High mobility group protein D


HMG-D binding to DNA

HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila. During overexpression and purification of HMG-D from E. coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide. Oxidation of this critical residue decreases the affinity of HMG-D for DNA by three-fold, altering the structure of the HMG-D-DNA complex without affecting the structure of the free protein. This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins (Dow, 1997).

Identification of Met13 as the oxidized residue in HMG-D explains why multiple binding modes of the protein are observed; whereas the putative DNA intercalation function of Met13 explains why the affinity of the protein is altered by such a minor modification. Sequence alignment of HMG-box proteins reveals that Met13 is equivalent to the isoleucine of SRY and to the methionine of LEF-1 that undergo minor groove partial intercalation. If Met13 does perform a comparable partial intercalation function in HMG-D, then the additional oxygen atom would be expected to alter DNA binding as observed (Dow, 1997).

The effect of oxidation of Met13 on DNA affinity of HMG-D is small, as expected, because the additional oxygen perturbs the structure or hydrophobicity of the methionine residue very little. Inspection of the sequences of HMG-box proteins, with the exception of UBF, reveals that several aliphatic amino acids and phenylalanine are consistently found in a position comparable to Met13. Even in non-HMG-box proteins, such as the purine repressor, PurR, and the TATA binding protein, TBP, leucine and phenylalanine act as minor groove intercalating residues. Mutational studies on PurR show that replacing the critical intercalating leucine with lysine, serine, tryptophan, threonine, or arginine results in decreased repression of the purF-lacZ gene, whereas mutation to a methionine has little effect. In contrast, the sex reversal mutation of SRY, I68T, where the equivalent to HMG-D Met13 has been replaced by threonine, decreases DNA binding by nearly two orders of magnitude. Therefore, hydrophobic intercalating residues are important for DNA binding by HMG-box and other proteins. The behavior of the conservative substitution of L>M in PurR is comparable to the change in affinity observed between the two forms of methionine in HMG-D. These studies suggest that amino acid substitution at the intercalating position may be used to fine tune DNA binding affinity and shape of the DNA-protein complex (Dow, 1997).

Oxidized methionines present at protein/protein or protein/ligand interfaces may not affect the structure of the free protein, but they can interfere with the interaction of the protein with its target molecules. In order to locate and assess the importance of such residues, several oxidizing agents have been used as effective tools to oxidize proteins. Hydrogen peroxide has been used to measure the kinetics of oxidation in a number of proteins. Oxidation of a key methionine at the small alphaß interface of hemoglobin completely destabilizes the T state. Treatment of proteins with progressively higher concentrations of an oxidizing agent can distinguish between surface, partially exposed, and buried methionine residues. Finally, another highly selective oxidant affects only surface exposed methionines. Selective oxidation can provide insight into the position of methionine residues in a protein and their effects on biological activity (Dow, 1997).

In the case of HMG-D, Met13 is consistently oxidized whereas Met46 is not. Therefore, Met13 must either be intrinsically more susceptible to oxidation or less easily reduced by E. coli methionine sulfoxide reductases than Met46. Clearly, chemical oxidants can distinguish between surface methionines and their buried counterparts. Met46 resides in a well packed hydrophobic core while Met13 is in an exposed helix. The surface area of the sulfur atom in Met13 is more than 25 times more exposed to a 1.4 Å probe than the sulfur of Met46 as determined using the exposed surface area algorithm implemented in the program ACCESS. Thus, Met13 is a much more likely candidate for oxidation. While the position of Met13 in the concave region of the L-shaped protein leaves it susceptible to small oxidizing agents, it may also restrict the entry of enzymes such as E. coli methionine sulfoxide reductases which serve to reverse the oxidation process (Dow, 1997).

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. The DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster have been compared. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, a series of DNA molecules containing modified bases were used to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favors the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally, coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase (David-Cordonnier, 1999).

The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), the changes have been determined in the end-to-end distance, consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported for the bend induced by a single domain. The induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). It is suggested this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif (Lorenz, 1999).

