HMGA proteins: multifaceted players in nuclear function

J. Zlatanova and S.H. Leuba (Eds.) Chromatin Structure and Dynamics: State-of-the-Art ß 2004 Elsevier B.V. All rights reserved DOI: 10.1016/S0167-7306(03)39007-6

CHAPTER 7

HMGA proteins: multifaceted players in nuclear function Raymond Reeves1 and Dale Edberg Washington State University, Biochemistry and Biophysics, School of Molecular Biosciences, Pullman, WA 99164-4660, USA 1 Tel.: 509-335-1948; E-mail: [email protected]

1. Introduction In contrast to the well established biological functions of the histone proteins, until recently our understanding of the roles played by the ‘‘high mobility group’’ (HMG) of nonhistone chromatin proteins in nuclear processes has been meager but tantalizing. Fortunately for one group of these proteins, the HMGA family, this situation has now dramatically changed. The reasons for this recent tidal shift in perception are many and reflect the realization by many workers that the nucleus consists of more than just DNA, histones and various enzymes. It also contains several classes of nonhistone proteins that participate in multiple functions ranging from serving as structural components of nuclear architecture to participating as ancillary players in such processes as transcription, replication, and DNA repair. The HMG proteins are the most abundant of these chromatin proteins and the HMGA subfamily is perhaps the best understood in terms of the multiple roles these proteins play in the nucleus. Members of the HMGA group of proteins, and the genes that code for them, possess a unique constellation of biochemical, biophysical, and biological attributes that enables them to participate in a diverse variety of activities not normally accessible to more specialized components of the nucleus. Foremost among these distinguishing characteristics are their remarkable degree of intrinsic flexibility and their ability to undergo extensive and complex patterns of in vivo biochemical modifications in response to external and internal stimuli. Although much is now known about these remarkable proteins, only future research will reveal the full extent, and nature, of involvement of the HMGA proteins in nuclear and cellular functions.

2. Biological functions of HMGA proteins Attesting to the current state of interest, the HMGA family of proteins (formerly known as the HMGI (Y) family [1]), and the genes coding for them, has been the subject of numerous recent reviews [2–13]. The HMGA proteins are coded for by two different genes: the HMGA1 gene, whose alternatively spliced mRNA

156 transcripts give rise primarily to the HMGA1a (a.k.a., HMG-I) and HMGA1b (a.k.a., HMG-Y) proteins, and the HMGA2 gene whose primary product is the HMGA2 (a.k.a., HMGI-C) protein. The HMGA proteins participate in, or are targets of, a wide variety of normal and pathological biological events. For example, HMGA proteins are the down-stream targets of a number of external and internal signal transductions pathways that affect both the types and levels of secondary biochemical modifications on the proteins and, as a consequence, regulate their substrate binding characteristics and their biological functions. Furthermore, by acting as ‘‘architectural transcription factors’’ the HMGA proteins participate in both the positive and negative regulation of a large number of eukaryotic and viral genes and are also thought to participate in such processes as DNA replication, amplification, and repair. Evidence further suggests that they are also involved in regulating cell proliferation, differentiation and apoptotic cell death. The HMGA genes are bona fide oncogenes and their induced over-expression in cells promotes both cancerous transformation and metastatic progression. Elevated levels of HMGA proteins are among the most consistent biochemical features of naturally occurring human tumors with the protein concentrations being a diagnostic marker for increasingly malignant and metastatic cancers. A likely explanation for why over-expression of HMGA proteins is found in so many different types of human cancers comes from recent experiments that demonstrate that expression of the HMGA1 gene is under negative transcriptional regulation by certain tumor-suppressing proteins and is also exquisitely sensitive to positive regulation by exposure of cells to numerous oncogenic growth factors as well as tumor promoting chemicals. The ability to participate in such varied biological processes, and to respond to so many different external and internal signaling events, has led to the HMGA genes and proteins being referred to as ‘‘hubs’’ of nuclear function [12]. A cardinal position of the HMGA proteins in normal nuclear activity is supported by the fact that homozygous knockouts of the Hmga1 gene in mice results in embryonic lethality [13] and homozygous knockouts of the Hmga2 gene results in the diminutive pygmy (or ‘‘mini-mouse’’) phenotype in mice [14]. The HMGA genes and proteins possess a number of distinguishing features that contribute to their ability to play vital roles in nuclear metabolism. For example, the HMGA1 gene has multiple promoters [15] that are regulated by different signal transduction pathways [16–18] and are responsive to a wide variety of external stimuli (reviewed in Ref. [12]). Furthermore, both the HMGA1 [15,19,20] and HMGA2 [21,22] genes produce a number of different isoform HMGA proteins as a result of alternative splicing of precursor mRNAs. As free molecules, the HMGA proteins are quite flexible with little intrinsic structure [12,23–26] but undergo disordered-to-ordered transitions upon binding DNA substrates [23,25]. The highly conserved DNA-binding regions of the HMGA proteins, the so-called AT-hook motifs, not only preferentially bind to the minor groove of AT-rich sequences [23], but also interact with non-B-form DNA structures such as four-way-junctions [27] and the distorted forms of DNA located at specific regions on the surface of

157 isolated nucleosome core particles [28]. The HMGA proteins also possess the ability to specifically interact with many other protein partners. They have, for example, been demonstrated to physically associate with at least 20 different transcription factors via localized peptide regions that specifically interact with restricted areas of the transcription factors (reviewed in Ref. [12]). This combination of characteristics enables the HMGA proteins to choreograph the transcriptional activation of a number of inducible genes by participating in the formation of stereo-specific multiprotein–DNA complexes called ‘‘enhanceosomes’’ [29] on their promoter/ enhancer regions. Another distinctive feature of the HMGA proteins is that they are among the most highly modified proteins in the cell nucleus being subject to complex patterns of in vivo phosphorylation, acetylation, and methylation. These secondary biochemical modifications are generally reversible and, in some cases, cell cycle dependent. In other cases the modifications are a consequence of the HMGA proteins being the down-stream targets of signal transduction pathways that are activated by extra-cellular stimuli (reviewed in Refs. [10,12,30,31]). The intricate patterns of in vivo modification found on the HMGA proteins have been demonstrated to not only affect their interactions with various molecular substrates but also to influence their biological functions [32–34]. Analogous to the modifications found on histone proteins in vivo [35], it has recently been suggested that the patterns of modifications found on the HMGA proteins function as a biochemical ‘‘code’’ that regulates, or coordinates, the many different biological activities of the HMGA proteins in living cells [10].

