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At least 37 records · Page 2Linked to original sources
PubMed1982

Identification of a single-stranded DNA binding protein from rat liver with high mobility group protein 1.

The rat liver single-stranded DNA binding protein, S25 and HD25, isolated by differential DNA cellulose affinity chromatography was compared to the high mobility group proteins, HMG1 and HMG2, isolated from rat liver chromatin by the technique of Goodwin et al. (Goodwin, G. H., Sanders, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14-19). Analysis of their amino acid composition, electrophoretic mobility, and tryptic peptide map reveal the identity of the single-stranded DNA binding protein with HMG1 protein, implying that the rat liver HMG1 protein becomes able both to destabilize a double helix of DNA and to stimulate homologous DNA polymerases only when rat liver cells enter a phase of DNA synthesis, possibly after a specific modification.

Amino Acids↗
PubMed2002

A panel of monoclonal antibodies against human polycomb group proteins.

Polycomb-group (PcG) proteins are chromatin-associated proteins that heritably repress gene activity in many organisms, including man. Two distinct human PcG complexes have been identified. The HPC/HPH PcG complex I contains the HPC, HPH, RING1, and BMI1 proteins, the EED/EZH2 PcG complex II contains the EED, EZH2, and YY1 proteins. Previously we found that the relative expression levels of proteins of the human PcG complexes I and II are severely deregulated in human tumors. These findings signify an important role for antibodies against human PcG proteins as diagnostic tools. To be able to produce standardized anti-human PcG antibodies, we developed a panel of five mouse monoclonal antibodies (MAbs) against the human PcG proteins HPC2, BMI1, RING1A, EED, and EZH2. All MAbs can be used for Western blot analysis and immunofluorescence labeling of tissue culture cells. With the exception of the MAb against HPC2, all MAbs can also be used in immunoprecipitation experiments and immunohistochemistry of human tissues. The novel MAbs are therefore valuable tools for the cell biological, biochemical, and pathological analysis of human PcG proteins.

Animals↗
PubMed2004

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Cell Line↗
PubMed1996

[Effect of phosphorylation by protein kinase C on the DNA binding activity of high mobility group protein I].

High mobility group protein I (HMG-I) is a nonhistone chromosomal protein. The present study aims to examine phosphorylation of HMG-I by protein kinase C (PKC) and its effect on HMG-I's DNA binding activity. HMG-I, extracted and purified from rat brain was phosphorylated in vitro equally well by PKC alpha, beta, gamma and delta. Phosphoamino acid analysis indicated that both serine and threonine residues were phosphorylated. The nonphosphorylated HMG-I was shown to bind specifically to the fragment of DNA containing bp -708 to -458 of RC3 genomic DNA, which is abundant in A-T sequences. In contrast, phosphorylation of HMG-I by PKC resulted in an attenuation of binding to the DNA fragment. It is suggested that phosphorylation of HMG-I by PKC may regulate DNA binding activity of HMG-I, thereby possibly altering its biological functions.

Animals↗
PubMed1999

Cellular memory of transcriptional states by Polycomb-group proteins.

The Polycomb-group constitutes an important, widely conserved group of transcriptional repressors best known for their function in stably maintaining the inactive expression patterns of key developmental regulators, including homeotic genes. Together with the counteracting trithorax-group proteins, they establish a form of cellular memory by faithfully maintaining transcription states determined early in embryogenesis. Besides being crucial for the correct execution of developmental programs, Polycomb-group mediated silencing also appears to be involved in controlling cell proliferation. Here we discuss several aspects of Pc-G function: target gene specificity and recognition as well as propagation of inactive chromatin states to subsequent cell generations.

Animals↗
PubMed1992

Insect proteins homologous to mammalian high mobility group protein 1. Characterization and DNA-binding properties.

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

Amino Acid Sequence↗
PubMed1994

Insect proteins homologous to mammalian high mobility group proteins I/Y (HMG I/Y). Characterization and binding to linear and four-way junction DNA.