The high mobility group (HMG) chromosomal proteins, which are common to all eukaryotes, bind DNA in a non-sequence-specific fashion to promote chromatin function and gene regulation. They interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in V(D)J recombination and in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. The X-ray crystal structure, at 2.2 Å resolution, of the HMG domain of the Drosophila melanogaster protein, HMG-D, bound to DNA provides the first detailed view of a chromosomal HMG domain interacting with linear DNA and reveals the molecular basis of non-sequence-specific DNA recognition. Ser10 forms water-mediated hydrogen bonds to DNA bases, and Val32 with Thr33 partially intercalates the DNA. These two 'sequence-neutral' mechanisms of DNA binding substitute for base-specific hydrogen bonds made by equivalent residues of the sequence-specific HMG domain protein, lymphoid enhancer factor-1. The use of multiple intercalations and water-mediated DNA contacts may prove to be generally important mechanisms by which chromosomal proteins bind to DNA in the minor groove (Murphy, 1999).

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. This study investigates the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. It has been shown by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, the HMG-D - bulged DNA complex was modelled. This model predicts that two residues, Val32 and Thr33, in the loop between small alpha-helices I and II are inserted deep into the 'hole' in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirms that both are required for the efficient binding and bending of DNA by HMG-D. The role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins is discussed, along with the role of the basic tail in stabilizing the induced DNA bend (Payet, 1999).

HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single 'prebent' site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. It is concluded that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail (Dow, 2000).

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them 'structure-specifically' with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, the binding to 88 bp and 75 bp DNA minicircles was studied of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail). In some cases cooperative binding was seen of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails (Webb, 2001).

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, while the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0°C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, while binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box (Dragan, 2003).

Ubiquitous high-mobility-group (HMGB) chromosomal proteins bind DNA in a non-sequence-specific fashion to promote chromatin function and gene regulation. Minor groove DNA binding of the HMG domain induces substantial DNA bending toward the major groove, and several interfacial residues contribute by DNA intercalation. The role of the intercalating residues in DNA binding, bending and specificity was systematically examined for a series of mutant Drosophila HMGB (HMG-D) proteins. The primary intercalating residue of HMG-D, Met13, is required both for high-affinity DNA binding and normal DNA bending. Leu9 and Tyr12 directly interact with Met13 and are required for HMG domain stability in addition to linear DNA binding and bending, which is an important function for these residues. In contrast, DNA binding and bending is retained in truncations of intercalating residues Val32 and Thr33 to alanine, but DNA bending is decreased for the glycine substitutions. Furthermore, substitution of the intercalating residues with those predicted to be involved in the specificity of the HMG domain transcription factors results in increased DNA affinity and decreased DNA bending without increased specificity. These studies reveal the importance of residues that buttress intercalating residues and suggest that features of the HMG domain other than a few base-specific hydrogen bonds distinguish the sequence-specific and non-sequence-specific HMG domain functions (Klass, 2003).

In addition to intercalation by Met13, the structure of HMG-D-bound to DNA shows partial DNA intercalation by a pair of residues, Val32 and Thr33, and the possibility of an additional DNA intercalation by Ala36 at the adjacent base step. To investigate the importance of these residues, mutations to glycine were made at all three positions, and two additional mutations, V32A and V32T, were made to examine the importance of Val32 intercalation in more detail. CD spectra and melting analyses on each of these mutants confirmed the stability of the HMG domain. The spectra were virtually indistinguishable from the wild-type protein, and the melting temperatures fell into three distinct ranges. The V32A mutant was significantly more stable than the wild type with a melting temperature of 44.7°C, the A36G mutant was less stable than the wild type with a melting temperature of 38.3°C, and V32T, V32G and V33G mutants had comparable stability to the wild-type protein. Residues Val32 and Thr33 are found near the N-terminus of helix 2, and Ala36 is in the middle of helix 2. The difference in melting temperature follows the expected trend based on the alpha helix-forming propensity of the amino acid substitutions for A36G and V32A, but not for the others. This observation is consistent with the position of Ala36 in the middle of the helix already having a good helix former, and residues Val32 and Thr33 that are already rather poor helix formers residing at the end of the helix. There are no other residues within the van der Waals interaction distance of Val32, which suggests that the stability of helix 2 is important in the stability of the overall protein fold. HMG-D has a destabilizing residue at this position, which suggests that there is a balance between its particular role in DNA bending and protein stability (Klass, 2003).