3. HMGA proteins: flexible players in a structured world Unlike most other proteins, members of the HMGA protein family are characterized by having little, if any, detectable secondary structure while free in solution [12,23–26] with individual proteins exhibiting greater than 75% random coil characteristics when analyzed by either circular dichroism or nuclear magnetic resonance (NMR) spectrometry [10,12]. Nevertheless, when bound to substrates such as DNA or other proteins, subdomains of the HMGA proteins undergo disordered-to-ordered transitions assuming defined conformations [23,25]. This structural transition has been most convincingly demonstrated by NMR studies of co-complexes of the HMGA1 protein with an AT-rich synthetic oligonucleotide substrate [25]. As illustrated in Fig. 1, the highly conserved palindromic core peptide sequence of the DNA-binding domain of the HMGA proteins, Pro–Arg–Gly–Arg–Pro, is disordered prior to substrate binding (panel A) but assumes a planar, crescentshaped configuration (the AT-hook motif [23]; panels B and C) which, when bound to the minor groove of AT-rich sequences (panels D and E), associates with about 5 bp of DNA, or about half a turn of B-form DNA (panel D). The peptide backbone on either side of an AT-hook bound to DNA retains a great deal of plasticity (Fig. 1E). Each HMGA protein has three independent AT-hook motifs

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Fig. 1. Schematic diagrams based on the solution NMR structure of a complex of the second AT-hook motif of the human HMGA1a protein bound to the minor groove of an AT-rich synthetic duplex DNA fragment [25]. Various projected views of either the AT-hook peptide, or a co-complex of the peptide with DNA are shown (see text for details). Modified from Ref. [7].

that are separated by highly flexible peptide sequences. This arrangement and flexibility allows the AT-hooks of an individual protein to associate with the minor groove of either long, contiguous stretches of AT-rich sequence (>
15 bp) or bind to three shorter stretches (4–7 bp) of sequence that are separated from each other by variable distances [23,36]. The intrinsic flexibility of HMGA proteins enables the AT-hooks of a single protein to not only bind simultaneously to AT-rich stretches on different DNA molecules, thereby forming a peptide bridge between separate DNA substrates [25], but to also bind to quite variable arrangements of AT-rich binding sites on a single DNA molecule. These unique binding capabilities facilitate a variety of biological functions including the regulation of gene transcription initiation. As the diagram in Fig. 2 illustrates, the promoter regions of most genes regulated by HMGA proteins contain variably arranged stretches of AT-rich sequence that have been postulated to represent a sort of ‘‘bar code’’ that is ‘‘read’’ by the binding of the AT-hooks of the HMGA proteins during the process of transcription activation [10]. In the majority of cases, as shown by the proximal promoter regions of the human IL-2, IL-2R, IL-4, iNOS, IFN- , and E-selectin genes, the unique patterns of AT-rich binding sites occur within about 300 bp 50 -upstream of the transcription start site. In some instances, however, as illustrated by the mouse TNF- promoter in Fig. 2, the HMGA binding sites can occur at much greater

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Fig. 2. The pattern of AT-rich, HMGA protein bindings sites (shown as black boxes; not to scale) in gene promoter regions form a unique ‘‘bar code’’ potentially involved with gene-specific transcriptional regulation ([10]; see text for details). Gene promoter sequence references: huIL-2, human interleukin-2 [38]; huIL-2R, human IL-2 receptor alpha subunit [60]; huIFN- , human interferon beta [120]; huE-Selectin, human E-selectin [120]; huIL-4, human IL-4 ([121]; GenBank Accession No. M23442); hu iNOS, human inducible nitric oxide synthase ([122]; GenBank Accession No. AF045478); muTNF- , murine tumor necrosis factor beta (a.k.a., lymphotoxin) [123]. Abbreviations: ARRE-1, -2, antigen regulated response elements-1 and -2; NFIL-2, -2B, nuclear factor interleukin-2 and -2B; CD28RE, CD28 response element; PRRII, positive regulatory region-II; PRDI-IV, positive regulatory domains I–IV.

distances 50 upstream of the start site or can even be located 30 downstream of the start site within intronic sequences [37]. Two additional features also contribute further combinatorial complexity to the postulated gene-specific ‘‘bar code’’ recognized by HMGA proteins. First, as illustrated by the arrows located beneath the human IL-2R promoter in Fig. 2, the HMGA proteins have been demonstrated to bind to AT-rich DNA sequences in an orientation-, or direction-specific manner [25,60]. And, second, the minor groove binding of the HMG proteins on gene promoters usually over-lap, or are near quite to, the major groove binding sites for transcription factors that interact physically with HMGA proteins during induction of gene transcription [10,12]. Together, the pattern and directionality of substrate binding, combined with specific interactions of the HMGA proteins with DNA, chromatin and other protein substrates, constitutes a collection of ‘‘determinants’’ that potentially allows these proteins to uniquely recognize and regulate individual gene promoter/enhancer regions among the immense number of other AT-rich binding sites present in the eukaryotic genome [60].

