Invertebrate HMG-1 homologs

dHMG-Z is a second Drosophila HMG1 homolog. The protein is 65% identical to Drosophila HMG-D, and also contains a single HMG-box as the DNA recognition motif. Analgous to HMG-D, two transcripts are observed for dHMG-Z; differently regulated, each is the product of zygotic transcription, unlike the HMG-D transcripts that arise from both maternal and zygotic transcription. The genes for HMG-D and dHMG-Z are located on adjacent loci in the genome; each contains two introns. The position of the second intron in the coding region is conserved between the two genes, suggesting a common origin via gene duplication. The reason for the presence of two HMG 1/2 related proteins in Drosophila is unknown. Two such genes have been identified in the insect Chironomus thummi. Perhaps this is a general feature in dipterans. The yeast S. cerevisiae also contains two highly related HMG-1/2 proteins, NHP6a and NHP6b, as does Tetrahymena (HMG-B and HMG-C) (Ner, 1993 and references).

In Caenorhabditis elegans, Wnt signaling is involved in several aspects of development such as cell fate specification during embryogenesis, vulval formation, somatic gonad development, and tail patterning. Some components are shared by several or all of these processes. During embryogenesis, Wnt signaling pathway genes, mom-2 (Wnt), mom-5 (Fz), wrm-1 (beta-catenin), and pop-1 (TCF/LEF), mediate interactions between the P2 and EMS cells. Signaling from the P2 cell promotes asymmetric division of the EMS cell to produce an anterior MS fate and a posterior E fate. Inactivation of the Wnt signaling pathway results in an E-to-MS fate transformation. The Wnt pathway also plays a role in vulval development. Mutants of several Wnt pathway components show various vulva lineage defects: for example, lin-17 (Frizzled), bar-1 (beta-catenin), and mom-1 and mom-3 mutants. In somatic gonad development, lin-17 has also been shown to control several asymmetric cell divisions. In the tail region, a Wnt signal LIN-44 and its putative receptor LIN-17 control the polarity of several asymmetric cell divisions, including B, T, F, and U. lin-44, lin-17, and bar-1 also are involved in the specification of P12 fate in the tail region. A common feature of most of these Wnt signaling events in C. elegans appears to be that Wnt signaling is required for posterior cell fates, and inactivation of signaling results in posterior to anterior fates transformation (Jiang, 1999 and references).

The TCF/LEF class HMG box proteins have been shown to regulate Wnt target gene expression on pathway activation. The C. elegans TCF/LEF class protein is encoded by the pop-1 gene. pop-1 negatively regulates Wnt signaling, as loss-of-function mutation in pop-1 confers a phenotype opposite that of inactivation of Wnt signaling. Moreover, the expression level of POP-1 protein appears to be up-regulated on inactivation of Wnt signaling. Therefore, it was proposed that activation of the Wnt signaling pathway relieves the inhibitory effects of POP-1 on gene transcriptional regulation. Yet it is not clear how Wnt target gene expression is regulated and what other unidentified factors are involved in this process (Jiang, 1999 and references).

Cell-cell interactions and cell fate specification during C. elegans male spicule development has been studied. Cell fate specification in spicule development is mediated by multiple signaling pathways. The C. elegans male spicule is generated by a single male specific B blast cell. The first division of B cell is asymmetric and is controlled by the Wnt signaling pathway genes, lin-44/Wnt and lin-17/Frizzled. At the early third larval stage (L3; B lineage 10-cell stage), the specification of B cell progeny involves a RAS signaling pathway and a C. elegans Notch homolog lin-12. During the L3 stage, those cells continue to divide to generate spicule neurons, non-neuronal sheath and socket cells, and connective tissues. For example, zeta sublineage generates the neuron SPD and its associated sheath (SPDsh) and socket (SPDso) cells (Jiang, 1999 and references).

