This article is based on a presentation at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research held 7-8 October 1999 in Research Triangle Park, North Carolina, USA.
Address correspondence to R. Reeves, School of Molecular Biosciences, Washington State University, PO Box 644660, Pullman, WA 99164-4660 USA. Telephone: (509) 335-1958. Fax: (509) 335-9688. E-mail: reevesr@mail.wsu.edu
This work was supported in part by National Institutes of Health grant RO1-GM46352.
Received 23 February 2000; accepted 14 June 2000.
The HMGI(Y) family of high-mobility group (HMG) nonhistone proteins [a.k.a., HMGI(Y) family] is a founding member of a new category of mammalian chromosomal protein referred to as architectural transcription factors, whose role in gene transcriptional regulation appears to be the recognition and modulation of both DNA and chromatin structure [reviewed in (
1,2)]. Rather than having a distinctive
in vivo function of their own, as architectural components of chromatin, the HMGI(Y) proteins serve as structural elements that, by interacting with other nuclear proteins and certain types of DNA substrates, facilitate the formation of specific multicomponent complexes that perform a particular biologic function. The mammalian
HMGI(Y) gene family consists of three known members: HMG-I [107 amino acids (aa); ~11.7 kDa], HMG-Y (96 aa; ~10.6 kDa), and HMGI-C (109 aa; ~12 kDa). The HMG-I and HMG-Y proteins are identical in sequence, except that the shorter HMG-Y isoform protein has an internal deletion of 11 aa, and is translated from alternatively spliced mRNA coded for by the
Hmgi-(y) gene located at chromosomal locus 6p21 in humans (
3) and the
Hmgi gene in mice located in the t-complex region of chromosome 17 (
4). The related HMGI-C protein is coded for by a separate gene,
Hmgi-c, located at chromosomal locus 12q14-15 in man (
5) and at the pygmy (pg or "mini-mouse") locus on chromosome 10 in mice (
6). The HMGI-C protein has high aa sequence homology (~55% overall) with the HMG-I and HMG-Y proteins, including the presence of three conserved DNA-binding domains (see below) but has the internal deletion of 11 aa characteristic of the HMG-Y protein (Figure 1B).
In vivo, members of the HMGI(Y) protein family exhibit considerable additional heterogeneity as the result of secondary biochemical modifications (e.g., reversible phosphorylations; Figure 1B) that affect their function and are initiated by signal transduction pathways and/or are cell cycle regulated (
1,7).
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Figure 1. The HMGI protein family. (A) Diagrammatic comparison of the structure of the proteins in the HMGI family. The HMG-I protein (107 aa) and HMG-Y (96 aa) proteins are produced by translation of alternatively spliced transcripts from the same gene, whereas the HMGI-C protein (109 aa) is translated from mRNA transcriptions from a different but closely related gene. (B) Comparison of the aa sequences of members of the mammalian HMGI(Y) family of nonhistone chromatin proteins. Both the HMG-Y and the HMGI-C proteins are missing an internal stretch of 11-12 aa residues (......) that is present in the HMG-I protein. The DNA-binding domains (BD-I, -II, and -III), also called the AT hooks, of the HMG-I, HMG-Y, and HMGI-C proteins are indicated, as is the consensus aa sequence for these motifs. The sites of in vivo phosphorylation of the human HMG-I, HMG-Y, and HMGI-C proteins by cdc2 kinase are indicated by the * symbol and the sites of in vitro phosphorylation by casein kinase II by the * symbol (1,7).