160 Even though the AT-hook peptide has no detectable secondary structure prior to substrate binding, there are inherent features of its conserved palindromic amino acid sequence that allow it to undergo a distinctive type of disordered-toordered transition. NMR studies have demonstrated that, as originally predicted [23], the proline residues of all three of the AT-hook peptides exist in a transconfiguration while the protein is free in solution [24,25]. The trans-configuration of the prolines restricts the flexibility of the peptide back bone on either side of a freely mobile central glycine residue (Fig. 1, panels A–C) and, thereby, predisposes the AT-hook peptide to adopt dynamic, turn-like configurations in solution [24]. When these flexible, but somewhat restrained, peptides turns encounter AT-rich stretches they are apparently ‘‘trapped’’ after assuming an energetically favorable planar, convex configuration that makes optimal molecular contacts with both the sides and bottom of the narrow minor groove and stabilizing ionic contacts with the phosphodiester backbones of the DNA. Major contributors to the specificity of AT-DNA binding are the side chains of the arginine residues which orient parallel to the minor groove and extend toward the central axis of the DNA (Fig. 1C), thereby allowing their guanidino groups to make hydrogen bond contacts with the O2 position of thymines (Fig. 1D). Owing to the hydrophobic interactions between the inward projecting arginine side chains and the adenine bases, the AT-hook binds in only one direction in the minor groove. The critical importance of the prolines in the conserved AT-hook motif in facilitating both the initial DNA contacts and the subsequent conformational changes in the peptide backbone is attested to by the fact that if these residues are either changed to other amino acids, or if their position in the peptide is altered, the resulting mutant peptides will no longer preferentially bind to AT-rich DNA sequences [23]. Proteins containing such mutations act as in vivo dominant negative competitors for HMGA function when introduced into mammalian cells [38]. Their extreme degree of intrinsic flexibility, combined with their ability to undergo substrate-induced conformational changes, sets the HMGA proteins apart from most other highly structured nuclear proteins and plays a critical role in enabling them to participate in a wide variety of biological processes. However, the importance of intrinsically disordered regions in proteins, and transitions from disordered-to-ordered structures, is now becoming widely recognized as a significant and general feature of many different biological systems [10,39–41]. Labile transitions between disordered and ordered configurations of the HMGA proteins, most likely mediated by reversible secondary biochemical modifications (see below), are likely to regulate the formation of functional HMGA complexes in cells and, thereby, control the biological activity of these proteins in vivo.

4. HMGA biochemical modifications: a labile regulatory code Over the last several years a substantial body of evidence has accumulated indicating the types and patterns of secondary biochemical modifications present on histones [42,43], transcriptional co-activators [44] and the HMGA proteins [33]

161 modulate their binding to DNA, to other proteins and to protein–DNA complexes. These modifications are often reversible and are employed by cells to precisely regulate the biological activity of proteins. The HMGA proteins are among the most highly modified proteins in the mammalian nucleus exhibiting complex patterns of phosphorylations, acetylations, methylations and possibly other covalent adductions [33,45]. These secondary biochemical modifications are both cell cycle-dependent and responsive to various environmental stimuli that activate specific signal transductions pathways (reviewed in Refs. [10,12]). Many years ago it was demonstrated that the HMGA proteins undergo cell cycle-dependent phosphorylations as a result of cdc 2 kinase activity in the G2/M phase of the cycle and that such modifications markedly reduce the affinity of binding of the proteins to AT-rich DNA in vitro [32]. More recent studies have shown that HMGA proteins are also the downstream targets of a number of signal transduction pathways whose activation results in phosphorylation of specific amino acid residues distributed throughout the length of the proteins. In mammalian cells, the in vivo signaling pathways that activate casein kinase 2 (CK-2; [46–49]) and protein kinase C (PKC; [33]) result in phosphorylation of HMGA proteins within 15–30 min of their stimulation. The HMGA1 homolog protein of the insect Chironomous has also been shown to be phosphoryated in vivo by stimulation of the mitogen-activated protein (MAP) kinase signaling pathway [50]. Interestingly, agents that activate signaling pathways leading to programmed cell death (apoptosis) also affect the phosphorylation state of HMGA proteins. Sgarra et al. [51] demonstrated, for example, that treatment of cells with drugs (etoposide, camptothecin) or viruses (herpes simplex virus type-I) that induce apoptosis also induce hyper-phosphorylation and mono-methylation of the HMGA1a protein soon after exposure to these agents followed a few hours later by a marked de-phosphorylation of the proteins. Since these hyper- and de-phosphorylation events occurred on the majority of the HMGA1a proteins in the cell, the authors propose that the modifications are causally connected to the global changes in chromatin structure that occur during the early and later phases of apoptotic cell death. Recent advances in mass spectrometry (MS) technology have provided researchers with an unparalleled ability to identify the types and patterns of secondary biochemical modifications found on proteins in living cells. Matrixassisted laser desorption/ionization-MS (MALDI-MS) analyses have shown, for example, that HMGA proteins in vivo are simultaneously subject to complex patterns of phosphorylation, acetylation and methylation and that, within the same cell type, different isoforms of these proteins can exhibit quite different modification patterns [33]. Furthermore, these in vivo modifications have been demonstrated to markedly alter the binding affinity of HMGA proteins for both DNA and chromatin substrates in vitro [33]. Nevertheless, due to their number and complexity, it has been difficult to determine the actual biological function(s) played by these biochemical modifications in living cells. The use of MALDI-MS analysis alone to study in vivo protein modifications has several limitations, especially when it comes to identifying the specific amino acid

162 residues in the HMGA proteins that are modified. To overcome these shortcomings, we employed a strategy in which MALDI-MS is combined with tandem mass spectrometry (MS/MS) analysis to specifically identify both the types and sites of modifications found on HMGA proteins in vivo [52]. This experimental approach is outlined in Fig. 3. The HMGA and other acid-soluble proteins are first isolated from cells and purified to >90% homogeneity by reverse-phase high performance liquid chromatography (RP-HPLC) employing standard techniques [53]. Enzymatic digests of the RP-HPLC purified proteins are then assessed by

Fig. 3. Strategy for analyzing the patterns of native secondary biochemical modifications found on HMGA proteins in living cells using mass spectrometry techniques. The upper left side of the figure shows steps of a standard protocol for determining both the amino acid sequence and sites of biochemical modification of native HMGA proteins isolated from cells. The upper right side of the figure shows the profile of a reverse-phase HPLC chromatogram of acid soluble proteins isolated from living cells, the initial fractionation step for isolating in vivo modified HMGA proteins. The lower left side of the figure shows an example of a restricted region of a MALDI/MS spectrum of a HMGA1 peptide digest containing the same peptide fragment with varying degrees of in vivo secondary biochemical modifications. Peaks: a, unmodified peptide; 1, the di-methylated peptide; 2, the tri-phosphorylated peptide; and, 3, the tetra-phosphorylated plus di-methylated peptide. The table on the lower right hand side shows the sequence and types of modifications present on the peptides shown in the chromatograph. See text for details.