Using a molecular marker that is specifically expressed in zeta-derived SPD neurons, neuronal versus non-neuronal cell fate specification during male spicule development has been studied. Although the cell division pattern of the zeta sublineage resembles the Drosophila sensory organ precursor (SOP) lineage in which the Notch pathway plays a crucial role, lin-12/Notch is not involved in the specification of the SPD neuron fate. Rather, the lin-17-mediated Wnt signaling pathway plays an important role in this neuronal versus non-neuronal fate decision in zeta lineage. Loss-of-function mutants of lin-17 display an SPDsh (SPD associated sheath cell) to neuron fate transformation. To identify new genes involved in this cell fate decision, a genetic screen was carried out and a mutant, son-1(sy549), was discovered which shows the same cell fate transformation. Moreover the son-1 (sheath-to-neuron fate transformation) mutation enhances the phenotypes of a lin-17 hypomorph, n698, to lin-17 null-like, with respect to vulval development, somatic gonad development, and male tail patterning. The son-1 locus was cloned and found to encode an HMG1/2-like DNA-binding protein, which is distinct from the TCF/LEF class HMG proteins. Disruption of son-1 function through RNA-mediated interference (RNAi) leads to defects in several Wnt pathway-mediated developmental processes. Overexpression of POP-1 causes the same spicule defect as loss-of-function son-1. These results provide in vivo evidence that the HMG1/2-like protein encoded by son-1 plays a specific role in Wnt signaling. SON-1 and POP-1 may both act in the Wnt-responding cells, but in opposite directions, to regulate gene transcription. Taken together, these data provide evidence for further complexity in Wnt responses during development (Jiang, 1999).

Although the HMG1/2 class proteins are traditionally considered as architectural components of chromatin, the specific phenotypes of son-1(sy549) suggest that this gene may be specifically involved in Wnt signaling for four reasons: (1) son-1(sy549) causes a similar SPDsh to neuron fate transformation as does a lin-17 hypomorph. The vulva and gonad defects observed in son-1(sy549) mutants, although at low penetrance, do resemble what are seen in lin-17 mutants. (2) Specific postembryonic phenotypes have been observed that resemble lin-17 mutant phenotypes from son-1 RNAi experiments. (3) son-1(sy549) enhances lin-17(n698) phenotypes to lin-17 null-like but does not enhance phenotypes of other unrelated mutants such as let-2(mn114). (4) Overexpression of POP-1 gave similar spicule phenotype as disruption of son-1 function. Taken together, son-1 is involved in several Wnt-mediated developmental processes, including vulva formation, somatic gonad development, and male spicule development. These results provide the first evidence for the specific functions of the HMG1/2 class proteins in vivo (Jiang, 1999 and references).

The HMG1/2-like protein encoded by son-1 has several interesting features that are different from what is known about the HMG1/2 class proteins. The HMG1/2 class of proteins is classified as sequence nonspecific HMG proteins in comparison to the sequence-specific HMG proteins, such as TCF/LEF. The canonical HMG box found in sequence nonspecific HMG proteins and the generalized HMG box found in sequence-specific HMG proteins share substantial sequence homology and identical protein structure. The sequence specificity is thought to reside in the first 12 amino-terminal residues and the last 25 carboxy-terminal residues of the HMG box. In particular, the amino acids at positions 7 and 12 have been suggested to be crucial for sequence-specific binding. The residues at positions 7 and 12 are valine (V) or isoleucine (I) and asparagine (N) in all sequence-specific HMG boxes. Instead, all presently known sequence nonspecific HMG boxes have proline (P) and serine (S) at these two positions. The box B of SON-1 protein appears to be consistent with this finding (positions 102 and 107 of the protein). Interestingly, the box A of SON-1 protein has a valine (V) residue at position 7 (position 14 of the protein). This raises the possibility that the C. elegans HMG1/2-like protein SON-1 has more specific functions than the HMG1/2-like proteins in other organisms. Furthermore, the data presented here data suggest that both HMG boxes are required in vivo for SON-1 functions, in contrast to the in vitro observations that each individual HMG box is able to recognize bent DNA structure and to interact with certain transcription factors in a similar fashion as is the whole protein. These features of SON-1 may be indicative of its specific functions (Jiang, 1999 and references).

The TCF/LEF class sequence-specific HMG proteins have been implicated in Wnt signaling. They can either positively or negatively regulate Wnt target gene expression depending on their transcription cofactors, with beta-Catenin acting as a co-activator and CBP acting as a co-repressor. The C. elegans TCF/LEF homolog, pop-1, appears to negatively regulate Wnt signaling because loss-of-function in pop-1 causes an opposite phenotype as inactivation of the Wnt signaling during C. elegans embryogenesis. In the specification of SPD neuron fate versus SPDsh fate during male spicule development, overexpression of POP-1 results in the same defect as loss-of-function mutation in lin-17 and son-1. Therefore, a POP-1-like activity also negatively regulates Wnt signaling during C. elegans postembryonic development. Both the sequence-specific TCF/LEF and sequence-nonspecific HMG1/2 class of proteins are involved in lin-17-mediated Wnt signaling and they play opposite roles in regulating Wnt pathway activity (Jiang, 1999 and references).