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Clones coding for the human
Hmgi(y) (
3) and
Hmgi-c (
5) genes have been isolated and characterized. The genomic organization of the
Hmgi(y) gene (Figure 2A) and the
Hmgi-c gene (Figure 2B) exhibit both remarkable similarities and differences. Among the similarities are the fact that in both genes the three independent DNA-binding domains (the AT-hook motifs; see below) are each located on separate exons, as are the regions coding for the acidic C-terminal domains of the two proteins. In contrast, the
Hmgi(y) gene is approximately 10.1 kilobases (kb) in length and has seven introns and eight exons, whereas the
Hmgi-c gene is much larger (> 36 kb) and has four introns and five exons. Furthermore, the
Hmgi(y) gene has four different promoter/enhancer regions and four different transcription start sites and exhibits a complex pattern of alternative transcript splicing (particularly in the 5´ untranslated region and in the coding region where alternative splicing gives rise to the HMG-I and HMG-Y protein isoforms; Figure 1A), whereas the
Hmgi-c gene has only one promoter/enhancer region with one major transcription start site and does not exhibit alternative transcript splicing. In the
Hmgi(y) gene, the three AT-hook motifs are located on exons V, VI, and VII and are separated from exon VIII containing the C-terminal tail of the protein by an intron that is 1.3 kb long. In the
Hmgi-c gene, the three AT-hook motifs are located on exons II, III, and IV and are separated from the C-terminal domain-containing exon V by an intron that is ~25 kb long. As discussed below, chromosome rearrangements involving all three AT-hook motifs of the HMGI(Y) genes occur frequently in certain types of benign human tumors. Rearrangements of the
Hmgi-c gene occur more frequently than with the
Hmgi(y) gene, a fact consistent with the relative lengths of the introns separating the DNA-binding regions of these genes from their C-terminal tail domains.
Figure 2. Human HMG-I(Y) gene structures (6p21 and 12q15). (A) Diagrammatic representation of the genomic organization of the human Hmgi(y) gene (3). (B) Diagrammatic representation of the genomic organization of the human Hmgi-c gene (5). The exon sequences are indicated by Roman numerals and the intron sequences by Arabic numbers.
The HMG-I, HMG-Y, and HMGI-C proteins share many biochemical, biophysical, and biologic properties, including possessing three independent AT-hook DNA-binding domains that allow these proteins to preferentially bind to the minor groove of stretches of AT-rich sequence and to recognize DNA structure rather than sequence (reviewed in (
1)]. The palindromic core sequence of the AT-hook motif, Pro-Arg-Lys-Arg-Pro, is the most highly conserved peptide sequence found in the HMGI(Y) family of proteins and is also evolutionarily conserved as a DNA-binding unit in many other proteins and transcription factors found in organisms ranging from bacteria to humans (
8).
The physical basis for recognition of the minor groove of AT-rich sequences by the HMGI(Y) proteins became clear with the recent nuclear magnetic resonance (NMR) determination of the structure of a cocomplex of the AT-hook motifs with a DNA substrate (9). These structural studies indicate that in solution the HMG-I protein is unstructured, but upon substrate association the DNA-binding regions of the protein (i.e., the AT hooks) undergo a disordered to ordered structural transition, assuming a specific planar, crescent-shaped structure that confers selectivity for the narrow minor groove of AT-rich sequences. The reason for the AT specificity of HMGI(Y) resides in the conserved core aa P-R-G-R-P sequence, which assumes a crescent, or hooklike, configuration upon binding to appropriately structured AT-DNA substrates (Figure 3). Nevertheless, even when the protein is free in solution, all of the proline residues present in the AT-hook motifs exist in a trans configuration. This structural restriction on the proline residues, combined with the intrinsic flexibility of the central glycine residue, allows the peptide backbone of the motif to assume a narrow concave structure that fits deep into the narrow minor groove of AT sequences without perturbation of the DNA structure. In this hooklike configuration the side chains of the arginine core residues are oriented parallel to the minor groove and extend away from the central AT base pair, with their quanidino groups making hydrogen bonds to the O2 atoms of thymidines and other residues (Figure 3). The snug fit of the hooked core peptide backbone, together with the inward-projecting arginine side chains, displaces water molecules from the narrow minor groove of AT sequences, thereby allowing numerous hydrophobic interactions to form that further stabilize the overall molecular interactions. The aa side chains of the arginines and lysines in the AT-hook peptide that flank either side of the core residues also make electrostatic contacts with the phosphates on the surface of the groove, providing even more stability to the protein-DNA complex. Furthermore, the AT hook binds in only one orientation in the minor groove due to hydrophobic interactions of the arginine side chains with the adenine bases. Optimal van der Waals packing is achieved when the adenine bases contacting the aliphatic portions of the arginine side chains are located on opposite strands of AT tracts. Thus, although the tightest binding of the intact HMGI(Y) proteins appears to be to long stretches of DNA sequence with a consensus (TATT)n or (AATA)n repeat, the optimal binding site for the core peptide appears to have the sequence AA(T/A)T at its center. This snug, stable, and directionally oriented fit of the AT hook into the narrow minor groove of AT sequences provides the substrate binding specificity of the HMGI(Y) proteins; it excludes the presence of a GC base pair within the core DNA-binding region, which would place a bulky 6-NH2 group of guanine in the minor groove and thereby disrupt numerous stabilizing protein-DNA interactions (Figure 3).