163 MALDI-MS to determine the types and extent of modifications found on different peptides fragments. These digests are also analyzed by ion trap MS/MS to directly obtain the sequence, types and sites of specific amino acid modifications present on individual peptides. These MS analytical techniques are very rapid, extremely accurate and require only small amounts of protein to obtain peptide sequences and amino acid modification information [54]. As an example, the restricted region of a MALDI/MS chromatograph illustrated in the lower left side of Fig. 3 shows peaks corresponding to the same peptide fragment with either no modifications (labeled ‘‘a’’) or containing the various types of secondary modifications (peaks 1–3) listed in the table on the lower right side of the figure. Figure 4 shows some of the sites and types of in vivo modifications found by MALDI/MS on the HMGA1a protein isolated from human MCF7 mammary epithelial cells [33]. The sequence of the HMGA1a protein is shown in the center of the figure and shaded boxes indicate the three AT-hook motifs (I, II, and III) in the HMGA1a protein and the clear box indicates the 11 internal amino acid residues that are deleted from the HMGA1b protein as a consequence of alternative mRNA splicing [15]. The types of secondary modifications found on the various amino acid residues are shown above the diagram and, where known, the enzymes thought to be responsible for these modifications (e.g., cdc-2, PKC, CK-2, etc.) are indicated above the sequence [10,33]. The lines below the sequence indicate the regions of the HMGA1 proteins that have been demonstrated to interact physically with other transcription factors [12]. It is important to note that in vivo the most highly modified part of the HMGA1 proteins is located between the second (II) and third (III) AT-hooks and corresponds to the region of the protein that has the most identified interacting protein partners [12]. The concurrence of numerous in vivo sites of reversible biochemical modifications with those of direct physical association with other proteins suggests that this region of the HMGA1 proteins is important for regulation of their biological function(s) in cells. Support for the suggestion that biochemical modifications regulate the biological function of HMGA1 proteins in cells comes from experiments that demonstrate isolated native HMGA proteins exhibit markedly different affinities and specificities, compared to unmodified recombinant proteins, for binding to various DNA and nucleosomal substrates in vitro [33]. This point is illustrated by the results of the in vitro electrophoretic mobility shift assays (EMSAs) shown in Fig. 5. Panel A shows the profile of bands obtained when increasing concentrations of unmodified recombinant HMGA1a protein are bound to a radio-labeled DNA substrate containing multiple (>7) AT-rich binding sites for the protein whereas panel B illustrates the results obtained when identical concentrations of in vivo modified protein are added to the DNA probe. It is obvious from these results that the modified HMGA1a proteins bind to the DNA probe with considerably less affinity than does the unmodified recombinant protein. Likewise, as shown in panels C and D, a similar marked reduction is observed in the affinity of binding of in vivo modified HMGA1b proteins, compared to unmodified proteins, to isolated nucleosome core particles. Additional evidence that the biochemical modifications

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Fig. 4. Diagram of the human HMGA1 protein showing, previously identified sites of in vivo biochemical modifications [10], sites of modification confirmed by mass spectrometry and the regions of the proteins identified as minimal areas required for specific interactions with other proteins. The upper line illustrates the full-length HMGA1a protein and the rectangular boxes (shaded) indicate the positions of the three AT-hook DNA-binding domains (I, II, III). The elliptical box (clear) indicates the position where an 11 amino acid residue deletion occurs that gives rise to the HMGA1b isoform protein as a result of alternative mRNA splicing. The amino acid sequence and numbering of the HMGA1a protein are shown in the middle of the diagram. The sites of in vivo modifications of the HMGA1 protein that have been confirmed by MALDI-MS and MS/MS are depicted by black boxes with white lettering or white lettering within the three AT-hooks. Serine and threonine residues are the sites of labile phosphorylation whereas variable acetylation occurs exclusively on lysine residues. The existence of methyl groups on HMGA1 proteins has previously been reported in the literature; however, the specific sites of such modifications have not yet been identified. Enzymes that are known to modify HMGA1 are indicated between the sequence and the diagram of the protein. The lines below the amino acid sequence show the areas of the protein that have been identified as the minimal required for specific protein–protein interactions with other transcription factors. The amino acid residues involved in these protein–protein interactions are indicated by numbers following the colons. The original sources demonstrating these physical protein interactions are: NF-B p50/p65 heterodimer, ATF-2/c-Jun heterodimer and IRF-1, Yie et al. [113]; NF-Y, Currie [114]; SRF (serum response factor), Chin et al. [115]; NF-B p50 homodimer, Zhang and Verdine [116]; Tst-1/Oct-6, Leger et al. [117]; HIPK2, Pierantoni et al. [118]. Figure modified and updated from Ref. [12].

165

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Fig. 5. Secondary in vivo biochemical modifications of HMGA proteins reduce the binding affinity of HMGA proteins for both free AT-rich DNA substrates (shown on left side of figure) and random sequence nucleosome core particles (right hand side of figure). Electrophoretic mobility shift assays (EMSAs) using radio-labeled free DNA or isolated nucleosome substrates were reacted with either unmodified recombinant human HMGA1 proteins (upper half of figure) or with native HMGA proteins isolated from cells containing complex patterns of secondary biochemical modifications (lower half of figure). See text for further details.

found on HMGA proteins may serve specific regulatory functions in cells comes from experiments by Munshi et al. [34] who investigated the role of acetylation of specific amino acid residues on the inducible regulation of the human interferon- (IFN- ) gene. These workers demonstrated that acetylation of the HMGA1a protein at residue Lys71 by the P/CAF acetyltransferase facilitates transcriptional activation of the IFN- by promoting formation of an enhanceosome on the gene’s promoter region in cells infected with viruses. In contrast, acetylation of the nearby residue Lys65 by a different acetyltransferase enzyme, CBP, was shown to turn off transcription of the IFN- gene by promoting destabilization and disassembly of a previously assembled enhanceosome. The cumulative data, therefore, support not only a role for secondary modifications in regulating the biological function of HMGA proteins but also suggest that the complex patterns of such modifications provide the cell with mechanisms for exerting exquisitely fine control over their in vivo activities.