pop-1 has been shown to act downstream of the Wnt receptor and its expression is negatively regulated by Wnt signaling. Inactivation of the Wnt pathway up-regulates POP-1 protein level. son-1 also acts in the Wnt responding cells. Therefore, both the POP-1-like protein and SON-1 act in the same cell nucleus to regulate gene transcription. The POP-like activity inhibits gene transcription, and SON-1 facilitates gene transcription. Since SON-1 is an HMG-1/2-like protein, it is thought that SON-1 could activate gene transcription in two ways: (1) SON-1 could alter chromatin conformation in favor of gene expression, and (2) SON-1 could physically interact with other transcription factors and help stimulate specific gene expression. Proteins of the HMG1/2 class have been shown to interact with POU domain proteins or HOX proteins in vitro and enhance their DNA-binding and transcriptional activities. The present data suggest that son-1 function may not be rate limiting for Wnt signal transduction. Moreover, son-1::GFP expression does not seem to be altered in a lin-17 mutant background. Therefore, it is possible that SON-1 acts to activate gene transcription after the inhibitory effect of POP-1 is relieved by activation of the Wnt pathway (Jiang, 1999 and references).

The signaling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signaling downregulates POP-1 activity in one posterior daughter cell called E. The genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. It is concluded that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, since TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1 (Meneghini, 1999).

Two chromosomal high mobility group (HMG) proteins from larvae of Chironomus thummi (Diptera) and from an epithelial cell line of Chironomus tentans were purified to homogeneity and chemically characterized. cDNA clones encoding these proteins were isolated from an expression library using an immunoscreening approach and were sequenced. The deduced amino acid sequences reveal their homology to HMG protein 1 of vertebrates. These insect proteins have therefore been designated cHMG1a and cHMG1b. They have a molecular mass of 12,915 and 12,019 kDa, respectively, and preferentially bind to AT-rich DNA. Indirect immunofluorescence microscopy with a polyclonal antibody has shown the presence of cHMG1a and cHMG1b in condensed chromomeres but not in puffs, nucleoli, and cytoplasm. The cHMG1a and cHMG1b genes were both localized to a single band in region 14 of chromosome 1 of C. tentans and appear to be single copy genes. An immunologically related protein was purified from Drosophila melanogaster Kc cells. Its size and amino acid composition indicate that it is an HMG1 of Drosophila (Wisniewski, 1992).

The mammalian high mobility group proteins HMGI/Y and HMG1/2 are thought to play an architectural role in assembly of nucleoprotein structures. Counterparts to these proteins have recently been found in the cells of the Dipteran insect Chironomus. The distribution of three abundant HMG proteins was examined in interphase giant chromosomes of the midge, Chironomus. By means of the indirect immunofluorescence technique the cHMG1b and cHMGI proteins were localized in chromosomal puffs, suggesting their involvement in the organization of transcriptionally active chromatin. In contrast, the highly abundant protein cHMG1a is rather uniformly distributed in the chromosomes. The cHMGI protein, but not cHMG1a or cHMG1b, is detected in nucleoli, which may indicate a role in the transcription of ribosomal genes. The regions of the interphase chromosomes containing AT-rich DNA do not contain higher levels of the cHMGI and cHMG1b proteins. A correlation between the specific location of these proteins in chromatin and their synthesis and turnover rates has been observed (Ghidelli, 1997).

HMG-1 and HMG-2 proteins include three structural domains. Two of these domains (termed A and B) have significant homology with one another, and the third comprises an acidic carboxyl-terminal tail that can interact with histone H1 in vitro. There is a repeat region of 85 amino acids that acts as a novel DNA binding domain. This region is called the HMG domain. The HMG domain consists of three alpha-helices between residues 8-24, 34-48 and 50-74. HMG-1/2 proteins can be divided into two subfamilies according to the number of HMG domains present in the protein, their specificity of sequence and their evolutionary relationships. One subfamily contains the abundant HMG-1 and -2 proteins. They are found in all cell types, have multiple HMG domains (usually 2) and recognize DNA with no obvious, or low, sequence specificity. The second subfamily includes DNA-binding proteins with a single HMG domain. These have a restricted cell-type distribution and interact with specific nucleotide sequences. HMG proteins have the ability to bend linear DNA. The HMG proteins bend DNA through either of two mechanisms: 1) By acting alone through a simple bending of the helix or 2) by physically interacting with other regulatory proteins. HMG-I(Y) acts by the second mechanism, interacting with transcription factors and thus promoting bending through protein-protein contacts (Grosschedl, 1994)