Figure 3. Schematic diagrams based on the solution NMR structure of a cocomplex of the second AT-hook motif of the human HMG-I protein bound to the minor groove of an AT-rich segment of DNA (9). Various projection views of the peptide bound to DNA (side, frontal, and along the long axis) are shown. Artwork courtesy of G. Banks (Washington State University).
HMGI(Y) transcripts and proteins are rapidly induced in cells after exposure to factors that stimulate metabolic activation and growth, and therefore have been postulated to be involved in the control of cell proliferation (
3,10-12). Accordingly, Lanahan et al. (
13) have placed the HMGI(Y) genes in the category of delayed early-response genes, whose transcriptional expression is induced within 1-2 hr of exposure of quiescent cells to growth stimulatory factors and which are necessary for subsequent DNA synthesis to occur. Consistent with this proposal, mutations in the
Hmgi-c gene that give rise to the pg phenotype in mice (
6) and the dwarf phenotype in chickens (
14) have demonstrated the direct involvement of the HMGI(Y) proteins in regulation of cellular growth and proliferation during embryonic development (6).
The HMGI(Y) proteins influence cellular function by participating in the regulation of gene transcription in either a positive or negative manner. For example, the HMGI(Y) proteins have been implicated in the positive in vivo regulation of genes coding for the tumor necrosis factor/lymphotoxin, interferon (IFN)-ß, interleukin (IL)-2, IL-2R*, granulocyte-macrophage colony-stimulating factor, major histocompatability complex II, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin proteins, among others, and in the negative regulation of genes coding for IL-4, TCR (T-cell receptor), IFN-*, and ß-globin [reviewed in Bustin and Reeves (1)]. The HMGI(Y) proteins are thought to influence positive gene transcriptional regulation by participation in the formation of multiprotein complexes on the promoter/enhancer regions of the genes they regulate. Formation of such stereospecific regulatory complexes, which in the case of the IFN-ß gene promoter is called an enhanceosome (15), is thought to require both specific protein-DNA and protein-protein interactions involving the HMGI(Y) proteins. Figure 4 schematically illustrates how the HMG-I protein is proposed to participate in the regulation of the transcription of the gene coding for the *-subunit of the human IL-2 receptor (IL-2R*) by initiating the formation of an enhanceosome on the proximal promoter of the gene following T-lymphocyte activation (16). Regulation of IL-2R* gene expression in vivo is the result of a combination of specific protein-DNA interactions between HMG-I and AT-rich sequences in the promoter of the gene and between HMG-I and other transcriptions factors, [e.g., Elf-1, nuclear factor kappa B (NF-*B), and serum response factor] that also bind specifically to this region of the promoter (16). In contrast, negative regulation of gene transcription has been proposed to result when the HMGI(Y) proteins bind to the promoter of the gene and prevent enhanceosome formation (1).
Figure 4. Enhanceosome formation on the human interleukin (IL)-2R* promoter. Diagrammatic model of the promoter region of the human IL-2R* chain gene before (resting T cells) and after (activated T cells) mitogen stimulation, indicating direct interactions between NF-*B, Elf-1, and HMGI(Y) proteins. The striped box depicts positive regulatory region I (PRRI) and the stippled box positive regulatory region II (PRRII) in the proxomal promoter/enhancer region of the gene. Redrawn with modification from John et al. (16).
