma cell lines but not in normal thyroid cells [142]. These ¢ndings suggest that new therapeutic approaches to cancer treatment based on selective inhibition of the HMGA proteins may be feasible. Intri-

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

guingly, in this connection, a new class of minor, groovebinding mitosene-based anti-tumor drugs currently in clinical trials chemically cross-links the HMGA proteins to DNA in vivo, suggesting that this is likely to be one of the modes of biological action of these drugs [143]. Another remarkable correlation with tumor formation is the fact that chromosomal rearrangements in which the AT hook DNA-binding domains of the HMGA proteins are fused to ectopic peptide sequences are among the most common lesions in non-malignant human tumors. Such chimeric oncogenic proteins bearing multiple AT hooks are observed in a high percentage of benign mesenchymal neoplasias including lipomas, leiomyomas, ¢broadenomas, aggressive myxomas, pulmonary hamartomas and endometrial polyps (Table 3) (reviewed in [136,144,145]). When considered together, the above data present a compelling case for involvement of the HMGA proteins, or their AT hook DNA-binding motifs, in both benign and malignant tumor transformation, tumor progression and metastasis of cancers. Nevertheless, evidence directly supporting a causal role for the HMGA proteins in tumorigenesis has only recently been obtained and comes from experiments in which transgenes coding for either full-length or truncated forms of the HMGA proteins were introduced into cells and their e¡ect on the cell's phenotype monitored. The results of these various studies can be brie£y summarized as follows. (1) Overexpression of either full-length [133,146], or truncated/chimeric forms [147], of the HMGA proteins induces anchorage-independent cell growth in soft agarose in vitro, one of the early indicators of neoplastic transformation. (2) Transgenic mice expressing a truncated form of the HMGA2 protein missing its C-terminal end exhibited a giant phenotype with marked adiposity and an abnormally high incidence of benign lipomas [148,149]. (3) Nude mice injected with non-malignant rat cells or human cells overexpressing full-length HMGA1 proteins developed highly malignant and metastatic tumors [133,146]. Together these in vivo experiments present a compelling case that the HMGA gene family is a causal agent in neoplastic transformation and tumor progression and therefore represents a new family of oncogenes important in the pathogenesis of several types of human cancer. 5.4. HMGA overexpression in£uences multiple genes involved in tumor progression Insights into the molecular events underlying tumor promotion induced by overexpression of the full-length HMGA proteins come from studies in which vectors carrying a tetracycline-responsive promoter driving the expression of either the HMGA1a (i.e., HMG-I) or the HMGA1b (i.e., HMG-Y) cDNAs were introduced into non-tumorigenic human breast epithelial cells [146]. In this tetracycline-regulated expression system the epithelial cells acquired the ability to form metastatic tumors in

BBAEXP 93527 29-5-01

25

nude mice only when the HMGA1 transgenes were `on' and actively expressing the HMGA1 proteins. Unexpectedly, it was found that the HMGA1b (i.e., HMG-Y), rather than the HMGA1a (i.e., HMG-I), isoform of these proteins was the most e¡ective elicitor of tumor formation and metastasis. Oligonucleotide array analysis of gene transcription pro¢les in the transgenic cells demonstrated that the overexpressed HMGA1a and HMGA1b isoform proteins each modulated the expression of distinctive constellations of genes known to be involved in signal transduction, cell proliferation, tumor initiation, invasion, migration, induction of angiogenesis and colonization. Whether the overexpressed HMGA1 proteins directly, or indirectly, in£uenced the transcription of these downstream responsive genes is unknown. Nevertheless, detailed analyses of the transcription pro¢les suggested that HMGA1 induction of integrins and their signaling pathways play a signi¢cant molecular role in the tumor promotion process [146]. Indeed, these ¢ndings supported the notion that HMGA1 plays the role of a `master' gene whose overexpression impacts multiple steps involved in neoplasia. If so, the question naturally arises as to what initial events might `turn on' the overexpression of this putative master oncogene. To address this critical issue, a number of disparate pieces of information must be considered. For example, it is known that the promoter regions of both the human HMGA1 gene [7,16] and the mouse [133] Hmga1 genes contain binding sites for the transcription factors AP-1 and c-Myc. Phorbol esters, tumor-promoting substances that activate AP-1 transcription factors, have also been demonstrated to activate transcription of the human HMGA1 gene [121] and to induce HMGA1b protein overexpression and neoplastic transformation in certain tumor promoter-sensitive mouse cell lines [128]. It has also been shown recently that the c-Myc protein, along with its partner Max, regulates transcription of the mouse Hmga1 gene in vivo [133] and that overexpression of either c-Myc or HMGA1 proteins leads to cancerous transformation of ¢broblasts [133]. Likewise, it is known that mutations in the tumor suppressor gene `adenomatous polyposis coli' (APC) leads to overexpression of the c-Myc protein and uncontrolled cell growth [150]. Therefore, when considered together, these data logically lead to a number of plausible mechanisms that could explain the widespread involvement of the HMGA1 gene in the multi-step process of neoplastic transformation and tumor progression. In one experimentally testable scenario, the initial transforming lesion is a mutation in a tumor suppressor gene (e.g., APC) that permits the aberrant or overexpression of one or more proto-oncogenes (e.g., c-Myc) coding for transcription factors that activate the promoter of the HMGA1 gene. This mutation event, by itself, might not directly result in the neoplastic transformation of the cells. However, if these mutant cells are subsequently exposed to substances that promote tumor progression (e.g., phorbol