The cells of the dipteran insects Chironomus and Drosophila contain high mobility group (HMG) 1 proteins that are homologous to the HMG1 protein of mammals but comprise one instead of two DNA-binding HMG boxes. Mobility shift assays have revealed that Chironomus cHMG1a and cHMG1b bind double stranded and four-way junction DNA in a similar way at apparent dissociation constants in the range of 7.5-20 x 10(-9) M. Both proteins are monomeric and highly asymmetric molecules in solution. cHMG1a and cHMG1b exhibit Stokes' radii of 2.4 and 2.3 nm, respectively, and both show a frictional ratio of 1.5. Despite these similarities in their hydrodynamic properties, the binding site of cHMG1a on DNA is approximately 1.5 of the size found for the cHMG1b. Enzymatically and chemically prepared peptides of cHMG1a as well as bacterially expressed cHMG1a with terminal deletions and point substitutions shows that sequences flanking the folded domain, which constitutes the HMG box, are essential for the interaction of the HMG box with DNA. In particular, changes in the number of positive and negative charges, respectively, within basic and acidic domains modulate the DNA binding affinity of the cHMG1a protein. The alteration of fluorescence of the Trp residues suggests that this modulation is due to interaction of the acidic domain with the positively charged HMG box (Wisniewski, 1994).

Several in vitro studies have suggested that high mobility group (HMG) protein 1 has a role in gene regulation as a trans activator or quasi-transcription factor. However, data on the molecular functions of HMG1 protein in these reactions are contradictory or obscure. In order to assess whether HMG1 protein does, in fact, have transcriptional activation potential, two assay systems in cultured cells were employed. HMG1 protein introduced into COS-1 cells as a complex with a reporter plasmid carrying the lacZ gene enhances the level of the gene expression. Cotransfection of an expression plasmid carrying HMG1 cDNA into the cells with the reporter plasmid enhances the activity of beta-galactosidase 2-3-fold in comparison with that of the control effector plasmid. The enhancement has proved to be dependent not on the replication but on the transcription of the reporter plasmid. In the cotransfection experiments, the HMG1 molecule lacking the acidic carboxyl terminus represses the expression of the reporter gene. The binding of an HMG1 protein variant lacking the acidic carboxyl terminus to DNA gives an extremely large shift of gel retardation in comparison with the complete HMG1 molecule. Together, these results indicate that HMG1 protein can enhance expression in cells in culture at the step of gene transcription and that the DNA binding domains comprising two-thirds of the HMG1 protein molecule are responsible for the inhibition property. Also, the acidic terminus of the HMG1 molecule is essential for the enhancement of gene expression in addition to elimination of the repression caused by the DNA binding (Aizawa, 1994).

HMG-D is one of the Drosophila counterparts of the vertebrate HMG1/2 class of abundant chromosomal proteins and contains three domains: an HMG domain followed by a basic region and a short acidic carboxyterminal tail. The HMG domain of HMG-D does not bind to deformed DNA structures such as DNA bulges, cis-platinated DNA or four-way junctions but does bind tightly to DNA microcircles, suggesting that in vivo the natural ligands of this domain are tightly bent DNA loops. The flanking basic region substantially increases the DNA-binding activity of the HMG domain to DNA ligands other than microcircles. The acidic tail alters the structural selectivity of DNA binding by increasing the affinity for deformed DNA and decreasing the affinity for linear B-DNA. The acidic tail increases the efficiency of constraining preformed negative supercoils but conversely decreases the efficiency of supercoiling relaxed DNA in the presence of topoisomerase I (Payet, 1997).

