高迁移率族蛋白成员HMG20a/b的研究进展

高迁移率族(HMG)蛋白是一类广泛存在的非组蛋白型染色体蛋白,能通过诱导染色质结构的变化影响DNA表达。HMG20a和HMG20b是一对高度同源的HMG家族蛋白,均含有一个结构保守的HMG-box结构域和一个coiled-coil结构域,在生物体内广泛表达。HMG20a/b在细胞核内参与组蛋白去甲基酶复合物LSD1-Co REST的形成及一系列与细胞分裂分化相关的生理进程,如神经细胞核红细胞的分化、细胞质分裂以及EMT过程。研究发现,HMG20a/b一些功能的发挥是通过LSD1-Co REST复合物来实现的;在神经分化过程中,HMG20a、HMG20b具有相互拮抗的作用;而HMG20a促进EMT过程反映它很可能是一个促癌因子。本文对HMG20a/b的结构和体内分布及生物学功能进行综述。     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain

High-mobility-group (HMG) proteins are a family of non-histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP-1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP-1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin-sepharose and sulfatide-octyl-sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP-1. Characterization by high performance liquid chromatography--mass spectrometry (HPLC-MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG-1 and HMG-2. Isoelectric focusing, HPLC-MS and peptide sequencing data also suggested that HMG-1 and SBP-1 were identical. Similar to SBP-1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC-immuno-overlay and solid-phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti-Leu7 hybridoma (HNK-1) and anti-SBP-1 peptide antibodies, similar to SBP-1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG-1 and -2 proteins have similar biochemical and biological properties and HMG-1 is most likely identical to SBP-1.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase

All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle.     

Nucleosome cores containing H2B,H3, H4 and HMG2 are reconstituted

Nucleosome cores mixed with the high mobility group proteins, HMG1 and HMG2, in 2 M NaCl, 5 M urea, 0.2 mM EDTA and 10 mM Tris pH 7.0, have been reconstituted by salt gradient dialysis. The reconstituted material, in 10 mM Tris pH 7.0, had a sedimentation peak at the same position as that of control nucleosome cores in sucrose density gradient ultracentrifugation. The SDS polyacrylamide gel electrophoresis of the reconstituted nucleosome cores demonstrated that they contain H2B, H3, H4 and HMG2 and are selectively deficient in H2A. The circular dichroism of DNA of the reconstituted cores was indistinguishable from that of control nucleosome cores. The results suggest that HMG2 replaces H2A as a component of the nucleosome histone core during reconstitution.     

Constitutive phosphorylation of the acidic tails of the high mobility group 1 proteins by casein kinase II alters their conformation, stability, and DNA binding specificity.

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. Here, we report that essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses revealed that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, it weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, we propose that the detected phosphorylation is important for the proper function and turnover rates of these proteins. As the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, our results are probably of general significance.     

A poxvirus host range protein, CP77, binds to a cellular protein, HMG20A, and regulates its dissociation from the vaccinia virus genome in CHO-K1 cells

Vaccinia virus does not grow in Chinese hamster ovary (CHO-K1) cells in the absence of a viral host range factor, cowpox protein CP77. In this study, CP77 was fused to the C terminus of green fluorescence protein (GFP-CP77) and a series of nested deletion mutants of GFP-CP77 was constructed for insertion into a vaccinia virus host range mutant, VV-hr, and expressed from a viral early promoter. Deletion mapping analyses demonstrated that the N-terminal 352 amino acids of CP77 were sufficient to support vaccinia virus growth in CHO-K1 cells, whereas the C-terminal residues 353 to 668 were dispensable. In yeast two-hybrid analyses, CP77 bound to a cellular protein, HMG20A, and GST pulldown analyses showed that residues 1 to 234 of CP77 were sufficient for this interaction. After VV-hr virus infection of CHO-K1 cells, HMG20A was translocated from the nucleus to viral factories and bound to the viral genome via the HMG box region. In control VV-hr-infected CHO-K1 cells, binding of HMG20A to the viral genome persisted from 2 to 8 h postinfection (h p.i.); in contrast, when CP77 was expressed, the association of HMG20A with viral genome was transient, with little HMG20A remaining bound at 8 h p.i. This indicates that dissociation of HMG20A from viral factories correlates well with CP77 host range activity in CHO-K1 cells. Finally, in cells expressing a CP77 deletion protein (amino acids 277 to 668) or a DeltaANK5 mutant that did not support vaccinia virus growth and did not contain the HMG20A binding site, HMG20A remained bound to viral DNA, demonstrating that the binding of CP77 to HMG20A is essential for its host range function. In summary, our data revealed that a novel cellular protein, HMG20A, the dissociation of which from viral DNA is regulated by CP77, providing the first cellular target regulated by viral host range CP77 protein.     

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.     

Differential expression of the chromosomal high mobility group proteins 14 and 17 during the onset of differentiation in mammalian osteoblasts and promyelocytic leukemia cells

The expression of chromosomal proteins HMG 14 and HMG 17 during proliferation and differentiation into the osteoblast and monocyte phenotypes was studied. Cellular levels of HMG 14 and HMG 1 7 mRNA were assayed in primary cultures of calvarial-derived rat osteoblasts under conditions that (1) support complete expression of the mature osteocytic phenotype and development of a bone tissue-like organization; and (2) where development of osteocytic phenotypic properties are both delayed and reduced in extent of expression. HMG 14 and HMG 17 are preferentially expressed in proliferating osteoblasts and decline to basal levels post-proliferatively at the onset of extracellular matrix mineralization. In contrast, under conditions that are not conducive to extracellular matrix mineralization, HMG 14 is maximally expressed following the downregulation of proliferation. Consistent with previous reports by Bustin and co-workers [Crippa et al., 1990], HMG 14 and HMG 17 are expressed in proliferating HL-60 promyelocytic leukemia cells and downregulated post-proliferatively following phorbol ester-induced monocytic differentiation. However, differentiation into the monocyte phenotype is accompanied by reinitiation of HMG 17 gene expression. The results indicate that the levels of HMG 14 and HMG 17 mRNA are selectively down-regulated during differentiation.     

Stability of the maize chromosomal high-mobility-group proteins,HMGa and HMGb,in vivo

Chromosomal non-histone high-mobility-group (HMG) proteins represent essential components of eukaryotic chromatin and have also been isolated from a variety of plants. In maize, studies on structure and function of the two larger of the four major HMG proteins have recently been performed and are now extended by analysis of theirin vivo stability using pulse-chase experiments in a cell suspension culture. The half-life of the analyzed HMGa and HMGb proteins was found to be 65 h or more than 78 h, respectively.     

Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil

The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons.     

DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain.

We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.