A chromosomal high mobility group (HMG) protein from an epithelial cell line of Chironomus tentans (Diptera) was purified to homogeneity and chemically characterized. cDNA clones encoding this protein were isolated and sequenced. The deduced amino acid sequence revealed a high similarity to HMG protein I of mammalia. This insect protein has therefore been designated cHMGI. It has a deduced molecular mass of 10,371 kDa and appears to be a product of a single gene copy. Similarly to mammalian HMGI/Y proteins the insect cHMGI protein has three putative DNA-binding motifs with a sequence K/RXRGRP that are each encoded by one exon of the gene. Using synthetic peptides we have shown that the first and the second motif are necessary for high-affinity binding of the protein to DNA. Protein cHMGI binds preferentially to AT-rich DNA with a half-saturation value of 1.1 nM. Both cHMGI and human HMGI proteins recognize specifically a four-way junction DNA. We have also purified a related protein with similar physico-chemical properties from Drosophila melanogaster Kc cells. The identification and characterization of HMGI proteins in insects with polytene chromosomes and with the cytologic and genetic potential of Chironomus and Drosophila opens new possibilities for studying function(s) of this group of chromosomal proteins.

Amino Acid Sequence↗
PubMed1994

Stimulation of DNA-dependent protein kinase activity by high mobility group proteins 1 and 2.

After incubation of high mobility group (HMG) proteins 1 and 2 with DNA-dependent protein kinase (DNA-PK) and [gamma-32P]ATP in the presence of double-stranded DNA, not only phosphorylation of HMG proteins but also enhancement of autophosphorylation of the catalytic polypeptide of 350 kDa in DNA-PK was observed. DNA-PK activity determined with a synthetic peptide and alpha-casein as substrates was stimulated several-fold by HMG1, HMG2, and the DNA-binding domains. The stimulation was decreased at higher concentrations of HMG proteins, and DNA-PK activity was inhibited by histone H1. Electrophoretic mobility shift analysis suggests that HMG proteins facilitate the binding of DNA-PK to DNA.

Adenosine Triphosphate↗
PubMed1985

General method for isolation of DNA sequences that interact with specific nuclear proteins in chromosomes: binding of the high mobility group protein HMG-T to a subset of the protamine gene family.

A general method is described for the isolation of the DNA with which specific nuclear proteins interact in chromosomes. This method is based on the covalent photo-cross-linking of nuclear proteins to the DNA sequences, to which they normally bind, by means of irradiation with UV light and the selective retrieval of specific subsets of protein-DNA adducts by using specific antibodies. The application of this procedure to isolate the DNA sequences with which the trout high mobility group protein (HMG-T) interacts has shown that in trout liver this protein associates specifically with DNA sequences in proximity to a subset of the family of protamine genes but not with the histone or vitellogenin genes. From these observations, it appears that the HMG-T protein may be associated with inactive gene sequences.

Animals↗
PubMed1998

The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes.

The trithorax group gene brahma (brm) encodes an activator of Drosophila homeotic genes that functions as the ATPase subunit of a large protein complex. To determine if BRM physically interacts with other trithorax group proteins, we purified the BRM complex from Drosophila embryos and analyzed its subunit composition. The BRM complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the BRM complex are not encoded by trithorax group genes. Furthermore, a screen for enhancers of a dominant-negative brm mutation identified only one trithorax group gene, moira (mor), that appears to be essential for brm function in vivo. Four of the subunits of the BRM complex are related to subunits of the yeast chromatin remodeling complexes SWI/SNF and RSC. The BRM complex is even more highly related to the human BRG1 and hBRM complexes, but lacks the subunit heterogeneity characteristic of these complexes. We present biochemical evidence for the existence of two additional complexes containing trithorax group proteins: a 2 MDa ASH1 complex and a 500 kDa ASH2 complex. These findings suggest that BRM plays a role in chromatin remodeling that is distinct from the function of most other trithorax group proteins.

Amino Acid Sequence↗
PubMed1997

Cdc2 and mitogen-activated protein kinases modulate DNA binding properties of the putative transcriptional regulator Chironomus high mobility group protein I.