Cyaan Magenta Geel Zwart

26

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

esters that activate AP-1 transcription factors), the result is likely to be constitutive HMGA1 gene activation and overexpression of HMGA1 proteins leading to subsequent activation of downstream genes involved in tumor progression and metastasis. Such wholesale aberrant expression of downstream genes will probably be extremely toxic and kill most cells but those that do survive will have an altered, malignant phenotype. 6. Conclusion : the importance of being £exible The collection of characteristics that distinguishes the HMGA proteins from other chromatin proteins and transcription factors includes their possession of multiple AT hook DNA-binding domains, their preferential recognition of structural features of DNA rather than nucleotide sequence, their intrinsic lack of structure and inherent £exibility, their ability to undergo reversible disorderedto-ordered structural transitions when binding substrates and their induction of conformational changes in bound DNA and protein substrates. This unique set of traits has undoubtedly been selected for in evolution so that the HMGA proteins can participate in diverse biological processes in ways that more structured proteins can not. When multiple types of secondary biochemical modi¢cations that alter their substrate-binding properties are superimposed on these other characteristics, the HMGA proteins are endowed with an almost limitless repertoire of functional possibilities. Their possession of multifunctional capabilities is perhaps best illustrated by the many di¡erent roles that have been suggested for the HMGA proteins in the various steps associated with the transcriptional activation of genes. These range from proposals that they out-compete inhibitory proteins and `open up' chromatin domains, that they participate in nucleosome-remodeling processes, that they recognize structural `bar codes' unique to di¡erent gene promoter/enhancer regions to the suggestion that they control the formation and disassembly of enhanceosome structures. No other family of eukaryotic proteins has been implicated in such a variety of nuclear processes, perhaps because all others lack the remarkable structural, biochemical and biological £exibility of the HMGA proteins.

References [1] M.E. Bianchi, M. Beltrame, EMBO Rep. 1 (2000) 1^6. [2] R. Grosschedl, K. Giese, J. Pagel, Trends Genet. 10 (1994) 94^100. [3] M. Bustin, R. Reeves, Prog. Nucleic Acid Res. Mol. Biol. 54 (1996) 35^100. [4] M. Bustin, Mol. Cell. Biol. 19 (1999) 5237^5246. [5] R. Reeves, Environ. Health Perspect. 108 (2000) 803^809. [6] D. Thanos, T. Maniatis, Cell 83 (1995) 1091^1100. [7] M. Friedmann, L.T. Holth, H.Y. Zoghbi, R. Reeves, Nucleic Acids Res. 21 (1993) 4259^4267.