These mutants had nearly the same trends in binding affinity for the pre-bent and linear DNA fragments, and had overall smaller effects on DNA affinity compared to the substitutions of the primary intercalating site in the protein, Met13. Therefore, the intercalating positions 32, 33 and 36, at the edge of the protein-DNA interface, are not as sensitive to mutation as Met13, which is in the center of the binding site. The T33G mutation resulted in nearly 2-fold weaker binding to both linear and pre-bent DNA. In contrast, V32G binds to both DNA fragments similarly to the wild type. V32A binds well to pre-bent DNA, but it binds with almost 2-fold lower affinity to linear DNA than the wild-type protein. Interestingly, the V32T protein has significantly increased affinity for the pre-bent DNA, which suggests that it may form additional stabilizing contacts with the DNA perhaps through hydrogen-bonding interactions. The difference between residues 32 and 33 in DNA binding suggests a more important role for Thr33 than had been appreciated from structural studies and comparison to the HMGB1-boxA protein, for which intercalation by residue 32 dominates DNA binding in this region of the protein (Klass, 2003).

DNA bending analysis clearly shows again that there are two modes of DNA bending. The residues that cluster in the wild-type DNA bending group are the mutants V32A, V32T and A36G, and the bending deficient proteins are the glycine mutants of Val32 and Thr33. The results of this bending study for Val32 are consistent with DNA bending studies conducted with other HMG domain proteins. However, the results and conclusions regarding the HMG-D V32A mutant differ slightly from those obtained in another study that found V32A was not the same as the wild type in bending; this difference could be due to the different DNA sequences used in the assay, which resulted in larger circle sizes of 75 bp in contrast to the 55 bp circles that were observed in this study. Because T33G and V32G failed to produce the wild-type pattern of DNA circles in the bending assay, intercalation or van der Waals DNA interactions must be required for wild-type DNA bending for both of these residues. It is interesting to note that although DNA bending was compromised, DNA-binding affinity was only minimally (<2-fold) affected in these two mutants, which indicates that the additional energetic cost of bending the DNA may only just be compensated by favorable DNA interactions. Finally, Ala36 appeared to intercalate, just barely, in the HMG-D-74-DNA complex crystal structure; mutation to glycine had no effect on DNA bending in the bending propensity assay. The slight effects on linear and pre-bent DNA binding, indicate instead that the beta carbon of Ala36 is most likely involved in DNA interactions by increasing van der Waal's contacts between the protein and the DNA (Klass, 2003).

The involvement of this secondary intercalation wedge in DNA binding and bending by HMG-D differs from the behavior of the comparable regions in the well studied NHP6A and HMGB1-box A proteins, which both have a phenylalanine at position 32 followed by a glycine, alanine or serine. The specificity of HMG domain proteins for cisplatin-modified DNA and four-way junction DNA appears to be related to the size of the residue at position 32, because HMGB1 boxA and NHP6A also have a greater preference for cisplatinated DNA than HMGB1 boxB and HMG-D, which have smaller residues at position 32. The importance of both Val32 and Thr33 in DNA bending reveals one mechanism by which the HMG-D domain achieves such high bending propensity when compared to other HMG proteins. The HMG-D HMG domain appears to bend DNA better than other HMG domains, as judged by its unique ability to circularize 55 bp DNA, and from solution and crystallographic studies. It is suggested that Thr33 intercalation is responsible for this additional bending, and it is noted that HMG-D is the only well studied HMG protein with an intercalating residue at both positions 32 and 33; other HMG proteins with a small intercalating residue at position 32 usually have a non-intercalating glycine or alanine at position 33 (Klass, 2003).