Cells of the dipteran insect Chironomus contain a high mobility group protein that is homologous to the mammalian high mobility group proteins I/Y (HMGI/Y). These proteins facilitate the assembly of higher order nucleoprotein complexes. In proliferating cells, >30% of Chironomus HMGI was found to be phosphorylated. The phosphorylation sites were mapped to Ser3, Ser22, and Ser72 and were found to be substrates for the kinases Cdc2 (and mitogen-activated protein (MAP)), MAP, and Ca2+/phospholipid-dependent protein kinase, respectively. In mitotically arrested cells, the extent of phosphorylation at Ser3 increased, whereas phosphorylation at Ser22 remained unchanged. In nondividing cells, phosphorylation at Ser3 and Ser22 was strongly reduced. The DNA binding affinity of Chironomus HMGI was not influenced by single phosphorylation at Ser3 or Ser22. In contrast, phosphorylation at both of these sites resulted in a 10-fold weakening of the binding activity and altered the mode of protein-DNA interaction. Since both human and murine HMGI/Y proteins, similarly to the insect HMGI protein, possess phosphorylation sites for Cdc2 and MAP kinases that intersperse the AT-hook DNA-binding motifs, our results may reflect a general mechanism that regulates the properties and function of this class of putative transcriptional regulators.

Amino Acid Sequence↗
PubMed1998

Protein footprinting reveals specific binding modes of a high mobility group protein I to DNAs of different conformation.

The high mobility group proteins I and Y (HMGI/Y) are abundant components of chromatin. They are thought to derepress chromatin, affect the assembly and activity of the transcriptional machinery, and associate with constitutive heterochromatin during mitosis. HMGI/Y protein molecules contain three potential DNA-binding motifs (AT-hooks), but the extent of contacts between DNA and the entire protein has not been determined. We have used a protein-footprinting procedure to map regions of the Chironomus HMGI protein molecule that are involved in contacts with DNA. We find that in the presence of double-stranded DNA all AT-hook motifs are protected against hydroxyl radical proteolysis. In contrast, only two motifs were protected in the presence of four-way junction DNA. Large regions that flank the AT-hook motifs were found to be strongly protected against proteolysis in complexes with interferon-beta promoter DNA, suggesting amino acid residues outside the AT-hooks considerably contribute to DNA binding.

Animals↗
PubMed1995

Functional interaction between the POU domain protein Tst-1/Oct-6 and the high-mobility-group protein HMG-I/Y.

The POU domain protein Tst-1/Oct-6 is a transcriptional activator of human papovavirus JC virus in transient transfections. Because of its endogenous expression in myelinating glia, Tst-1/Oct-6 might also be an important determinant for the glia specificity of JC virus in vivo. Activation of viral early and late genes depends on the ability of Tst-1/Oct-6 to interact with an AT-rich element within the viral regulatory region. Here, we show that this element not only is bound by Tst-1/Oct-6 but, in addition, serves as a binding site for the high-mobility-group protein HMG-I/Y. In the presence of HMG-I/Y, Tst-1/Oct-6 exhibited an increased affinity for this AT-rich element. The specificity of this effect was evident from the fact that no stimulation of Tst-1/Oct-6 binding was observed on a site that did not allow binding of HMG-I/Y. In addition, both proteins interacted with each other in solution. Direct contacts were identified between the POU domain of Tst-1/Oct-6 and a short stretch of 10 amino acids in the central portion of HMG-I/Y. These results point to an accessory role for HMG-I/Y in the activation of JC viral gene expression by the POU domain protein Tst-1/Oct-6. In agreement with such a role, HMG-Y synergistically supported the function of Tst-1/Oct-6 in transient transfections, measured on the early promoter of JC virus or on an artificial promoter consisting of only a TATA box and the common binding element for Tst-1 and HMG-I/Y.

Base Sequence↗
PubMed1999

Intracellular assembly of Kell and XK blood group proteins.