BBAEXP 93527 29-5-01

[8] S. Nagpal, C. Ghosn, D. DiSepio, Y. Molina, M. Sutter, E.S. Klein, R.A. Chandraratna, J. Biol. Chem. 274 (1999) 22563^22568. [9] K.R. Johnson, S.A. Cook, M.T. Davisson, Genomics 12 (1992) 503^ 509. [10] K.R. Johnson, D.A. Lehn, T.S. Elton, P.J. Barr, R. Reeves, J. Biol. Chem. 263 (1988) 18338^18342. [11] R. Reeves, M.S. Nissen, Prog. Cell Cycle Res. 1 (1995) 337^347. [12] K.Y. Chau, U.A. Patel, K.L. Lee, H.Y. Lam, C. Crane-Robinson, Nucleic Acids Res. 23 (1995) 4262^4266. [13] H.R. Ashar, M.S. Fejzo, A. Tkachenko, X. Zhou, J.A. Fletcher, S. Weremowicz, C.C. Morton, K. Chada, Cell 82 (1995) 57^65. [14] C.P. Ruyter-Spira, A.J. de Groof, J.J. van der Poel, J. Herbergs, J. Masabanda, R. Fries, M.A. Groenen, J. Hered. 89 (1998) 295^300. [15] J.R. Wisniewski, U. Grossbach, Int. J. Dev. Biol. 40 (1966) 177^187. [16] M.L. Pedulla, N. Tre¡, L. Beckerbauer, L.M. Resar, R. Reeves, GenBank accession No. AF285780 (2000). [17] J. Liu, J.F. Schiltz, P.C. Shah, K.F. Benson, K.K. Chada, Gene 246 (2000) 197^207. [18] G. Man¢oletti, A. Rustighi, F. Mantovani, G.H. Goodwin, V. Giancotti, Gene 167 (1995) 249^253. [19] F.M. Schoenberg, H.R. Ashar, K.S. Krauter, W.L. Powell, M.S. Rein, S. Weremowicz, S.J. Yoon, R.S. Kucherlapati, K. Chada, C.C. Morton, Genes Chromosomes Cancer 17 (1996) 1^6. [20] G. Goodwin, Int. J. Biochem. Cell Biol. 30 (1998) 761^766. [21] P. Claus, E. Schulze, J.R. Wisniewski, J. Biol. Chem. 269 (1994) 33042^33048. [22] A.B. Krech, D. Wul¡, K.D. Grasser, G. Feix, Gene 230 (1999) 1^5. [23] T. Laux, J. Seurinck, R.B. Goldberg, Nucleic Acids Res. 19 (1991) 4768^4768. [24] R. Gupta, C.I. Webster, J.C. Gray, Plant Mol. Biol. 35 (1997) 987^ 992. [25] R. Gupta, C.I. Webster, J.C. Gray, Plant Mol. Biol. 36 (1998) 897^ 907. [26] M. Kanemaki, Y. Makino, T. Yoshida, T. Kishimoto, A. Koga, K. Yamamoto, M. Yamamoto, V. Moncollin, J.M. Egly, M. Muramatsu, T. Tamura, Biochem. Biophys. Res. Commun. 235 (1997) 64^68. [27] G. Tjaden, G.M. Coruzzi, Plant Cell 6 (1994) 107^118. [28] S. Yamamoto, T. Minamikawa, Plant Mol. Biol. 33 (1997) 537^544. [29] J. Nieto-Sotelo, P.H. Quail, Biochem. Soc. Symp. 60 (1994) 265^275. [30] J. Nieto-Sotelo, A. Ichida, P.H. Quail, Nucleic Acids Res. 22 (1994) 1115^1116. [31] K.D. Grasser, Plant J. 7 (1995) 185^192. [32] R. Gupta, C.I. Webster, J.C. Gray, Plant Mol. Biol. 35 (1997) 987^ 992. [33] R. Reeves, M.S. Nissen, J. Biol. Chem. 265 (1990) 8573^8582. [34] G.C. Banks, B. Mohr, R. Reeves, J. Biol. Chem. 274 (1999) 16536^ 16544. [35] D.A. Hill, R. Reeves, Nucleic Acids Res. 25 (1997) 3523^3531. [36] D.A. Hill, M.L. Pedulla, R. Reeves, Nucleic Acids Res. 27 (1999) 2135^2144. [37] G.C. Banks, Y. Li, R. Reeves, Biochemistry 39 (2000) 8333^8346. [38] R. Schwanbeck, M. Gerharz, A. Drung, P. Rogalla, A. Piekielko, C. Blank, J. Bullerdiek, J.R. Wisniewski, Biochemistry 39 (2000) 14419^ 14425. [39] M.S. Nissen, R. Reeves, J. Biol. Chem. 270 (1995) 4355^4360. [40] R. Reeves, A.P. Wol¡e, Biochemistry 35 (1996) 5063^5074. [41] L. Aravind, D. Landsman, Nucleic Acids Res. 26 (1998) 4413^4421. [42] F.J. Nicolas, M.L. Cayuela, I.M. Martinez-Argudo, R.M. RuizVazquez, F.J. Murillo, Proc. Natl. Acad. Sci. USA 93 (1996) 6881^ 6885. [43] K.W. Pwee, C.I. Webster, J.C. Gray, Plant Mol. Biol. 26 (1995) 1907^1920. [44] C.I. Webster, L.C. Packman, K.H. Pwee, J.C. Gray, Plant J. 11 (1997) 703^715. [45] K.Y. Chau, N. Munshi, A. Keane-Myers, K.W. Cheung-Chau, A.K. Tai, G. Man¢oletti, C.K. Dorey, D. Thanos, D.J. Zack, S.J. Ono, J. Neurosci. 20 (2000) 7317^7324.