There is a remarkably high correlation between elevated levels of
HMGI(Y) gene expression and neoplastic transformation of normal cells and/or increased metastatic potential of tumor cells [reviewed in (
1,17-19)]. In normal, nondividing, or differentiated somatic cells, the levels of expression of HMGI(Y) mRNAs and proteins are usually low or undetectable in most tissues (
20-22) but can be induced in cells in response to various growth stimulatory factors (
1,3,10-13). In contrast, in neoplastically transformed cells, as well as in embryonic cells that have not yet undergone differentiation, levels of
HMGI(Y) gene products are often exceptionally high (
1,23-27), with increasing concentrations being correlated with increasing degrees of metastatic potential and poor prognosis (
28-32). It also appears that the HMG-Y isoform of the HMGI(Y) proteins may be differentially induced by tumor-promoting agents during the process of metastatic progression (
33). In any event, it is important to note that the elevated HMGI(Y) product levels found in tumors at different stages of metastatic progression appear to be both independent of normal cell cycle controls and not directly related to the actual rate of cellular proliferation (
23,28). The correlation between cancerous transformation and high constitutive levels of
HMGI(Y) gene products is so striking that it has been suggested that their eleveated levels are a characteristic and diagnostic feature of the transformed cellular phenotype (
23-25). Likewise, increasing concentrations of HMGI(Y) products have been correlated with the increased metastatic potential and have been successfully used as indicators of the state of malignancy of tumors (
28-32). Not surprisingly, given these findings, transfection experiments have recently demonstrated that truncated and chimeric forms of the HMGI-C genes can induce neoplastic transformation of NIH3T3 murine fibroblasts (
34,35) and that transfection of antisense HMGI-C constructs into cells can prevent their transformation by oncogenes (
36).
The human
HMGI(Y) genes located at chromosomal loci 12q14-15 (
Hmgi-c) and 6p21 [
Hmgi(y)] are frequently rearranged by chromosomal translocations in many common solid mesenchymal human tumors including lipomas, leiomyomas, fibroadenoma, pleomorphic adenomas, aggressive angiomyxomas, and pulmonary hamartomas [reviewed in (
19,37)]. In these benign tumors, as shown in Table 1, the three AT-hook motifs of the HMGI(Y) genes are fused "in frame" to the N-terminal end of an ectopic protein or peptide sequence of variable length (ranging from 3 to > 120 aa residues) to form a chimeric protein whose fusion partner can be derived from a number of different chromosomes. It is interesting that these benign tumors, in contrast to neoplasms associated with overexpression of full-length HMGI(Y) proteins from apparently unrearranged and unmutated genes in other cells and tissues, seldom progress to an overtly malignant state. This fact has naturally raised questions concerning the actual role, if any, that these HMGI(Y)-ectopic protein fusion chimeras play in the process of tumorigenesis in solid mesenchymal tissues. For example, there is considerable debate as to whether the appearance of hybrid HMGI(Y)-fusion proteins represents primary or secondary events in the tumorigenesis process. Similarly, there is doubt about whether the ectopic fusion partner plays an active role in tumorigeneis or whether it is merely the absence of the negatively charged C-terminal end of the HMGI(Y) proteins that somehow deregulates the specificity and/or activity of the AT hooks themselves. Little information is available, however, about whether the chimeric fusion proteins have any functional activity in the transformed cells, for example, by retaining the ability to specifically bind to AT-rich DNA sequences and/or by interacting with other proteins. Nevertheless, it has been suggested (
38) that the large LIM/homeodomain transcription factor (LIM) protein sequences fused to the AT hooks of the HMGI-C protein in benign lipomas probably function
in vivo as transactivation domains, probably altering the biological activity of wild-type HMGI-C and leading to deregulation of downstream target genes. Similarly, the three AT hooks of the human acute lymphoblastic leukemia (ALL)-1 [mixed lineage leukemia (MLL)] gene are commonly fused to even larger ectopic protein partners in chromosomal translocations that occur in highly malignant multilineage human leukemias [(
39); Table 1], and these chimeric proteins have also been suggested to alter the expression of downstream target genes
in vivo, perhaps by the mistargeting of fused transactivation/repression domains via the AT-hook motifs (
39-42). Such mechanistic suggestions assume a priori that the AT hooks will still specifically function in AT-DNA substrate binding when fused to large protein partners [> 127 aa residues in the case of the LIM peptide (
38); 861 aa residues in the case of AF4 (
41); up to about 2,175 aa residues in CREB-binding protein histone acetyltransferase (CBP) (
42); and approximately 2,523 aa residues in the case of ENL (a nuclear protein with trnascriptional activation potential) (
40)]. This might not necessarily be the case, as large peptide or protein fusion partners could well inhibit the function of the AT-hook motifs by not allowing them to fold into the proper configuration for AT-substrate recognition (Figure 3), by steric blockage of their access to DNA substrates, and/or by otherwise incorporating the AT-hook aa into nonfunctional secondary or tertiary folded structures in the chimeric proteins.