5. HMGA proteins, AT-hooks and chromatin remodeling As well as being accessory regulators of gene transcription, HMGA proteins are also integral components of chromatin and are thought to be involved with controlling the mechanics of chromosome structure, function, and dynamics (reviewed in Refs. 2,10,30). In contrast to these global influences on chromosome architecture, a second, much more restricted, effect of HMGA proteins on altering

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Fig. 6. Purified recombinant HMGA proteins bind to four regions of DNA on random sequence nucleosome core particles. Panel A: The results of EMSA gel assays in which increasing concentrations of either purified nonhistone HMGN2 (a.k.a., HMG-17, which binds to two sites on nucleosome core particles) or recombinant human HMGA1a protein were bound nucleosome core particles isolated from chicken erythrocytes [57]. Panel B: Two different views of the nucleosome taken from the X-ray structure of Luger et al. [119] showing the sites of binding of HMGA proteins (dashed circles) determined by DNA foot-printing analyses and other techniques (see text for details).

localized nucleosome structure and function has also been proposed. Indeed, one of the first biological activities suggested for the HMGA proteins (originally referred to as  proteins) was to induce positioning of nucleosomes on the AT-rich -satellite DNA sequences of chromosomes in monkey cells [55,56]. It was later discovered, however, that the highly repetitive -satellite DNA sequences are capable of positioning nucleosomes in vitro independent of HMGA proteins. Nevertheless, as shown in Fig. 6, HMGA proteins are among only a few known transcription factors that can bind directly to DNA on the surface of nucleosome core particles [57]. Panel A (see also Fig. 5C) shows the results of EMSA analyses that indicate HMGA proteins form four discrete complexes when bound to random sequence core particles isolated from chicken erythrocytes [57], whereas another well characterized nuclear protein, HMGN2 (a.k.a., HMG-17), forms only two complexes [58]. As illustrated by the schematic diagram in panel B, DNA footprinting and other analyses have demonstrated that these four sites are located at the entrance and exits of DNA from the nucleosome and at the junctions of the over- and under-wound regions of DNA flanking either side of the dyad axis of the core particle [57,59]. In addition to these four sites, HMGA proteins are also able to bind to AT-rich stretches located on the surface of nucleosomes that have been reconstituted in vitro using core histones and cloned fragments of DNA of defined nucleotide sequence [59,60]. Protein domain-swap experiments have, furthermore, demonstrated that it is the AT-hook regions of the HMGA proteins that are responsible for nucleosome core particle binding [28]. Importantly, HMGA binding to either random sequence or defined sequence core particles has been shown to induce localized changes in the rotational setting of DNA on

168 the surface of nucleosome [59], thus mediating a restricted form of chromatin remodeling. The HMGA proteins have been proposed to participate in the localized chromatin remodeling events that accompany transcriptional activation of the promoters of certain inducible gene such as those coding for the human cytokine interleukin-2 (IL-2) [38,61] and the regulatory  subunit of its receptor, IL-2R [60,62]. For example, it has been demonstrated that a nucleosome is positioned over the important PRRII regulatory sequence in the promoter of the human IL-2R gene (see Fig. 2) in unstimulated lymphoid cells and that this core particle undergoes a ‘‘remodeling’’ process during transcriptional activation of the gene in stimulated cells [60]. Importantly, additional experiments demonstrated that it is possible to reconstituted a positioned nucleosome at this same position over the PRRII element on an isolated fragment of the IL-2R promoter DNA in vitro and, most remarkably, that the HMGA1 protein binds to this reconstituted core particle with a direction-specific polarity. This directional binding of the HMGA1 protein has been proposed to impart a stereo-specificity to the positioned nucleosome and thus ‘‘tag’’ or uniquely identify it for subsequent disruption by ATPdependent chromatin remodeling complexes during the process of transcriptional activation of the IL-2R promoter [60]. The ability of AT-hook peptide motifs to bind to, and induce localized changes in the structure of, nucleosome core particles is not restricted to HMGA proteins. Significantly, it has also recently been discovered that other proteins that contain AT-hook motifs are essential components of the multi-protein, ATP-dependent chromatin remodeling complexes or ‘‘machines’’ (CRMs) found in yeast, Drosophila and mammalian cells. For example, the Swi2p/Snf2p protein, which is the ATPase component of the SWI chromatin remodeling complex in yeast, contains two AT-hook motifs [63] and the ISWI ATPase component of the Drosophila chromatin remodeling complex NURF (nucleosome remodeling factor) contains a single AT-hook peptide [64]. Likewise, studies have demonstrated that the AT-hooks present in chromatin remodeling proteins are critically important for the biological activity of CRM complexes. For instance, the Rsc1 and Rsc2 subunits of the RSC (remodeling the structure of chromatin) complex in the yeast S. cerevisiae each contain a single AT-hook motif that, when mutated or deleted, destroys the chromatin remodeling activity of the RSC complex and results in cell lethality [65]. Similarly, mammalian SWI/SNF-like CRM complexes contain one or the other of two essential and closely related ATPases, known as brm/SNF2 (also called BAF) and BRG-1/SNF2 (also called PBAF) [66,67], each of which contains a single AT-hook motif in their C-terminal region. Yaniv and his colleagues [68] have demonstrated that when the AT-hook is deleted from brm/SNF, the CRM complex looses its in vivo functional chromatin remodeling activity and also can no longer bind to chromatin substrates. And, finally, Xiao et al. [69] discovered that the N-terminal end of the largest subunit of the Drosophila NURF complex, NURF301, contains two AT-hook peptide motifs and an acidic region that have high sequence similarity to the mammalian HMGA proteins. Intriguingly, the amino acid sequence of the N-terminal end of NURF301 more