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29 [46] B.R. Cairns, A. Schlichter, H. Erdjument-Bromage, P. Tempst, R.D. Kornberg, F. Winston, Mol. Cell 4 (1999) 715^723. [47] B. Bourachot, M. Yaniv, C. Muchardt, Mol. Cell. Biol. 19 (1999) 3931^3939. [48] J.R. Huth, C.A. Bewley, M.S. Nissen, J.N. Evans, R. Reeves, A.M. Gronenborn, G.M. Clore, Nat. Struct. Biol. 4 (1997) 657^665. [49] R. Reeves, W.J. Leonard, M.S. Nissen, Mol. Cell. Biol. 20 (2000) 4666^4679. [50] J.N. Evans, J. Zajicek, M.S. Nissen, G. Munske, V. Smith, R. Reeves, Int. J. Pept. Protein Res. 45 (1995) 554^560. [51] B.H. Geierstanger, B.F. Volkman, W. Kremer, D.E. Wemmer, Biochemistry 33 (1994) 5347^5355. [52] A. Slama-Schwok, K. Zakrzewska, G. Leger, Y. Leroux, M. Takahashi, E. Kas, P. Debey, Biophys. J. 78 (2000) 2543^2559. [53] A.K. Dunker, E. Garner, S. Guilliot, P. Romero, K. Albrecht, J. Hart, Z. Obradovic, C. Kissinger, J.E. Villafranca, Pac. Symp. Biocomput. (1998) 473^484. [54] K.W. Plaxco, M. Gross, Nature 386 (1997) 657^659. [55] P.E. Wright, H.J. Dyson, J. Mol. Biol. 293 (1999) 321^331. [56] A.K. Dunker, D. Lawson, C.J. Brown, P. Romero, J. Oh, C.J. Old¢eld, A.M. Campen, C.M. Ratli¡, K.W. Hipps, J. Ausio, M.S. Nissen, R. Reeves, C.H. Kang, C.R. Kissinger, R.W. Bailey, M.D. Griswold, W. Chiu, E.C. Garner, Z. Obradovic, J. Mol. Graph. Model. 19 (2001) 1^65. [57] R. Reeves, M.S. Nissen, Methods Enzymol. 304 (1999) 155^187. [58] S.R. Himes, R. Reeves, J. Attema, M. Nissen, Y. Li, M.F. Shannon, J. Immunol. 164 (2000) 3157^3168. [59] D.A. Lehn, T.S. Elton, K.R. Johnson, R. Reeves, Biochem. Int. 16 (1988) 963^971. [60] J.V. Falvo, D. Thanos, T. Maniatis, Cell 83 (1995) 1101^1111. [61] M.B. Chase, S.B. Haga, W.D. Hankins, D.M. Williams, Z. Bi, J.W. Strovel, C. Obriecht, P.E. Berg, Am. J. Hematol. 60 (1999) 27^ 35. [62] R. Bagga, S. Michalowski, R. Sabnis, J.D. Gri¤th, B.M. Emerson, Nucleic Acids Res. 28 (2000) 2541^2550. [63] L. Li, K. Yoder, M.S. Hansen, J. Olvera, M.D. Miller, F.D. Bushman, J. Virol. 74 (2000) 10965^10974. [64] R. Reeves, T.S. Elton, M.S. Nissen, D. Lehn, K.R. Johnson, Proc. Natl. Acad. Sci. USA 84 (1987) 6531^6535. [65] M.Z. Radic, M. Saghbini, T.S. Elton, R. Reeves, B.A. Hamkalo, Chromosoma 101 (1992) 602^608. [66] J.F. Maher, D. Nathans, Proc. Natl. Acad. Sci. USA 93 (1996) 6716^ 6720. [67] J. Yie, S. Liang, M. Merika, D. Thanos, Mol. Cell. Biol. 17 (1997) 3649^3662. [68] O. Frank, R. Schwanbeck, J.R. Wisniewski, J. Biol. Chem. 273 (1998) 20015^20020. [69] J.K. Strauss, J. Maher, Science 266 (1994) 1829^1834. [70] T.K. Kim, T. Maniatis, Mol. Cell 1 (1997) 119^129. [71] R. Strick, U.K. Laemmli, Cell 83 (1995) 1137^1148. [72] F. Girard, B. Bello, U.K. Laemmli, W.J. Gehring, EMBO J. 17 (1998) 2079^2085. [73] M. Merika, A.J. Williams, G. Chen, T. Collins, D. Thanos, Mol. Cell 1 (1998) 277^287. [74] N. Munshi, M. Merika, J. Yie, K. Senger, G. Chen, D. Thanos, Mol. Cell 2 (1998) 457^467. [75] J. Yie, K. Senger, D. Thanos, Proc. Natl. Acad. Sci. USA 96 (1999) 13108^13113. [76] T. Agalioti, S. Lomvardas, B. Parekh, J. Yie, T. Maniatis, D. Thanos, Cell 103 (2000) 667^678. [77] J. Yie, M. Merika, N. Munshi, G. Chen, D. Thanos, EMBO J. 18 (1999) 3074^3089. [78] M.T. Chin, A. Pellacani, H. Wang, S.S. Lin, M.K. Jain, M.A. Perrella, M.E. Lee, J. Biol. Chem. 273 (1998) 9755^9760. [79] R.T. Lewis, A. Andreucci, B.S. Nikolajczyk, J. Biol. Chem. 276 (2001) 9550^9557. [80] N. Beaujean, C. Bouniol-Baly, C. Monod, K. Kissa, D. Jullien, N.