To test whether the AT-hook motif of the HMGI(Y) proteins could impose its substrate binding on a large, highly structured hybrid fusion partner, we performed a peptide domain-swap experiment (
49) between the extended N-terminal domain of the B box of the human HMG-1 protein and the second DNA-binding domain (i.e., AT-hook II) from the human HMG-I protein (Figure 5A). The B box of the HMG-1protein was chosen as the fusion partner for these experiments for a number of reasons. First, the tertiary structure of the 75 aa-long HMG-1 B box has been determined by solution NMR (
50,51); the structure is composed of three *-helices and an extended N-terminal peptide segment that together form an unusual twisted "L" or "V" shape consisting of two arms, one shorter (~31 Å) than the other (~36 Å), with an angle at the apex of between the arms of ~ 70-80° (Figure 5A). The longer arm, composed of the C-terminal third helix stacked against the extended N-terminal region, is referred to as the minor wing (
51). The minor wing, and in particular its extended N-terminal peptide, is the part of the B box making primary contacts with DNA and is mostly responsible for any substrate selectivity that a particular B-box motif might exhibit (
50,51). Additionally, the DNA-binding, extended N-terminal peptide of the B box and the AT-hook II peptide share a common tetrapeptide sequence (P-K-R-P), as well as three similarly spaced proline residues (all in the
trans configuration) (Figure 5A), suggesting that these peptides may have evolved from a common evolutionary ancestral sequence (
1). It is therefore not surprising that both HMG-1
and the HMGI(Y) proteins share similar abilities to bind to the DNA minor groove, to selectively bind to bent, supercoiled, or distorted DNA substrates, and to selectively bind to non-B-form DNA structures such as four-way junctions (FWJs) (
1,52). Nevertheless, there are also a number of significant differences between the substrate recognition characteristics of these two different DNA-binding motifs that can be used to quantitatively distinguish between them (
1). For example, whereas HMG-1
preferentially binds to single-stranded DNA substrates, the HMGI(Y) proteins specifically bind to double-stranded DNA substrates. Another major difference, as already mentioned, is that HMG-1
binds to double-stranded DNAs in a sequence-independent manner, whereas the HMGI(Y) proteins specifically bind to AT-rich substrates. Finally, whereas the HMGI(Y) proteins can bind to nucleosome core particles (
53,54), the HMG-1 protein cannot, preferring instead to bind to the linker DNA between adjacent nucleosomes in chromatin (
1). These similarities and differences in binding characteristics between the AT-hook motif and the B-box peptide of HMG-1 were experimentally exploited to characterize the substrate binding capabilities of an artificially produced recombinant hybrid protein. This chimeric protein was created by fusing a single AT-hook peptide motif, in-frame, to the three *-helices of the B box of HMG-1, thereby replacing its extended N-terminal peptide DNA-binding region with that of the HMGI(Y) proteins [(
49); Figure 5A]. For convenience, we will refer to this chimeric protein as the hybrid B box.
Figure 5. (A) Schematic diagram of a domain-swap experiment in which a single AT-hook motif (AT-hook II from the human HMG-I protein) is exchanged for the extended N-terminal peptide segment of the B box of the human HMG-1 protein. Also shown is a sequence comparison between the two exchanged peptide segments. (B) Computer model of the hybrid HMG-1 B box based on the known solution structures of both the HMG-I DNA-binding motif and the HMG-1 B box. See text for explanation. Artwork courtesy of G. Banks (Washington State University).
