Fractionation by high-performance liquid chromatography of the low-molecular-mass high-mobility-group (HMG) chromosomal proteins present in proliferating rat cells and an investigation of the HMG proteins present in virus transformed cells

The low-molecular-mass high-mobility-group (HMG) proteins from young rat thymus nuclei were fractionated by high-performance liquid chromatography. Two proteins analogous to calf HMG14 and HMG17 were found together with a third major component HMGI similar to that found in HeLa cells [Lund et al. (1983) FEBS Lett. 152, 163-167]. HMGI has as amino acid composition similar to but distinct from HMG14 and HMG17. The three proteins form a family of proteins with HMG14 having an amino acid composition intermediate between HMG17 and HMGI. HMGI is present in proliferating fibroblasts and embryos but is present in very low levels in rat liver, a non-dividing tissue, supporting the notion that HMGI is required for proliferating cells. Fibroblasts transformed with avian sarcoma virus have high levels of HMGI and an additional band HMGI' but the presence of HMGI and HMGI' is not dependent on a functional src gene product.     

Nuclear matrix localization of high mobility group protein I(Y) in a transgenic mouse model for prostate cancer

Nuclear shape and the underlying nuclear structure, the nuclear matrix in cancer cells. Since the NM composition is considered to maintain nuclear shape and architecture, nuclear matrix proteins (NMPs) may be involved in transformation. Our laboratory has recently characterized a subset of NMPs that are associated with prostate cancer development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. One of the identified NMPs, E3E, has a similar molecular weight (22 kDa) with a protein known as HMGI(Y). HMGI(Y) belongs to a group of non-histone and chromatin-associated proteins, high-mobility-group (HMG) proteins, and it has been shown to associate with the NM. HMGI(Y) has been reported to be elevated in different types of cancer including prostate cancer. In this study, we examined the expression of HMGI(Y) protein in the NMP composition of the TRAMP model during the progression from normal to neoplasia. The expression of HMGI(Y) in the NMP extracts of three prostatic epithelial cell lines derived from a 32-week TRAMP mouse: TRAMP-C1, TRAMP-C2, and TRAMP-C3 was also examined. Using both one-dimensional and high-resolution two-dimensional immunoblot analyses, we found that: (i) HMGI(Y) is a nuclear matrix protein expressed as two protein bands with MW of 22-24 kDa and (ii) HMGI(Y) expression is correlated with neoplastic and malignant properties in late stage TRAMP prostate tumors. Overall, these findings support the evidence that HMGI(Y) can be utilized as a marker and prognostic tool for prostate cancer.     

High mobility group-like proteins of the insect Plodia interpunctella

Nuclei from Plodia interpunctella larvae contain four major proteins, which are extracted by 5% perchloric acid and 0.35 M NaCl. The proteins have been designated PL1, PL2, PL3, and PL4. The amino acid analyses of these proteins show that they have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group (HMG) proteins isolated from vertebrate tissues. Immunological characterization of these proteins clearly shows that PL1, PL2, and PL4 are more closely related to HMG1 dipteran proteins, while PL3 is more closely related to HMGI dipteran proteins. The possible relatedness of these proteins to HMG proteins is discussed.     

Selective distribution of histone H1 variants and high mobility group proteins in chromosomes

Recent results on the differential distribution of sequence variants of histone H1, of proteins of the HMG 1/2 family, and of HMGI in polytene chromosomes are reviewed. Several organisms are known to contain two different HMG 1/2 proteins. InChironomus, one of them is restricted to decondensed puffs and may have a specific function. One of the H1 variants ofChironomusis found only in a minority of chromosome bands and differs from the other H1 proteins of the organism by genomic organization and by an inserted structural motif that is also present in single H1 variants of other organisms.     

Phosphorylation of high-mobility-group chromatin proteins by protein kinase C from rat brain

Chromosomal high-mobility-group (HMG) proteins have been examined as substrates for calcium/phospholipid-dependent protein kinase C. Protein kinase C from rat brain phosphorylated efficiently both HMG 14 and HMG 17 derived from calf thymus and the reactions were calcium/phospholipid-dependent. About 1 mol of ³²P was incorporated per mol of HMG 14 and HMG 17. Phosphopeptide mapping suggested that the same major site was phosphorylated in both proteins at serine. The apparent K_m values for HMG 14 and HMG 17 were about 5 μM. HMG 14, HMG 17 and the five histone H1 subtypes prepared from rat thymus, liver and spleen were phosphorylated by the kinase. HMG 14 and HMG 17 from transformed human lymphoblasts (Wi-L2) were also phosphorylated in a calcium/phospholipid-dependent manner. HMG 1 and HMG 2 from the tissues examined were found to be poor substrates for the kinase.     