Protein Interactions

Two kinds of binding are evident when HMG-D binds to DNA. At high concentrations of HMG-D, high-molecular-weight complexes are formed, while ladders are formed at lower concentrations, corresponding to one or more bound HMG-D molecules. HMG-D has a slight preference for, but is not limited to, A + T-rich DNA in vitro (Wagner, 1992).

In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques it has been found that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. A conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region (Churchill, 1995).



The same two HMG-2 transcripts can be seen throughout embryonic development, with peak mRNA levels occurring between 8 and 10 hours. The relative abundance of the two transcripts is similar in all samples except those from adult females and early embryos both of which show lower transcript levels. HMG-D mRNA levels begin to decrease in late embryonic development and drop dramatically in the first instar larval stage (Wagner, 1992).

HMG-D, an abundant chromosomal protein, is associated with condensed chromatin structures during the first six nuclear cleavage cycles of the developing Drosophila embryo. At the same time, histone H1 is absent from these same structures. HMG-D protein is found associated with mitotic chromosomes and the polar bodies. Subsequently, strong staining is evident in both the polyploid yolk nuclei and the pole cell nuclei. As H1 accumulates from nuclear division 7 onwards, the nuclei become more compact and transcriptionally active. This compaction is paralleled by a reduction in size of mitotic chromatin. In addition,there is a striking correlation between the switch in HMG-D:H1 ratios and the changes that occur between nuclear cycles 8 and 13 (collectively termed the mid-blastula transition). This transition is characterized by an increase in the nuclear cycle times, a change in the nucleo-cytoplasmic ratio, and a 5- to 20-fold decrease in nuclear volume. It is estimated that there are approximately 10,000 HMG-D molecules per nucleosome in the very earliest embryos. However, by the cellularization stage there are only from 2 to 5 molecules of HMG-D per nucleosome, and still later in embryogeneis less than 0.2 molecules per nucleosome. This absolute increase in the level of H1 and relative decrease in HMG-D correlates with the stages during which nuclei start to become competent for transcription, i.e. cycle 10. Exceptionally, the pole cell nuclei stain intensely until gastrulation, correlating with the late start of transcription in these nuclei. In addition, HMG-D, but not H1, is present in transcriptionally silent yolk nuclei. It is proposed that the switch in HMG-D:H1 ratios is a direct consequence of a re-organization of chromatin from a less condensed state with HMG-D to a more condensed state with H1. It is argued that HMG-D, either by itself or in conjunction with other chromosomal proteins, induces a condensed state of chromatin, both distinct from, and less compact than the H1-containing 30 nm fibre; this state of chromatin could facilitate rapid cycles of DNA synthesis and mitosis (Ner, 1994).

There is an unusual sequestering of HMG-D maternal mRNA, found within the periphery of oocytes during late oogenesis and zygotic expression, confined to the developing embryonic nervous system. Hence, rather than being ubiquitously expressed, HMG-D transcripts display a complex pattern of temporal and spatial localization implying a specialized rather than general role during early fly development (Stroumbakis, 1994).

High mobility group proteins are thought to have an architectural function in chromatin. Changes in titers, extent of phosphorylation, and cellular distribution are described of the three abundant HMG proteins during embryonic development of Drosophila. The titers of the HMG proteins HMGD, HMGZ, and D1 are highest in ovaries and at the beginning of embryonic development. They decrease continuously until cellularization of the embryo. Relative to the histone H1 titer, the levels of HMGD and D1 remain almost constant during gastrulation and organogenesis, whereas the titer of HMGZ increases during late organogenesis. Up to gastrulation, the development is accompanied by dephosphorylation of D1. In contrast, HMGD and HMGZ appear to be constitutively phosphorylated. As the high extent of phosphorylation of D1 is also characteristic in ovaries, it is likely that the posttranslational modifications of this protein observed in early embryonic stages are of maternal origin. Using site specific antibodies against helices I and III of HMGD and HMGZ and against the AT-hook motif of D1, protein-specific staining patterns have been observed during embryonic development. Despite high levels of HMG proteins at the beginning of embryonic development, none of these proteins were detected in nuclei of stage 2 embryos. The accumulation of the HMG proteins correlates with the onset of transcription in stage 3. These results argue against a proposal of a shared role of HMGD and histone H1 in Drosophila chromatin (Renner, 2000).