BBAEXP 93527 29-5-01

[81] [82] [83] [84] [85] [86] [87] [88] [89]

[90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117]

27

Aulner, C. Amirand, P. Debey, E. Kas, Dev. Biol. 221 (2000) 337^ 354. S. Janssen, O. Cuvier, M. Muller, U.K. Laemmli, Mol. Cell 6 (2000) 1013^1024. S. Janssen, T. Durussel, U.K. Laemmli, Mol. Cell 6 (2000) 999^1011. S. Heniko¡, D. Vermaak, Cell 103 (2000) 695^698. A.P. Wol¡e, Chromatin Structure and Function, Academic Press, Harcourt Brace and Co. Publ., New York, 1998. K.J. Pollard, C.L. Peterson, Bioessays 20 (1998) 771^780. J.D. Aalfs, R.E. Kingston, Trends Biochem. Sci. 25 (2000) 548^555. D. Guschin, T.M. Geiman, N. Kikyo, D.J. Tremethick, A.P. Wol¡e, P.A. Wade, J. Biol. Chem. 275 (2000) 35248^35255. K. Zhao, E. Kas, E. Gonzalez, U.K. Laemmli, EMBO J. 12 (1993) 3237^3247. A. Varshavsky, L. Levinger, O. Sundin, J. Barsoum, E. Ozkaynak, P. Swerdlow, D. Finley, Cold Spring Harb. Symp. Quant. Biol. 47 (1983) 511^528. R. Reeves, M.S. Nissen, J. Biol. Chem. 268 (1993) 21137^21146. S. John, R. Reeves, J.X. Lin, R. Child, J.M. Leiden, C.B. Thompson, L. Leoni, Mol. Cell. Biol. 15 (1995) 1786^1796. S. John, C.M. Robbins, W.J. Leonard, EMBO J. 15 (1996) 5627^ 5635. D.J. Fitzgerald, J.N. Anderson, J. Biol. Chem. 274 (1999) 27128^ 27138. R. Reeves, Curr. Opin. Cell Biol. 4 (1992) 413^423. M.S. Nissen, T.A. Langan, R. Reeves, J. Biol. Chem. 266 (1991) 19945^19952. R. Reeves, T.A. Langan, M.S. Nissen, Proc. Natl. Acad. Sci. USA 88 (1991) 1671^1675. J.S. Siino, M.S. Nissen, R. Reeves, Biochem. Biophys. Res. Commun. 207 (1995) 497^507. T. Lund, K.H. Dahl, E. Mork, J. Holtlund, S.G. Laland, Biochem. Biophys. Res. Commun. 146 (1987) 725^730. J. Palvimo, A. Linnala-Kankkunen, FEBS Lett. 257 (1989) 101^104. P. Ferranti, A. Malorni, G. Marino, P. Pucci, G.H. Goodwin, G. Man¢oletti, V. Giancotti, J. Biol. Chem. 267 (1992) 22486^22489. D.Z. Wang, P. Ray, M. Boothby, J. Biol. Chem. 270 (1995) 22924^ 22932. D. Wang, J. Zamorano, A.D. Keegan, M. Boothby, J. Biol. Chem. 272 (1997) 25083^25090. D.M. Xiao, J.H. Pak, X. Wang, T. Sato, F.L. Huang, H.C. Chen, K.P. Huang, J. Neurochem. 74 (2000) 392^399. R. Schwanbeck, J.R. Wisniewski, J. Biol. Chem. 272 (1997) 27476^ 27483. T.S. Elton, R. Reeves, Anal. Biochem. 157 (1986) 53^62. R. Schwanbeck, G. Man¢oletti, J.R. Wisniewski, J. Biol. Chem. 275 (2000) 1793^1801. V. Giancotti, A. Bandiera, C. Sindici, L. Perissin, C. Crane-Robinson, Biochem. J. 317 (1996) 865^870. C.S. Nirodi, S. NagDas, S.P. Gygi, G. Olson, R. Aebersold, A. Richmond, J. Biol. Chem. 276 (2001) 9366^9374. L.D. Wood, A.A. Farmer, A. Richmond, Nucleic Acids Res. 23 (1995) 4210^4219. H. Leger, E. Sock, K. Renner, F. Grummt, M. Wegner, Mol. Cell. Biol. 15 (1995) 3738^3747. B.C. Schaefer, E. Paulson, J.L. Strominger, S.H. Speck, Mol. Cell. Biol. 17 (1997) 873^886. C.A. Panagiotidis, S.J. Silverstein, Virology 256 (1999) 64^74. S.W. French, M.C. Schmidt, J.C. Glorioso, Mol. Cell. Biol. 16 (1996) 5393^5399. A. Henderson, M. Bunce, N. Siddon, R. Reeves, D.J. Tremethick, J. Virol. 74 (2000) 10523^10534. C. Farnet, F. Bushman, Cell 88 (1997) 483^492. L. Li, C.M. Farnet, W.F. Anderson, F.D. Bushman, J. Virol. 72 (1998) 2125^2131. P. Hindmarsh, T. Ridky, R. Reeves, M. Andrake, A.M. Skalka, J. Leis, J. Virol. 73 (1999) 2994^3003.