Point mutations within AT-hook domains of the HMGI homologue HMGIYL1 affect binding to gene promoter but not to four-way junction DNA

High-mobility group I/Y (HMGI/Y) proteins are chromosomal proteins involved in gene and chromatin regulation. Elevated levels of HMGI/Y proteins were reported in diverse malignant tumors, and rearrangements of their genes are casually involved in the development of benign tumors. In humans, the chromosomal locus Xp22 has been often found to be affected in diverse benign mesenchymal tumors. Recent studies revealed that this region contains a retropseudogene HMGIYL1 which potentially can be activated in a way of "exonization" upon aberrations involving this region. The coding sequence of the HMGIY-L1 is highly homologous to the HMGI(Y) gene. On the protein level, both HMGIYL1 and HMGI differ at few amino acid residues, including their putative DNA-binding domains (DBDs). Here we have approached the question of whether the HMGIYL1 product would be able to adopt a role of HMGI in the context of binding to gene promoters and chromatin. Comparative binding studies, employing protein footprinting technique, revealed that HMGIYL1 has lost the ability to bind to the promoter of the interferon beta gene, but retained its high affinity for the four-way junction DNA. Our results stress the importance of particular residues within the DBDs for DNA binding and demonstrate that tight binding of HMGI/Y proteins to the four-way junction DNA can be achieved in alternative ways. The binding of HMGIYL1 to four-way junction DNA suggests that activation of the HMGIYL1 gene would yield a protein sharing some binding properties with HMG1-box proteins and histone H1. Thus, the HMGIYL1 could interplay together with these components in chromatin regulation.     

Characterization of high mobility group protein binding to cisplatin-damaged DNA.

cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) is a widely used chemotherapeutic agent. While many tumors are highly responsive to CDDP, certain tumors are resistant to this drug, limiting its efficacy. The anti-tumor activity of CDDP is believed to result from its coordination bonding to chromosomal DNA. Alterations in tumor cell sensitivity to CDDP may result from the presence or absence of protein(s) which specifically recognize CDDP-damaged DNA. We have developed a damaged-DNA affinity precipitation assay that allows the direct identification of cellular proteins that bind to CDDP-damaged DNA. Using this procedure, we have identified several proteins which specifically bind to CDDP-damaged DNA. Two of these proteins have been identified as high mobility group proteins (HMG) 1 and 2 in the current report, we have characterized the binding of these proteins to CDDP-DNA. The calculated Kd of binding to CDDP-damaged DNA was 3.27 x 10(-10) for HMG1 and 1.87 x 10(-10) for HMG2. Using highly specific chemical modifying reagents, we have determined that Cys residues play an important role in protein binding. We also observed that HMG2 will bind to DNA modified with carboplatin and iproplatin although to a lesser extent than to DNA damaged with CDDP. Thus, our results indicate that HMG 2 binds with high affinity to DNA modified with therapeutically active platinum compounds. In addition, our findings suggest that thiol groups play an essential role in the binding of HMG1 and HMG2 to CDDP-DNA.     

Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites.

The primary structure of the Lewis lung carcinoma protein HMGY belonging to the nuclear group of proteins HMGI (high mobility group I) was determined using electrospray and fast atom bombardment mass spectrometry. It was demonstrated that the sequence of the tumor protein corresponds to the amino acid sequence derived from the cDNA from cultured cells and that the N-terminal serine residue is N-acetylated. Moreover, the two high performance liquid chromatography-purified forms Y1 and Y2 of the protein HMGY were shown to differ at the level of serine phosphorylation, since they contain three phosphate and two phosphate groups, respectively, in the C-terminal region. No other modification was detected in the remaining part of the molecule.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both thein vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using thein vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstitutedin vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or thein vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene’s expression.     

Dexamethasone-induced phosphorylation of high mobility group nonhistone proteins of aging rats

Phosphorylation of high mobility group (HMG) proteins and its modulation by dexamethasone were examined in vitro by incubating liver slices of young (15- ) and old (138-week) male rats with (³²P) orthophosphate. HMG proteins were extracted and analyzed by acid-urea polyacrylamide gel electrophoresis. Phosphorylation of HMG proteins, particularly of HMG 2, 14 and 17 decreases drastically in old rats. Dexamethasone stimulates the phosphorylation of total HMG proteins in both ages. Individual HMG proteins vary in the extent of ³²P incorporation. Such differential phosphorylation of HMG proteins and its modulation by dexamethasone may affect chromatin organization and gene expression during aging.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both thein vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using thein vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstitutedin vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or thein vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene’s expression.     

Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (相似文献