Kell, a 93 kDa type II membrane glycoprotein, and XK, a 444 amino acid multi-pass membrane protein, are blood group proteins that exist as a disulfide-bonded complex on human red cells. The mechanism of Kell/XK assembly was studied in transfected COS cells co-expressing Kell and XK proteins. Time course studies combined with endonuclease-H treatment and cell fractionation showed that Kell and XK are assembled in the endoplasmic reticulum. At later times the Kell component of the complex was not cleaved by endonuclease-H, indicating N-linked oligosaccharide processing and transport of the complex to a Golgi and/or a post-Golgi cell fraction. Surface-labeling of transfected COS cells, expressing both Kell and XK, demonstrated that the Kell/XK complex travels to the plasma membrane. XK expressed in the absence of Kell was also transported to the cell surface indicating that linkage of Kell and XK is not obligatory for cell surface expression.

Amino Acid Transport Systems, Neutral↗
PubMed1996

Human papillomavirus type 16 E6 protein up-regulates the expression of the high mobility group protein HMG-I(Y) gene in mouse 10T1/2 cells.

Using a differential hybridization technique, we have identified a mouse cellular gene, high mobility group protein HMG-I(Y), whose expression is up-regulated by the E6 protein of human papillomavirus (HPV) type 16. This gene was overexpressed in E6-expressing mouse 10T1/2 cells, but not in G418-resistant 10T1/2 cells. The expression of the HMG-I(Y) gene was up-regulated by the transient expression of E6 from a zinc-inducible human metallothionein-IIA gene promoter. Expression was found to be more efficient at a confluent cell density than at a subconfluent cell density. The up-regulation of HMG-I(Y) gene expression by E6, in particular at a confluent cell density, may be part of an altered genetic program in host cells infected with HPV-16.

Animals↗
PubMed2001

Differential expression of human Polycomb group proteins in various tissues and cell types.

Polycomb group proteins are involved in the maintenance of cellular identity. As multimeric complexes they repress cell type-specific sets of target genes. One model predicts that the composition of Polycomb group complexes determines the specificity for their target genes. To study this hypothesis, we analyzed the expression of Polycomb group genes in various human tissues using Northern blotting and immunohistochemistry. We found that Polycomb group expression varies greatly among tissues and even among specific cell types within a particular tissue. Variations in mRNA expression ranged from expression of all analyzed Polycomb group genes in the heart and testis to no detectable Polycomb group expression at all in bone marrow. Furthermore, each Polycomb group gene was expressed in a different number of tissues. RING1 was expressed in practically all tissues, while HPH1 was expressed in only a few tissues. Also within one tissue the level of Polycomb group expression varied greatly. Cell type-specific Polycomb group expression patterns were observed in thyroid, pancreas, and kidney. Finally, in various developmental stages of fetal kidney, different Polycomb group expression patterns were observed. We conclude that Polycomb group expression can vary depending on the tissue, cell type, and development stage. Polycomb group complexes can only be composed of the Polycomb group proteins that are expressed. This implies that with cell type-specific Polycomb group expression patterns, cell type-specific Polycomb group complexes exist. The fact that there are cell type-specific Polycomb group targets and cell type-specific Polycomb group complexes fits well with the hypothesis that the composition of Polycomb group complexes may determine their target specificity. J. Cell. Biochem. Suppl. 36: 129-143, 2001.

Blotting, Northern↗
PubMed1997

Structural and functional consequences of mutations within the hydrophobic cores of the HMG1-box domain of the Chironomus high-mobility-group protein 1a.

The high-mobility-group protein 1 box domain (HMG1-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. Its structure is dominated by three alpha-helices. The spatial arrangement of these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trp14 of the Chironomus HMG1a protein and have synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletion of the seven N-terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMG1a, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants still retained their ability to bind and bend DNA. Conformational analysis of wild-type cHMG1a and [W14A]cHMG1a showed that the proteins unfold at 2-4 M urea. In contrast, their DNA complexes persisted even at 6-8 M of the denaturant. Multiple contacts between the HMG1-BD and the DNA are probably responsible for the unusual stability of the complexes.

Amino Acid Sequence↗