Cyaan Magenta Geel Zwart

28

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29

[118] P. Hindmarsh, J. Leis, Microbiol. Mol. Biol. Rev. 63 (1999) 836^ 843. [119] E. Brin, J. Yi, A.M. Skalka, J. Leis, J. Biol. Chem. 275 (2000) 39287^39295. [120] K.R. Johnson, J.E. Disney, C.R. Wyatt, R. Reeves, Exp. Cell Res. 187 (1990) 69^76. [121] S.A. Ogram, R. Reeves, J. Biol. Chem. 270 (1995) 14235^14242. [122] L.T. Holth, A.E. Thorlacius, R. Reeves, DNA Cell Biol. 16 (1997) 1299^1309. [123] A. Lanahan, J.B. Williams, L.K. Sanders, D. Nathans, Mol. Cell. Biol. 12 (1992) 3919^3929. [124] X. Zhou, K.F. Benson, H.R. Ashar, K. Chada, Nature 376 (1995) 771^774. [125] A. Anand, K. Chada, Nat. Genet. 24 (2000) 377^380. [126] K.R. Johnson, D.A. Lehn, R. Reeves, Mol. Cell. Biol. 9 (1989) 2114^2123. [127] T.G. Ram, R. Reeves, H.L. Hosick, Cancer Res. 53 (1993) 2655^ 2660. [128] J.L. Cmarik, Y. Li, S.A. Ogram, H. Min, R. Reeves, N.H. Colburn, Oncogene 16 (1998) 3387^3396. [129] A. Pellacani, M.T. Chin, P. Wiesel, M. Ibanez, A. Patel, S.F. Yet, C.M. Hsieh, J.D. Paulauskis, R. Reeves, M.E. Lee, M.A. Perrella, J. Biol. Chem. 274 (1999) 1525^1532. [130] G. Giannini, C.J. Kim, L.D. Marcotullio, G. Man¢oletti, B. Cardinali, F. Cerignoli, E. Ristori, M. Zani, L. Frati, I. Screpanti, A. Guilino, Br. J. Cancer 83 (2000) 1503^1509. [131] S. Chakarov, L. Chakalova, Z. Tencheva, V. Ganev, A. Angelova, Life Sci. 66 (2000) 1725^1731. [132] Y.S. Ji, Q. Xu, J.F.J. Schmedtje, Circ. Res. 83 (1998) 295^304. [133] L.J. Wood, M. Mukerjee, C.E. Dolde, Y. Xu, J.F. Maher, T.E. Bunton, J.B. Williams, L.M. Resar, Mol. Cell. Biol. 20 (2000) 5490^5502. [134] T. Kinoshita, H. Shirasawa, Y. Shino, K. Shimizu, H. Moriya, B. Simizu, Virus Res. 42 (1996) 119^125. [135] T.A. Ayoubi, E. Jansen, S.M. Meulemans, W.J. van de Ven, Oncogene 18 (1999) 5076^5087. [136] G. Tallini, P. Dal Cin, Adv. Anat. Pathol. 6 (1999) 237^246. [137] V. Giancotti, E. Buratti, L. Perissin, S. Zorzet, A. Balmain, G. Portella, A. Fusco, G.H. Goodwin, Exp. Cell Res. 184 (1989) 538^545. [138] V. Giancotti, A. Bandiera, A. Fusco, R. Marzari, B. Coles, G.H. Goodwin, Eur. J. Biochem. 198 (1991) 211^216. [139] M.J. Bussemakers, W.J. van de Ven, F.M. Debruyne, J.A. Schalken, Cancer Res. 51 (1991) 606^611. [140] Y. Tamimi, H.G. van der Poel, M. Denyn, R. Umbas, H.F. Karthaus, F.M. Debruyne, J.A. Schalken, Cancer Res. 53 (1993) 5512^5516. [141] M.T. Berlingieri, G. Man¢oletti, M. Santoro, A. Bandiera, R. Visconti, V. Giancotti, A. Fusco, Mol. Cell. Biol. 15 (1995) 1545^ 1553. [142] S. Scala, G. Portella, M. Fedele, G. Chiappetta, A. Fusco, Proc. Natl. Acad. Sci. USA 97 (2000) 4256^4261. [143] L. Beckerbauer, J.J. Tepe, J. Cullison, R. Reeves, R.M. Williams, Chem. Biol. 7 (2000) 805^812. [144] J.L. Hess, Am. J. Clin. Pathol. 109 (1998) 251^261. [145] E. Jansen, M.R. Petit, E.F. Schoenmakers, T.A. Ayoubi, W.J. van de Ven, Gene Ther. Mol. Biol. 3 (1999) 387^395. [146] R. Reeves, D.D. Edberg, Y. Li, Mol. Cell. Biol. 21 (2001) 575^ 594. [147] M. Fedele, M.T. Berlingieri, S. Scala, L. Chiariotti, G. Viglietto, V. Rippel, J. Bullerdiek, M. Santoro, A. Fusco, Oncogene 17 (1998) 413^418. [148] S. Battista, V. Fidanza, M. Fedele, A.J. Klein-Szanto, E. Outwater, H. Brunner, M. Santoro, C.M. Croce, A. Fusco, Cancer Res. 59 (1999) 4793^4797. [149] P. Arlotta, A.K. Tai, G. Man¢oletti, C. Cli¡ord, G. Jay, S.J. Ono, J. Biol. Chem. 275 (2000) 394^14400.