169 closely resembles that of HMGA proteins than do the AT-hook containing domains of Rsc1, Rsc2, brm/SNF or any of the other CRM proteins. These workers also demonstrated that the only subunits of the NURF complex required for the induction of nucleosome sliding (i.e., remodeling) in an in vitro model system are NURF301 and the ISWI ATPase protein that also contains a single AT-hook peptide motif. Quite importantly, Xiao et al. [69] went on to show that the N-terminal end of the NURF301 is the region of the protein responsible for binding to nucleosome core particles in vitro and that when the two AT-hooks of this region are deleted, the ability of the truncated protein to both bind (‘‘tether’’) to core particles and induce sliding is inhibited [69]. This cumulative in vivo and in vitro evidence thus strongly supports an active role for the AT-hook motifs found in various CRMs in both nucleosome binding and ATP-dependent sliding/remodeling. Several mechanistic explanations have been advanced for explaining how AT-hook peptides in CRMs might be involved with nucleosome remodeling processes including the attractive suggestion that by selectively binding to distorted regions of DNA on core particles they induce, via ATP-driven reactions, dynamic localized rotational changes in DNA structure that are propagated in a screw-like manner to induce nucleosome translational sliding [12,69]. When considered in the context of activation of the human IL-2R gene discussed above, this information has also led to a proposal that the directional binding of HMGA1 proteins (via their AT-hook motifs) to the nucleosome positioned on the PRRII promoter region in unstimulated T-cells likely acts as a ‘‘marker’’ or ‘‘placeholder’’ for binding by the AT-hooks of CRM proteins during the subsequent ATP-dependent disruption of the core particle that occurs during transcriptional activation of the gene in vivo [10,60]. Considerable experimental support for this model has recently been obtained employing chromatin immunoprecipitation (ChIP) assays that demonstrated that in vivo the HMGA protein is bound to the positioned nucleosome on the IL-2R promoter in resting T lymphocytes and that, within 30 min of following cell stimulation, the HMGA1 protein dissociates from the nucleosome. In contrast, parallel ChIP assays showed that BRG-1, a subunit of the human SWI/SNF complex, is not bound to the positioned nucleosome in unstimulated lymphocytes but becomes associated with the core particle immediately after cell stimulation (within 5–15 min) at about the same moment that the HMGA1 protein is dissociating from the nucleosome, a time during which chromatin remodeling and transcriptional activation of the IL-2R gene is occurring [70]. Further experimental support for a functional cooperation, or active interplay, between the HMGA proteins and the AT-hook-containing CRM proteins during the process of chromatin remodeling comes from the work of Lomvardas and Thanos [71]. These workers demonstrated that an in vitro system composed of only purified recombinant HMGA1, SWI/SNF and TBP (TATA-binding protein) proteins is capable of efficiently inducing ATPdependent nucleosome sliding/remodeling. It is likely that such cooperative interactions between different AT-hook containing proteins are common and that many more examples of HMGA proteins being intimately, and actively,

170 involved in chromatin remodeling processes mediated by ATP-requiring CRM activities will be found.

6. HMGA proteins as potential drug targets Given their central role in such a variety of normal and pathological processes [12], the HMGA genes and proteins are attractive potential targets for the development of therapeutic drugs. The experimental strategies for development of such drugs fall into several categories. Some of the more promising approaches are to develop: (i) drugs that lower the effective concentration of HMGA proteins in cells; (ii) drugs that non-specifically compete with the AT-hooks of the proteins for binding to substrates; (iii) drugs that block specific binding of HMGA proteins to gene promoter regions; (iv) drugs that either specifically inactivate HMGA proteins or selective cross-link them to DNA in vivo. Many of these strategies have, with differing degrees of success, already been investigated while others remain to be explored. 6.1. Methods to lower the cellular concentrations of HMGA proteins There are several reports demonstrating that lowering the endogenous levels of HMGA proteins in cells by the introduction of either anti-sense or dominantnegative expression vector constructs results in the reversal or amelioration of certain pathologic conditions. These include the demonstration that anti-sense eukaryotic cell expression vectors can: (i) inhibit neoplastic transformation of normal rat thyroid cells infected with retroviruses [72]; (ii) suppress the growth rate of cancerous cells and decrease their ability for anchorage-independent growth [73]; and (iii) preferentially induce apoptotic cell death in anaplastic human thyroid carcinoma cells but not in normal thyroid cells [74]. On the other hand, there are also reports demonstrating that reduction of cellular levels of HMGA proteins interferes with normal cellular processes such as inhibition of the inducible expression of the unique-sequence genes coding for the human interferon- [75] and interleukin-2 [76,77] proteins. These examples give some encouragement to the notion that modulation of endogenous HMGA protein levels might be a fruitful target for drug development. However, the anti-sense and dominant negative studies reported so far have involved delivery of expression vectors to cells, either as transfected DNAs or via viral infections, processes that are inherently inefficient and therefore of limited general use. Alternative approaches of employing synthetic oligonucleotide-based strategies [80,81], such as the use of stable, anti-sense synthetic oligonucleotides [78] or anti-sense peptide-nucleic acids [79], in transfection experiments to inhibit HMGA protein expression seem feasible in principal but have not yet reported in the literature. Nevertheless, these procedures also suffer from similar limitations to those outlined for eukaryotic cell expression vectors. In order to overcome these shortcomings, there are a variety of other promising strategies (reviewed in

171 Refs. [82,83]) that can potentially be used to develop drugs that modulate the levels or functions of HMGA proteins in cells. 6.2. Drugs that non-specifically compete with AT-hooks peptides for DNA-binding As illustrated in Fig. 1, the highly conserved Pro–Arg–Gly–Arg–Pro peptide core region of the AT-hook DNA-binding domains assumes, following DNA-binding, a planar, crescent-shaped structure similar in conformation to the pyrrole antibiotics netropsin and distamycin A and to the fluorescent dye Hoechst 33258, all of which also reversibly bind to the minor groove of AT-rich sequences with high selectivity. In fact, the structural similarity between the AT-hook DNA-binding peptide motif and Hoechst 33258 is the basis for an extremely sensitive fluorescence competition assay employed to quantitative determine the affinity of binding of HMGA proteins to AT-rich DNA substrates in vitro [23]. Distamycin A, and its chemical derivatives, are highly cytotoxic and exhibit antiviral [84] and anti-cancer [85–87] activities. Distamycin A, and other minor groove binding drugs appear to exert their biological effects by interfering with cellular gene expression patterns through the alteration or disruption of DNA-binding by transcription factors [86] and, as a consequence, inhibiting the initiation step of transcription [87]. Interestingly, early in vitro protein–drug–DNA-binding studies by Wegner and his colleagues [88] led to the proposal that the cytotoxic effects of drugs that selectively bind to the minor groove of AT-rich stretches of DNA (such as distamycin A, netropsin and berenil) are likely to be a consequence of competitive displacement of HMGA proteins from their in vivo DNA-binding sites. In vivo support for displacement of HMGA proteins by such minor groove binding drugs also comes from studies by Radic et al. [89] who demonstrated that both Hoechst 33258 and distamycin A directly compete with HMGA proteins for binding to AT-rich satellite DNA sequences in mouse cells causing chromosome decondensation, particularly in heterochromatic regions. Similar cytological effects have recently been observed in human cells treated with these drugs [90]. The observed in vivo effects on chromosome structure of drugs that selectively bind to the minor groove of AT-rich sequences agree quite well with the predictions of models of global gene activation originally advanced by Laemmli and his colleagues [91,92]. These workers proposed that, during the early stages of cellular differentiation when developmental changes in chromatin domains are occurring, HMGA proteins out-compete inhibitory proteins (such as histone H1) for binding to AT-rich DNA sequences, called ‘‘scaffold attachment sites’’ (or SARs), that are distributed along the backbone of metaphase chromosomes and, as a consequence, ‘‘open up’’ selected regions of chromatin for active gene transcription [91,92]. Similarly, the results of minor groove-binding drug studies are consistent with the observation that artificially created ‘‘multiple AT-hook’’ (MATH) proteins that contain numerous AT-hook motifs separated by flexible peptide linkers have the ability to both condense chromatin and inhibit chromosome assembly when added to in vitro extracts of oocytes of the amphibian Xenopus laevis [93].

