Immunochemical detection of chromosomal protein HMG-17 in chromatin subunits

Chromosomal protein HMG-17, purified from calf thymus, has been used to elicit specific antibodies in rabbits. Specific serological reaction between the antigen and the antisera is demonstrated by solid-phase radioimmunoassay and by competitive inhibition assays. The antisera did not cross-react with histones or other chromosomal HMG proteins. The antisera bound specifically to chromatin subunits isolated from HeLa cells, demonstrating that it may be used to study the in situ organization of this chromosomal protein. Chromatin purified from HeLa nuclei was digested with micrococcal nuclease, and the resulting mono- and oligonucleosomes were fractionated on a sucrose gradient. Analyses of the content of chromosomal proteins HMG-1, HMG-17, and H4 in different size nucleosomal particles, by the solid-phase radioimmunoassay, reveal that the distribution of HMG-17 was the same as that of H4, but different from that of HMG-1.     

Developmental changes in the expression of high mobility group chromosomal proteins

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-14a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.     

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.     

Serological analysis of species specificity in the high mobility group chromosomal proteins.

The non-histone chromosomal protein of the high mobility group (HMG-1) present in mouse liver was purified to homogeneity. Antibodies against this protein as well as pure HMG-1 derived from calf thymus and HMG-E purified from duck erythrocytes were elicited in rabbits. The interaction between the antibodies and the immunogens was measured by passive hemoagglutination and by quantitative microcomplement fixation. Quantitative microcomplement fixation assays revealed that the immunological distance between HMG-1 from calf thymus and HMG-1 from mouse liver and duck erythrocytes was 15. This corresponds to 3% sequence differences. It was estimated that amino acid substitution occurred at about seven positions in the polypeptide chain. Thus, HMG-1 proteins display remarkable evolutionary conservation in their primary sequence, similar to that displayed by histones H4 and H3, suggesting that their biological function is dependent on stringent structural requirements. HMG-E protein is significantly different from both HMG-1 and HMG-2 derived from calf thymus. As such, it is a protein unique to avian erythrocytes.     

Persistence of chromosomal proteins HMG-14/-17 in myotubes following differentiation-dependent reduction of HMG mRNA.

The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both HMG-14 and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cycle-related event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein. These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis.     

Detection of monoclonal antibody to high-mobility-group protein 17 from chick oviduct.

Total chromosomal HMG (high-mobility-group) proteins have been isolated from oestrogen-stimulated chick oviduct. The antibodies against these proteins were induced in mice and subsequently their spleen cells were fused with myeloma cells to form hybridomas. A highly purified HMG protein, 17, was used to select for the hybridomas that produce antibody against HMG protein 17. The hybridomas were cultured and injected into mice to produce ascites. The antibody against HMG protein 17 in the IgG (immunoglobulin G) fraction of the ascites fluid was obtained by Protein A-Sepharose column chromatography. We have devised a solid-phase radioimmunoassay and enzyme-linked serological assay for the detection and characterization of this antibody directed against HMG protein 17. This anti-(HMG protein 17) IgG interacted only with HMG protein 17, but not with other chromosomal proteins, e.g. histone H1, "95K protein' (a chick oviduct-specific chromosomal protein) and HMG proteins 1, 2 and 14. The monospecific nature of this anti-(HMG protein 17) IgG fraction is confirmed.     

Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.     

Immunological relatedness of high mobility group chromosomal proteins from calf thymus.

The non-histone proteins HMG-1, HMG-2, HMG-3, HMB-8, HMG-14, and HMG-17 (Goodwin, G. H., SANDERS, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14) were purified from calf thymus. The apparent molecular weights on polyacrylamide gels run in the presence of sodium dodecyl sulfate of the high mobility group (HMB) proteins were determined. Those for HBG-1 and HMG-2 agreed with the molecular weights determined by sedimentation; that for HMG-17 was anomalously high. Antibodies against HMG-1 were elicited in rabbits. The interaction between HMG-1 and anti-HBG-1 was measured by quantitative precipitation and by the microcomplement fixation technique. Quantitative microcomplement fixation assays revealed that the indices of dissimilarity between HMG-1 and HMG-2, HMG-3, HMG-8, HMG-14, and HMG-17 were 2.0, 1.0, 3.8, 10.0, and 6.1, respectively. These correspond to 6%, 0%, 12%, 20%, and 16% sequence difference between HMG-1 and the other five HMG proteins, although the immunological distance between HMG-1 and HMG-14 may be too large to allow a good correlation between the sequence and the immunological reaction. Antibodies to HMB-1 bind to chromatin purified from calf thymus. Therefore, we suggest that the in situ organization of HMG proteins in chromatin and chromosomes may be studied by serological techniques.     

Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2

Monoclonal antibodies were prepared against the high mobility group (HMG) proteins 1, 2a, and 2b from hen erythrocyte chromatin. One antibody that recognized multiple sites along HMG-1, -2a, and -2b reacted strongly with HMG proteins from all vertebrates tested. In contrast, five antibodies that detected unique epitopes on chicken HMG-1 and -2a recognized antigenic sites that exhibited restricted phylogenic distributions. The differential reactivity of these antibodies on vertebrate proteins was in agreement with traditional taxonomy in that the avian HMGs were most closely related to those from reptiles and less related to those from mammals, amphibians, bonyfish, and especially the jawless fish. Mononucleosomes generated by mild digestion of erythrocyte chromatin with micrococcal nuclease were highly enriched in HMG-2a. One antigenic determinant located within the N-terminal domain of HMG-2a was freely accessible to its antibody when the protein was bound to these mononucleosomes. In contrast, two antibodies that recognized determinants in the central region of HMG-2a exhibited little chromatin binding activity. The masking of the central domain by DNA binding was presumably not responsible for these results because all three determinants were available for antibody binding when HMG-2a was bound to DNA in vitro. Therefore, the central region of HMG-2a may be masked from antibody binding by protein-protein interactions in chromatin.     

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.     

Fine structure and activity of discrete RAG-HMG complexes on V(D)J recombination signals

Two lymphoid cell-specific proteins, RAG-1 and RAG-2, initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences (RSSs). Although the RAG proteins themselves bind and cleave DNA substrates containing either a 12-RSS or a 23-RSS, DNA-bending proteins HMG-1 and HMG-2 are known to promote these processes, particularly with 23-RSS substrates. Using in-gel cleavage assays and DNA footprinting techniques, I analyzed the catalytic activity and protein-DNA contacts in discrete 12-RSS and 23-RSS complexes containing the RAG proteins and either HMG-1 or HMG-2. I found that both the cleavage activity and the pattern of protein-DNA contacts in RAG-HMG complexes assembled on 12-RSS substrates closely resembled those obtained from analogous 12-RSS complexes lacking HMG protein. In contrast, 23-RSS complexes containing both RAG proteins and either HMG-1 or HMG-2 exhibited enhanced cleavage activity and displayed an altered distribution of cleavage products compared to 23-RSS complexes containing only RAG-1 and RAG-2. Moreover, HMG-dependent heptamer contacts in 23-RSS complexes were observed. The protein-DNA contacts in RAG-RSS-HMG complexes assembled on 12-RSS or 23-RSS substrates were strikingly similar at comparable positions, suggesting that the RAG proteins mediate HMG-dependent heptamer contacts in 23-RSS complexes. Results of ethylation interference experiments suggest that the HMG protein is positioned 5' of the nonamer in 23-RSS complexes, interacting largely with the side of the duplex opposite the one contacting the RAG proteins. Thus, HMG protein plays the dual role of bringing critical elements of the 23-RSS heptamer into the same phase as the 12-RSS to promote RAG binding and assisting in the catalysis of 23-RSS cleavage.     

Affinity of HMG17 for a mononucleosome is not influenced by the presence of ubiquitin-H2A semihistone but strongly depends on DNA fragment size.

We have used a two-dimensional (deoxyribonucleoprotein leads to DNA) electrophoretic binding assay to study the interaction of the purified high mobility group protein HMG17 with isolated HeLa mononucleosomes as a function of their DNA fragment size and the presence of ubiquitin-H2A semihistone. No significant differences between affinities of HMG17 for ubiquitinated and non-ubiquitinated core mononucleosomes were observed. In striking contrast, the apparent affinity of HMG17 for a mononucleosome increases more than 100-fold upon an increase of the length of the mononucleosomal DNA fragment by as few as 3 to 5 bp over the core DNA length (integral of 146 bp). We suggest that the magnitude of this effect is sufficient to explain the preferential binding of HMG17 in vitro to mononucleosomes derived from actively transcribed genes.