Sedimentation and gel retardation studies show a stronger interaction of HMG 1 and 2 with negatively supercoiled DNA than with linear, nicked-circular, or positively supercoiled ds-DNA. An apparent unwinding angle of 58 degrees was obtained for HMG 1 and 2 when assayed by protection of negatively supercoiled DNA from topoisomerase I relaxation or when assayed by the supercoiling of nicked-circular DNA with T4 DNA ligase. The protection of negatively supercoiled DNA is linear up to molar ratios of about 250:1. There is little change in binding reactions or in the protection of supercoiled DNA at ratios above 250:1, indicating that both activities saturate and that HMG 1 and 2 have binding site sizes of about 20 bp. P1, the major tryptic fragment of HMG 1 or 2, which retains the two DNA binding HMG 1/2 boxes, displays a 2-fold increase in binding to all types of ds-DNA compared to intact HMG 1 or 2. However P1 protects negatively supercoiled DNA from topoisomerase I relaxation about 5-fold less than intact HMG 1 or 2. Complete protection with P1 occurs at a molar ratio 1040:1, indicating a DNA binding site size of about 4 bp and an apparent unwinding angle of 10 degrees. P1 binding to closed-circular ss-DNA also involves a binding site of about 4 bp. Adding the acidic C-terminal fragment to P1 reverses its binding and allows topoisomerase I to relax supercoiled DNA. These findings highlight the importance of the acidic C-terminal domains of HMG 1 and 2 in limiting electrostatic interactions of the HMG 1/2 boxes with ds- or ss-DNA. N-Ethylmaleimide inhibits the binding of intact HMG 1 or 2 to negatively supercoiled DNA, but does not inhibit the electrostatic binding of HMG 1 or 2 to ss-DNA, or of P1 to any form of DNA (ds or ss). These results suggest that cysteine residues are involved in the specific interaction of HMG 1 or 2 with negatively supercoiled DNA and that the acidic C-terminal domains modulate an intramolecular conformational change involving sulfhydryls within the HMG 1/2 boxes (Sheflin, 1999).

High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal. To understand the mode of HMG2 interaction with DNA, HMG2 peptides containing HMG1/2 box(es) were expressed in Escherichia coli cells and then purified. Gel retardation and DNA supercoiling assays indicate that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but is not sufficient alone. The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities. The intrinsic DNA binding affinity of box B is weaker, and these adjoining regions largely strengthen the affinity. In contrast, box A, even in the presence of the adjoining basic linker region, shows no such activities, indicating that boxes A and B differ in their DNA recognition mode. The computer modeling suggests that the side chain of Phe-102 in box B is inserted into the base stack to cause DNA conformational changes, while the side chain of Ala-16 in box A is too small to intercalate. These represent that boxes A and B have similar tertiary structures but their activities for DNA conformational changes obviously differ. Box B is the main region for DNA recognition and conformational changes, and box A must play an assistant to increase its DNA recognition (Yoshioka, 1999).

The maize HMGa protein is a typical member of the family of plant chromosomal HMG1-like proteins. The HMG domain of HMGa is flanked by a basic N-terminal domain characteristic of plant HMG1-like proteins, and is linked to the acidic C-terminal domain by a short basic region. Various derivatives of the HMGa protein were expressed in Escherichia coli and purified. The individual HMG domain can functionally complement the defect of the HU-like chromatin-associated Hbsu protein in Bacillus subtilis. The basic N-terminal domain that contacts DNA enhances the affinity of the protein for linear DNA, whereas it has little effect on the structure-specific binding to DNA minicircles. The acidic C-terminal domain reduces the affinity of HMGa for linear DNA, but does not affect to the same extent the recognition of DNA structure, which is an intrinsic property of the HMG domain. The efficiency of the HMGa constructs to facilitate circularization of short DNA fragments in the presence of DNA ligase is, like the binding to linear DNA, altered by the basic and acidic domains flanking the HMG domain, while the supercoiling activity of HMGa is only slightly influenced by the same regions. Both the basic N-terminal and the acidic C-terminal domains contribute directly to the self-association of HMGa in the presence of DNA. Collectively, these findings suggest that the intrinsic properties of the HMG domain can be modulated within the HMGa protein by the basic and acidic domains (Ritt, 1998).

The nuclear distribution of the non-histone HMG-I protein has been investigated by indirect immunofluorescence in several human and murine somatic cell lines and in growing mouse oocytes. HMG-I, a high mobility-group protein that interacts in vitro with the minor groove of AT-rich B-DNA, is found exclusively in the nucleus and this localization corresponds to a complex distribution. By comparing the HMG-I-dependent fluorescence signal with the chromatin density determined by Hoechst 33342 or propidium iodide staining, evidence is presented for the existence of three HMG-I sub-populations whose contribution to the total fluorescence can be determined using a newly developed quantitative co-localization image analysis program: foci that correspond to regions of heterochromatin, intense dots located within decondensed chromatin, and a more diffuse component extending throughout the nucleoplasm. These sub-populations differ in their sensitivity to nuclease digestion and in vivo displacement by the minor-groove binder Hoechst 33342. Double immunolabeling of RNA polymerase II-dependent transcription and HMG-I show that the intense dots are not correlated with sites of high transcriptional activity. Three sub-populations are likely to reflect distinct and separable biological functions of the HMG-I protein (Amirand, 1998).