172 MATH proteins have also been shown to regulate the in vivo transcription of endogenous host cell genes in differentiated adult tissues when transgenes coding for these proteins were introduced into the laraval stages of the insect Drosophila [94]. A major problem with using drugs such as netropsin, berenil or derivatives of distamycin as potential therapeutic agents, however, is their generalized binding to the minor groove of most AT-rich sequences. These promiscuous interactions result in non-specific toxicity of the drugs for all types of cells thus greatly limiting their use as selective anti-viral or anti-tumor agents. 6.3. Drugs that block binding of HMGA proteins to specific gene promoters One alternative that is beginning to be explored to overcome such generalized drug toxicity problems is to create membrane-permeable synthetic molecules that target only specific gene promoters that are naturally regulated by the HMGA proteins. Based on the known DNA-binding characteristics of the HMGA proteins and assuming, as previously discussed, that the promoter region of each HMGAregulated gene has a unique ‘‘bar code’’ of AT-rich binding sites (Fig. 2), it should be possible to create synthetic drugs that will selectively recognize portions of this ‘‘bar code’’. Such promoter-specific drugs would be expected to competitively inhibiting the binding of endogenous HMGA proteins to their natural target sites in this promoter and, thereby, reduce or eliminate the expression of only this particular gene in living cells. A number of experimental advances support the feasibility of this ‘‘designer’’ drug-based approach to inhibiting expression of specific HMGA-regulated genes. Principal among these has been the successful design and synthesis of small, minor groove binding molecules, called lexitropsins, that contain polymers of N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids and which bind with high affinity to predetermined DNA sequences (reviewed in Refs. [83,95–97]). Importantly, the structural basis and pairing rules have been developed to design rationally pyrrole–imidazole (Py–Im) polyamide dimers [98] that bind specifically to the minor groove of either AT- [99] or GC-sequences [100] with affinities and specificities comparable to native DNA-binding proteins. Such Py–Im polyamide molecules have been demonstrated to specifically inhibit the transcription of viral and genomic genes in living cells [101] by selectively binding to only certain promoter sequences and, thereby, interfering with both the binding of TATA-box binding protein (TBP) and basal transcription by RNA polymerase II [102]. The synthesis of a Py–Im polyamide molecule that specifically recognizes a short stretch of AT-rich sequence that constitutes part of the distinctive ‘‘bar code’’ of an individual gene promoter is certainly possible. However, targeting of a lexitropsin molecule to only a single site in a complex promoter is unlikely to provide the necessary degree sequence recognition specificity required for gene specific regulation in eukaryotic cells. Thus, at a minimum, the challenge for specific gene regulation will be to create chimeric molecules with Py–Im moieties that bind to

173 specific (but different) stretches of AT-DNA and which are separated from each other by a peptide linker of such length that the dimeric drug binds to only the appropriately spaced and directionally oriented AT-stretches present in a given target promoter. Encouragingly, experiments attesting to the feasibility of using such bipartite drugs as reagents for modulating gene expression in living organisms have recently been performed in the insect Drosophila. In an elegant series of experiments, Janssen et al. [103] synthesized a series of dimeric oligopyrrole drugs containing internal flexible peptide linkers of varying length that exhibited high binding affinity for large and bipartite AT-rich DNA tracks with the various drugs in the series specifically binding to different AT-rich satellite DNA sequences in Drosophila. When these drugs were fed to larvae they significantly modulated normal developmental gene expression patterns and caused both gain- and loss-of-function of phenotypes in adult flies. For example, one of the polyamide drugs suppressed position-effect variegation (PEV) of the white-mottled locus (a consequence of increased gene expression) whereas another drug mediated homeotic transformations (caused by loss of gene function) exclusively in the brown-dominant locus [103]. These are remarkable biological results and if analogous phenotypic effects can be obtained by the targeting of bipartite Py–Im polymide drugs to the promoters of specific HMGA-regulated genes in mammalian cells, the path could be opened for new types of therapeutic treatments for a wide range of pathological conditions ranging from viral infections and immune disorders to the formation of atherosclerotic plaques. Of course, the success of such a drug development strategy is dependent on identifying the promoters of those genes that are the direct targets of HMGA protein regulation in cells exhibiting various pathological conditions, a task that has already been initiated [73,104].

6.4. Drugs that specifically inactivate or cross-link HMGA proteins in vivo Drugs that recognize, and selectively interact with structural or other features of the HMGA protein themselves could potentially be used as therapeutic reagents to eliminate, for example, aberrant tumor cells that are constitutively over-express high levels of HMGA proteins. The power of combinatorial chemistry (reviewed in Ref. [105]) could, in principal, be employed to select for synthetic drugs that specifically interact with the unbound, unstructured forms of the HMGA proteins present in cells and prevent them from performing their endogenous biological functions. Likewise, cell permeable reagents that selectively react with structural features of the AT-hook peptide motif when it is directly bound to the minor groove of DNA (Fig. 1) might also prove to be an efficacious way of inactivating the HMGA proteins in vivo. In this connection, the drug FR900482 (4-formyl6,9-dohydroxy-14-oxa-1,11-diazatetracyclo [7.4.1.02,7,010,12]-tetradeca-2,4,5-triene-8-yl methyl carbamate [106]), and its chemical derivative FK317 (11-acetyl-8-carbamoyloxymethyl-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo [7.4.1.02,7,010,2]tetradeca-2,4,6-trien-9-yl acetate [107]) are two examples of new anti-cancer drugs