Both HMGI(Y) proteins, as well as the AT-hook motif itself, lack secondary structure while free in solution (
1,9). Therefore, one of the principal objectives of the artificial domain-swap experiments (Figure 5A) was to investigate the influence of the structure imparted by the helical frame of the B box on the substrate binding capabilities of the AT hook. It was originally predicted that a conformational shift of the peptide backbone of the AT hook would occur during DNA binding to allow for tighter protein-substrate interactions and that this change in peptide shape might be necessary to allow for AT-binding specificity (
1), a suggestion recently confirmed by NMR studies (
9). It seems reasonable, therefore, to expect that if the AT-hook motif were placed within a rigid peptide structural environment such as that artificially created in the hybrid B-box protein or likely to exist in certain of the naturally occurring chimeric turmor proteins, it might loose its specificity for AT-rich DNA substrates. Such a limitation, or abolishment, of the binding specificity of the AT hook in structured hybrid proteins would imply that the presence of these DNA-binding motifs in chimeric protein found in human tumors is probably fortuitous and not likely to be causally involved with the neoplastic condition. On the other hand, if the AT-hook motif could be demonstrated to retain its native DNA-binding specificity even as part of a rigid, structured hybrid protein, the case can be forcefully argued that it is the substrate-binding capabilities of the hybrid proteins that are probably important in the formation and/or maintenance of the tumor.
Employing standard recombinant DNA methodologies and following the strategy outlined in Figure 5A, a hybrid B-box protein was created in which the first 10 aa residues of the N-terminal end of the HMG-1 B-box peptide were replaced by an equal number of residues from the second DNA-binding domain (AT-hook II) of the human HMG-I protein (49). Two different quantitative experimental techniques, circular dichroism and intrinsic fluorescence, were then employed to demonstrate that the resulting recombinant B-box hybrid protein had refolded into the same type of *-helical and extended peptide domain structure as the recombinant wild-type B box of the HMG-1 protein (49). To demonstrate functionality of both of the properly folded hybrid B box and wild-type B-box proteins, electrophoretic mobility shift analyses (EMSAs) were performed with FWJ DNA as a substrate. Like the AT-hook motif, the hybrid B-box protein formed two specific complexes with this DNA substrate, whereas the wild-type B box formed only one, as expected (49). These initial results therefore suggested that the hybrid B box had assumed substrate binding characteristics more akin to that of the AT-hook motif than that of the wild-type B box (compare panels i and ii of Figure 5B). To confirm this interpretation, competition EMSAs were then performed on AT-rich B-form DNA substrates, as well as on isolated nucleosome core particles. The results of these experiments likewise confirmed the AT-hooklike substrate binding characteristics of the hybrid B-box protein (49). Finally, DNase-I footprinting analysis demonstrated (Figure 6) that the hybrid B box, but not the wild-type B box, bound specifically to the AT-rich regions in a DNA substrate in a manner that was indistinguishable from that produced by the native human HMG-I protein on the same substrate (49). We therefore conclude from these experiments that the AT-hook motif is functional in conferring DNA-binding specificity on a structure chimeric human protein in vitro, and suggest that a similar situation probably also exists for certain chimeric tumor-associated proteins in vivo (49).
Figure 6. DNase I footprinting demonstrates that the correctly folded hybrid B-box protein specifically binds to AT-rich sequences, whereas the wild-type B-box protein does not. (A) Correctly folded hybrid B-box protein (5 µM) footprinted on BLT (the 3´ tail region of the bovine IL-2 cDNA) substrate. The sites of specific protection of AT-rich sequences by the bound protein are indicated by the vertical lines adjacent to the panel. (B) Wild-type B-box protein (5 µM) incubated with BLT DNA. The correctly folded hybrid B-box protein (A) shows a pattern of protection of the AT-rich of the BLT substrate similar to that previously reported for the full-length HMGI(Y) protein on this DNA (49). However, as expected, the wild-type B box does not show specific protection of any sequences relative to naked DNA. The + indicates lanes in which protein samples were incubated with the BLT DNA, whereas the - indicates lanes with naked DNA alone. Lanes in which the BLT DNA has been chemically cleaved by Maxam-Gilbert G and G+A reactions are included in each panel as a reference [(49); reproduced with permission].