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. It previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. The association of HMG-D with chromatin has been studied using a cell-free system for chromatin reconstitution derived from Drosophila embryos. Association of HMG-D with chromatin, like that of histone H1, increases the nucleosome spacing indicative of binding to the linker DNA between nucleosomes. HMG-D interacts with DNA during the early phases of nucleosome assembly but is gradually displaced as chromatin matures. By contrast, purified chromatin can be loaded with stoichiometric amounts of HMG-D, and this can be displaced upon addition of histone H1. A direct physical interaction between HMG-D and histone H1 was observed in a Far Western analysis. The competitive nature of this interaction is reminiscent of the apparent replacement of HMG-D by H1 during mid-blastula transition. These data are consistent with the hypothesis that HMG-D functions as a specialized linker protein prior to appearance of histone H1 (Ner, 2001).

Histone H1 and HMGB1 proteins could influence chromatin structure in a similar manner by binding to linker DNA sequence. Histone H1 associates with linker DNA sequences and organizes nucleosomal arrays into higher order chromatin structures, such as the 30-nm chromatin fiber. However, little is known about how HMGB1 interacts with the nucleosome and about the consequences in structure and function. H1 and HMGB1 share important features; both protect linker DNA sequences from nuclease digestion, and both bind four-way junctions. Consistent with the idea that interaction of HMGB1 might replace histone H1, in the very early stages of Drosophila embryogenesis histone H1 is absent, but the high mobility group protein D (HMG-D) is present in vast excess. Based on the similarities between HMG-D and H1, a role for HMG-D as a linker protein compatible with and perhaps required for the fast condensation-decondensation cycles associated with the very rapid nuclear division cycles found in preblastoderm embryos has been suggested. An analogous role has been proposed for the Xenopus HMGB1 and B4 proteins; both proteins have been demonstrated to bind di-nucleosomal DNA (Ner, 2001).

The fact that recombinant HMG-D increases the nucleosome repeat length (NRL) in a cell-free chromatin assembly system strongly supports this hypothesis. The NRL is strongly dependent on the ionic environment such that polycations are particularly effective in increasing the average separation between adjacent nucleosomes. In accordance with these findings the data implicate the polycationic basic region (residues 85-99; net charge, +10) of HMG-D in this function. However, the HMG-D-dependent increase in NRL is mediated both by the full-length protein and by HMG-D100. These forms differ substantially in net charge +7 (for HMG-D) and +17 (for HMG-D100), suggesting that the chromatin DNA can compete effectively with the polyanionic acidic tail of HMG-D. Histone H1 and the HMGN1 and HMGN2 proteins (formerly HMG-14 and HMG-17) are the only other proteins reported to cause such a change, in the case of H1 presumably by binding to the linker DNA. The binding of H1 to the linker sequence appears to differ from that of HMG-D. Increasing concentrations of histone H1 added to the assembly reaction will continue to increase the NRL to well over 220 bp before the regular nucleosomal array is lost. HMG-D, on the other hand, increases the NRL to only ~180 bp. This may reflect the stoichiometry of binding to the linker sequence. Di-nucleosomal DNA reconstituted by dialysis has been shown to be able to bind two molecules of H1 but only a single molecule of HMGB1. Although the exact nature of the binding remains unknown, HMG-D binds ~14 bp of DNA, and consequently 1-2 molecules of HMG-D could potentially occupy the linker space (Ner, 2001).