174

Fig. 7. Diagram of the structure of the FR900482 and FK317 pro-drugs and the proposed scheme of their reductive activation via a miosene-like intermediate inside living cells (see the text for further details). Figure modified from Ref. [108].

that have recently been demonstrated to cross-link HMGA proteins to the minor groove of DNA in living cells [108,109]. As illustrated in Fig. 7, both FR900482 and FK317 are reductively activated inside cells through a scheme that involves the thiol-mediated two-electron reduction of the N–O bond in the presence of trace Fe(II) generating a transient ketone which rapidly cyclizes to a carbinolamine derivative, followed by expulsion of water to produce the reactive electrophilic mitosene derivative [110,111]. This requirement for reductive activation is thought to be the mechanism responsible for the preferential targeting of these pro-drugs to tumorgenic cells that, in general, exhibit a more anaerobic metabolism than normal cells. The presence, or lack, of reductive activation leads to different active compounds derived from the FR900482 and FK317 drugs in normal and cancerous cells. In tumorgenic cells both drugs are deacetylated, oxidized and then reduced to create a 4-alcohol derivative that has cytotoxic properties. In contrast, in normal cells FK90082 and FK317 are deacetylated and oxidized to form a 4-carboxylic acid, which is not cytotoxic [112]. While both the FR900482 and FK317 drugs, which differ by only a single methyl group in cells after they have been reductively activated, are highly toxic to tumor cells, the two drugs have other quite different secondary biological effects. One of the major differences between the drugs is that FR900482 induces a pathological condition in treated patients known as vascular leak syndrome (VLS) where as the K317 drug does not. As a consequence, FR900482 has recently been withdrawn from clinical studies whereas FK317 is currently in phase II clinical trials for cancer treatment in Japan. One possible explanation for the difference in the ability of the two drugs to induce VLS could be differences in their abilities to up-regulate expression of the IL-2 and IL-2R genes in lymphoid cells [109].

175 Although FR900482 and FK317 are not specific cross-linkers of HMGA proteins to DNA in living cells (i.e., they also cross-link other minor groove binding proteins in vivo), they are the first drugs demonstrated to specifically cross-link HMGA proteins to the minor groove of DNA in vivo and therefore present a major technical advance for studies aimed at examining the cellular dynamics of binding of these proteins to specific sequences of DNA in living cells [70]. And, importantly, the FK317 drug also potentially serves as a model for designing future therapeutic hybrid FK317-polyamide lexitropsin drugs that target specific AT-rich HMGA binding for selective killing of particular cell types. These future hybrid drugs are envisioned to consist of a cell permeable, reductively activated pro-drug (e.g., FK317) that is connected by a flexible linker to a bipartite AT-sequence recognizing Py–Im polyamide lexitropsin that specifically recognizes the promoters of specific HMGA-regulated genes. In principal, these hybrid drugs could be used to specifically treat individual pathological conditions in ways that are not currently possible. Additionally, such drugs could find use as new reagents to investigation the biological function(s) of HMGA proteins in normal processes such as embryogenesis and cell differentiation.

7. Conclusions A constellation of at least three characteristics distinguishes the HMGA proteins from almost all other cellular proteins: (i) the AT-hook DNA-binding motif that recognizes the structure, rather than the nucleotide sequence, of substrates; (ii) an unusually high degree of intrinsic flexibility that allows the proteins to undergo disordered-to-ordered structural transitions as part of their biological function; and (iii) complex patterns of secondary modifications that appear to function as a biochemical ‘‘code’’ to precisely regulate the biological activity of the protein in vivo. Structural simplicity and flexibility, combined with very sophisticated regulatory control mechanisms, are the biophysical and biochemical traits that allow the HMGA proteins to function as either ‘‘generalists’’ or as ‘‘specialists’’ in so many different nuclear activities. In this review we have briefly discussed how these features enable the proteins to participate in such diverse processes as chromosome dynamics during the cell cycle, transcriptional regulation of genes and both global and localized chromatin remodeling events. Their central role in nuclear metabolism makes the HMGA proteins attractive targets for therapeutic interventions to treat several different types of pathologies. A number of current and potential approaches appear to be promising in this area. The key problem for developing such useful therapeutics, however, is to create drugs with the requisite specificity to target only certain functions of the HMGA proteins inside cells. Unfortunately, the same characteristics that allow the HMGA proteins to perform multifaceted tasks within the nucleus are precisely those that present the most challenge in creating effective and specific anti-HMGA drugs. Nevertheless, important strides have been made in our understanding of the

176 structure and function of the HMGA proteins and thus a solid foundation has been laid for future advances in this area.

Abbreviations ARRE-1, -2 AT-hook bp CD28RE CK-2 CD ChIP CK2 CRM EMSA HMG HMGA (a.k.a., HMGI/Y) HMGB (a.k.a., HMG-1 and -2) HMGN (a.k.a., HMG-14 and -17) hu IFN- IL-2 IL-2R IL-4 iNOS ISWI MALDI/MS MAP kinase MATH mRNA mu MS MS/MS NFIL-2 NMR NURF PEV PKC PRD-I to -IV PRRII Py–Im

antigen regulated response elements-1 and -2 DNA-binding domain peptide of HMGA proteins base pair CD28 response element casein kinase 2 circular dichroism chromatin immunoprecipitation assay casein kinase 2 chromatin remodeling machine electrophoretic mobility shift assay ‘high mobility group’ nonhistone chromatin proteins the ‘AT-hook’ containing family of HMG proteins the ‘‘B box’’ containing family of HMG proteins the ‘Nucleosome-binding’ family of HMG proteins human interferon beta interleukin-2 alpha subunit of the IL-2 receptor interleukin-4 inducible nitric oxide synthase imitation SWI remodeling complex matrix-assisted laser desorption ionization/mass spectrometry mitogen activated protein kinase synthetic multi-AT hook proteins messenger ribonucleic acid murine mass spectrometry tandem mass spectrometry–mass spectrometry nuclear factor interleukin-2 nuclear magnetic resonance spectrometry nucleosome remodeling factor position effect variegation protein kinase C positive regulatory domains I to IV positive regulatory region II pyrrole–imidazole polyamides

177 SRF SWI/SNF TCR TNF- SAR TPA

serum response factor chromatin remodeling complexes the T cell receptor -chain tumor necrosis factor- scaffold attachment region phorbol ester 12-O-tetradecanoylphorol-13-acetate

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