These peptide domain-swap experiments unambiguously demonstrate that the core aa residues of the AT-hook motif (i.e., P-R-G-R-P) retain the ability to assume a structural configuration sufficient for specific recognition of the narrow minor groove of AT-rich DNA sequences, even when they are placed within the confines of the rigid three-dimensional framework of the hybrid B-box protein (
1,9,49). Thus, the inherent flexibility exhibited by the AT-hook peptide in solution is not an absolute prerequisite for substrate-binding specificity as long as the core of the motif can assume its functional conformation in the hybrid. Computer modeling studies of the hybrid B box based on the known structures of both the HMG-1 B box and the AT-hook motif (Figure 5B) suggest why the hybrid B box, but not the wild-type B box, can exhibit such HMGI(Y)-like specific binding. In the hybrid B box, because the core of AT hook is localized in the extended N-terminal domain of the properly folded protein molecule, there is no steric hindrance from the rigid *-helices to prevent the peptide backbone of the motif core from bending into the hook-shaped structure necessary for fitting into the narrow minor groove of AT-rich sequences. Likewise, this configuration allows for projection of the side chains of the arginine residues so they can form a flat, crescent-shaped peptide extension that lies approximately in the same plane as (or about parallel to the linear axis of) the minor wing of the B box itself, thus allowing for interaction with the minor groove. Such a conformation of the core of the AT-hook motif in the hybrid would allow for specific interactions of the arginine side chains with the AT base pairs in the DNA, conferring their substrate specificity on the chimeric protein (c.f., Figure 3). In contrast, although the N-terminal-extended domain of the wild-type B box has a bent or curved peptide backbone somewhat analogous to the core sequence in the hybrid protein (
50,51), the positively charged side chains of the lysine and arginine residues project laterally from the plane of the minor wing so they are in a position to make electrostatic associations with the phosphates on the DNA backbones rather than extending into the groove (
55). In addition, in the wild-type B box, the side chains of these lysine and arginine residues do not form the planar, crescent-shaped structure necessary for specific AT base pair recognition, thus partially explaining the lack of sequence-binding specificity of the HMG-1 protein.
There are numerous implications with regard to the possible etiology and maintenance of human tumors associated with chromosomal translocations that an HMGI(Y)-like DNA-binding specificity can be conferred on a heterologous, structured protein produced by chimeric fusion with one or more AT-hook motifs. The first is that many of the chimeric AT-hook-containing fusion proteins probably retain HMGI(Y)-like DNA-binding properties in vivo. However, these tumor-associated hybrid fusion proteins may well have lost other important properties of the HMGI(Y) proteins necessary for normal in vivo functioning of these proteins. Among the normal properties of the HMGI(Y) proteins that may be aberrant in these chimeric proteins are loss of controlled, cell cycle-regulated transcriptional expression of the hybrid gene; loss of a linkage of expression of the hybrid gene to the differentiated state of the cell; loss of the ability of the hybrid protein to make specific associations with other proteins; loss of specific sites for secondary biochemical modifications in the hybrids; loss of normal mechanisms regulating stability and/or translational efficiency of the hybrid transcripts; and finally, alterations in the stability and/or intracellular localization of the hybrid proteins. Thus, overexpression of AT-DNA-binding, but otherwise defective, hybrid fusion proteins would be expected to lead to aberrant gene transcriptional expression, as well as major alterations in chromatin structure, in tumor cells. Whether these chimeric protein-induced changes in cellular nuclear function are primary or secondary events during the process of tumorigenesis may well depend on the individual tumor. However, because many different chromosomal translocations give rise to chimeric proteins containing AT-hook motifs fused to various types of ectopic partners, it seems likely that such chromosomal changes are a secondary event within tumors. The most consistent correlation for tumors containing either aberrantly expressed full-length HMGI(Y) proteins or AT-hook-containing hybrid fusion proteins is the overexpression and/or disregulation of expression of the AT-hook motif itself. Thus the process of overexpression of the AT-hook motif in whatever context, rather than the nature of the ectopic fusion partner per se, may be the important issue for chimeric tumor proteins resulting from chromosome translocations.
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Last Updated: October 3, 2000