A tight correlation between nucleosome spacing and the folding of the nucleosomal fiber into a 30-nm fiber has been observed, which led to the suggestion that different NRLs would correspond to particular fiber geometries and, therefore, compaction states. Accordingly, increased nucleosome spacing is indicative of more compacted chromatin. The observation that HMG-D does not increase the NRL beyond 185 bp as H1 may indicate that HMG-D-containing chromatin is folded but is less compacted (Ner, 2001).

Like other HMG domain proteins such as LEF-1 and SRY, HMG-D can introduce sharp bends or kinks into DNA. The current estimates of the magnitude of the DNA kinks induced by HMG-D range from 100-120° for the full-length protein to 60° to >90° for HMG-D100. These values are substantially greater than the average curvature of DNA wrapped around the histone octamer and indicate that HMG-D bound DNA is not smoothly curved. In the context of linker DNA, such a state would be consistent with both the lack of UV-induced thymine dimer formation in the linker and also, with evidence from electric dichroism studies, that the trajectory of linker DNA differs from that of DNA bound to the core histones. Of particular relevance are the observations that, in the presence of histone H1 derivatives containing a major proportion of the basic C-terminal domain, the linker DNA enters and leaves a single chromatosome as a straight rod approximately perpendicular to the superhelical axis. A similar structure has been observed in chromatin fibers. This organization implies that the DNA must bend sharply as it enters and leaves the chromatosome. A possible role for HMG-D would be to induce such sharp bends by kinking the DNA and thereby promoting a higher level of chromatin folding (Ner, 2001).

Evidence has been provided for an interaction of HMGB1 with the nucleosome and it has been suggested that it might replace histone H1 in the nucleosome. Evidence has been provided for interactions between histone H1 and HMGB1. The results are consistent with these observations. (1) In a Far Western analysis, H1 is the predominant protein identified when labeled HMG-D was used as a probe. (2) Using chromatin assembled on DNA attached to paramagnetic beads and preloaded with HMG-D protein, HMG-D is displaced upon titration of histone H1. It is noted that the full-length HMG-D and HMG-D100 both interact with H1 in a Far Western analysis. The alanine-lysine-rich region (amino acids 84-100, AKKRAKPAKKVAKKSKK) is very similar to a region found in histone H1. Far Western analysis suggests that this region, or possibly the region immediately preceding glycine-rich linker, is interacting with H1. In HMG-D this sequence contains a serine residue that is phosphorylated by casein kinase II (Ner, 2001).

Although it is possible to argue for a structural role for HMG-D and HMGB1 in early embryonic chromatin, in vitro observations show that in the absence of H1 HMG-D, although initially present at high levels, is displaced to below 1 molecule/10-20 nucleosomes as the reaction proceeds and the chromatin matures. This would argue against a purely structural role for HMG-D and suggest that the protein may fulfill a different role. One possibility is that HMG-D functions as a chaperone molecule and preconfigures the DNA to facilitate the chromatin assembly process. HMG-D could participate to bend the DNA at the exit and entry points to the nucleosome, and this bend is then stabilized by histone H1. Under such a scenario, as chromatin assembly proceeds and the core histones are recruited, HMG-D molecules are displaced. The linker sequences would be the only locations where the protein would persist for longer duration. However, this too would be displaced on the addition of other chromatin-associated proteins (transcription factors, assembly factors). Such a mechanism would be very similar to that proposed for the recruitment of transcription factors. Similarly the displacement and competition with histone H1 can be envisaged as part of a process in which the DNA is kinked by HMG-D, and then the binding of the linker histone stabilizes this kink (Ner, 2001).