BBAEXP 93527 29-5-01

[150] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin, B. Vogelstein, K.W. Kinzler, Science 281 (1998) 1509^1512. [151] L.C. Foster, P. Wiesel, G.S. Huggins, R. Panares, M.T. Chin, A. Pellacani, M.A. Perrella, FASEB J. 14 (2000) 368^378. [152] H. Lewis, W. Kaszubska, J.F. DeLamarter, J. Whelan, Mol. Cell. Biol. 14 (1994) 5701^5709. [153] M.Z. Whitley, D. Thanos, M.A. Read, T. Maniatis, T. Collins, Mol. Cell. Biol. 14 (1994) 6464^6475. [154] S.V. Allander, S.K. Durham, A.O. Scheimann, R.M. Wasserman, A. Suwanichkul, D.R. Powell, Endocrinology 138 (1997) 4291^ 4300. [155] M.A. Perrella, A. Pellacani, P. Wiesel, M.T. Chin, L.C. Foster, M. Ibanez, C.M. Hsieh, R. Reeves, S.F. Yet, M.E. Lee, J. Biol. Chem. 274 (1999) 9045^9052. [156] K. Luger, A.W. Maden, R.K. Richmond, D.F. Sargent, T.J. Richmond, Nature 389 (1997) 251^260. [157] D. Thanos, T. Maniatis, Cell 71 (1992) 777^789. [158] E. Bonnefoy, M.T. Bandu, J. Doly, Mol. Cell. Biol. 19 (1999) 2803^ 2816. [159] S.R. Himes, L.S. Coles, R. Reeves, M.F. Shannon, Immunity 5 (1996) 479^489. [160] C. Shang, J. Attema, D. Cakouros, P.N. Cockerill, M.F. Shannon, Int. Immunol. 11 (1999) 1945^1956. [161] S.J. Fashena, R. Reeves, N.H. Ruddle, Mol. Cell. Biol. 12 (1992) 894^903. [162] S.A. Abdulkadir, S. Krishna, D. Thanos, T. Maniatis, J.L. Strominger, S.J. Ono, J. Exp. Med. 182 (1995) 487^500. [163] S. Sato, H. Miura, H. Yamamoto, T. Takeuchi, Pigment Cell Res. 7 (1994) 279^284. [164] M.A. Lokuta, J. Maher, K.H. Noe, P.M. Pitha, M.L. Shin, H.S. Shin, J. Biol. Chem. 271 (1996) 13731^13738. [165] K. Jacobsen, N.B. Laursen, E.O. Jensen, A. Marcker, C. Poulsen, K.A. Marcker, Plant Cell 2 (1990) 85^94. [166] T.J. Pedersen, L.J. Arwood, S. Spiker, M.J. Guiltinan, W.F. Thompson, Plant Mol. Biol. 16 (1991) 95^104. [167] E. Czarnecka, J.C. Ingersoll, W.B. Gurley, Plant Mol. Biol. 19 (1992) 985^1000. [168] S. Chuvpilo, C. Schomberg, R. Gerwig, A. Hein£ing, R. Reeves, F. Grummt, E. Ser£ing, Nucleic Acids Res. 21 (1993) 5694^5704. [169] S. Klein-Hessling, G. Schneider, A. Hein£ing, S. Chuvpilo, E. Ser£ing, Proc. Natl. Acad. Sci. USA 93 (1996) 15311^15316. [170] D.G. Skalnik, E.J. Neufeld, Biochem. Biophys. Res. Commun. 187 (1992) 563^569. [171] R. Bagga, B.M. Emerson, Genes Dev. 11 (1997) 629^639. [172] M. Fedele, A. Bandiera, G. Chiappetta, S. Battista, G. Viglietto, G. Man¢oletti, A. Casamassimi, M. Santoro, V. Giancotti, A. Fusco, Cancer Res. 56 (1996) 1901. [173] D.H. Kim, Y.S. Park, C.J. Park, K.C. Son, E.S. Nam, H.S. Shin, J.W. Ryu, D.S. Kim, C.K. Park, Y.E. Park, Int. J. Cancer 84 (1999) 376^380. [174] Y. Tamimi, H.G. van der Poel, H.F. Karthaus, F.M. Debruyne, J.A. Schalken, Br. J. Cancer 74 (1996) 573^578. [175] B.V. Giancotti, M.T. Berlingieri, P.P. DiFiore, A. Fusco, G. Vecchio, C. Crane-Robinson, Cancer Res. 45 (1985) 6051^6057. [176] P. Rogalla, K. Drechsler, B. Kazmierczak, V. Rippe, U. Bonk, J. Bullerdiek, Mol. Carcinog. 19 (1997) 153^156. [177] W.M. Liu, F.K. Guerra-Vladusic, S. Kurakata, R. Lupu, T. KohwiShigematsu, Cancer Res. 59 (1999) 5695^5703. [178] A. Bandiera, D. Bonifacio, G. Man¢oletti, F. Mantovani, A. Rustighi, F. Zanconati, A. Fusco, L. Di Bonito, V. Giancotti, Cancer Res. 58 (1998) 426^431. [179] P. Rogalla, B. Kazmierczak, K. Meyer-Bolte, K.H. Tran, J. Bullerdiek, Genes Chromosomes Cancer 22 (1998) 100^104. [180] G. Tallini, P. Dal Cin, K.J. Rhoden, G. Chiapetta, G. Man¢oletti, V. Giancotti, A. Fusco, H. Van den Berghe, R. Sciot, Am. J. Pathol. 151 (1997) 37^43.