Preblastoderm embryonic chromatin clearly differs profoundly from post-blastoderm chromatin. Early syncytial nuclei are much larger and contain chromatin that is less compacted than later nuclei. In the early embryo HMG-D is highly abundant, although not all molecules are necessarily available for DNA binding. It is deposited in the egg by the mother but thereafter is maintained at an approximately constant level per embryo. Consequently, with each nuclear division the average number of HMG-D molecules per nucleus falls, although during nuclear cycles 7-14 the amount of H1 rapidly increases. Only during cycle 7 does the size of the nuclei begin to decrease. By cycles 10-12 a sufficient amount of histone H1 has accumulated to allow the reorganization of chromatin to a transcriptionally active state. Subsequently, increased zygotic transcription elevates histone H1 levels further. This exponential increase of histone H1 together with the increasing number of nuclei rapidly deplete HMG-D protein to levels that cannot have global effects on chromatin structure. What could be the physiological significance of different linker proteins? HMG-D- or H1-containing chromatin may differ profoundly in the degree or mode of compaction. The looser structure formed in the absence of H1 could facilitate the rapid condensation and decondensation required during the very short early cleavage cycles (Ner, 2001).

The switch from HMG-D- to H1-containing chromatin correlates with the acquisition of global transcriptional competence. Similar observations have been described in the Xenopus system in which B4, an H1 variant, and HMGB1 disappear during mid-blastula transition, again correlating with a change in the accessibility of embryonic chromatin to class III transcriptional machinery. The cell-free system employed in this study may facilitate the detailed analysis of this major switch in genome function during embryonic development (Ner, 2001).


Both transcripts are abundant in the adult female and in the O- to 2-hour embryo, suggesting maternal loading of the HMG-D transcripts into the oocyte (Wagner, 1992).

Effects of Mutation or Deletion

When the region 57D-58D (containing the HMG-D gene) is present in three copies, one sees a suppression of position effect variegation. This finding indicates that the chromatin of loci susceptible to position effect in a strain carrying the duplication is being packaged in a form more accessible to transcription (there is less sporadic silencing). It is an intriguing idea that HMG-D may be involved in packaging the DNA at a level that makes a distinction between euchromatin and heterochromatin (Wagner, 1992 and Wustmann, 1989).

HMG-D was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4221) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4221-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

A group of transcriptional regulators, group A, controls the size of the dendritic field of class I neurons. RNAi of 19 TFs resulted in reduction of the field size covered by ddaD and ddaE. A reduction of coverage could be the result of a net reduction in dendrite outgrowth, branching, or both. Group A TFs have effects on both primary dendrite growth and secondary dendrite growth. For example, RNAi of the PAS-domain TF trachealess (trh) caused a minor reduction in both primary branch outgrowth and the number of lateral branches and a more marked reduction in the overall length of lateral branches. Consequently, the most distal regions of the dendritic field, especially the regions covered by lateral branches, are not innervated. By contrast, RNAi of genes such as the zinc-finger TF pygopus or the BTB/POZ-domain TF cg1841 caused more severe reduction of primary branch outgrowth as well as lateral branching and lateral branch length, resulting in a more drastic reduction of receptive field. In an extreme case, RNAi of the high mobility group gene hmgD resulted in an almost complete block of primary dendrite extension and lateral branching. In general, the genes with the most severe effects on primary branch outgrowth also have the most severe effects on branching, suggesting that these genes may function to regulate dendritic arborization overall (Parrish, 2006).

Although the genes in this class all caused qualitatively similar defects in arborization, some notable phenotypic differences are suggestive of distinct functions for some of these genes in regulating dendrite arborization. RNAi of the nuclear hormone receptors ultraspiracle (usp) and ecdysone receptor (EcR) significantly reduced primary dendrite outgrowth, but caused only modest reduction of lateral branching and lateral branch outgrowth, suggesting that branching is not absolutely dependent on proper outgrowth. Since the Usp/EcR heterodimer is responsible for ecdysone-responsive activation of transcription, as well as ligand-independent transcriptional repression, it is likely that these genes function together to promote dendrite outgrowth (Parrish, 2006).

RNAi of many group A genes resulted in embryonic lethality at a significantly higher rate than control injections. Thus, many of these genes are likely essential for embryonic development, either due to their involvement in regulating neuronal morphogenesis or due to other aspects of their functions (Parrish, 2006).


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High mobility group protein D: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 October 2007

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