Cyaan Magenta Geel Zwart

R. Reeves, L. Beckerbauer / Biochimica et Biophysica Acta 1519 (2001) 13^29 [181] N. Abe, T. Watanabe, T. Masaki, T. Mori, M. Sugiyama, H. Uchimura, Y. Fujioka, G. Chiappetta, A. Fusco, Y. Atomi, Cancer Res. 60 (2000) 3117^3122. [182] F. Basolo, L. Fiore, A. Fusco, R. Giannini, A. Albini, G.R. Merlo, G. Fontanini, P.G. Conaldi, A. Toniolo, Int. J. Cancer 81 (1999) 956^962. [183] T.M. Fletcher, M.A. Soares, S. McPhearson, H. Hui, M. Wiskerchen, M.A. Muesing, G.M. Shaw, A.D. Leavitt, J.D. Boeke, B.H. Hahn, EMBO J. 16 (1997) 5123^5138. [184] S. Xiao, M.L. Lux, R. Reeves, T.J. Hudson, J.A. Fletcher, Am. J. Pathol. 150 (2001) 901^910. [185] B. Kazmierczak, Y. Hennig, S. Wanschura, P. Rogalla, S. Bartnitzke, W. Van de Ven, J. Bullerdiek, Cancer Res. 55 (1995) 6038^6039. [186] M.M. Petit, R. Mols, E.F. Schoenmakers, N. Mandahl, W. Van de Ven, Genomics 36 (1996) 118^129.

BBAEXP 93527 29-5-01

29

[187] P. Dal Cin, S. Wanschura, B. Kazmierczak, G. Tallini, T.A. Dei, J. Bullerdiek, I. Van den Berghe, P. Moerman, H. Van den Berghe, Genes Chromosomes Cancer 22 (1998) 95^99. [188] B. Kazmierczak, P. Dal Cin, S. Wanschura, L. Borrmann, A. Fusco, H. Van den Berghe, J. Bullerdiek, Genes Chromosomes Cancer 23 (1998) 279^285. [189] P. Dal Cin, D. Timmerman, I. Van den Berghe, S. Wanschura, B. Kazmierczak, I. Vergote, J. Deprest, P. Neven, P. Moerman, J. Bullerdiek, H. Van den Berghe, Cancer Res. 58 (1998) 2278^2281. [190] T.H. Rabbits, Nature 372 (1994) 143^149. [191] R. Vanni, P. Dal Cin, S. Marras, P. Moerman, M. Andrai, E. Valdes, J. Deprest, H. Van den Berghe, Cancer Genet. Cytogenet. 68 (1993) 32^33. [192] R. Vanni, U. Lecca, Cancer Genet. Cytogenet. 32 (1998) 33^34. [193] A.H. Ligon, C.C. Morton, Genes Chromosomes Cancer 28 (2000) 235^245.

Cyaan Magenta Geel Zwart