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M Bustin

Publications and source records attributed to M Bustin.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of transgenic mice with an increased content of chromosomal protein HMG-14 in their chromatin.

Chromosomal protein HMG-14 is a ubiquitous nuclear protein that may modulate the chromatin structure of transcriptionally active genes. To gain insights into the cellular function of the HMG-14 protein, we generated two transgenic mouse lines carrying either two or six copies of the human HMG-14 gene. The transgenic mice express human HMG-14 mRNA and protein in all tissues examined at a level reflecting the increased gene dosage, suggesting that the HMG14 transgene contains all the control regions necessary for regulated gene expression. Expression of the human HMG-14 protein does not alter the expression of the endogenous mouse HMG-14 protein or its close homolog, protein HMG-17. The intracellular distribution of the exogenous human protein is indistinguishable from that of the endogenous mouse protein, resulting in a three-fold increase in the level of the chromatin-bound HMG-14. The transgenic mice had a higher incidence of epithelial cysts in their thymus than did control animals. We conclude that the cellular levels of HMG-14/-17 are determined by gene copy number, that the DNA fragment containing the gene and about 1,000 bp flanking its 5' and 3' ends contain most of the elements necessary for gene expression, that the upper limits of HMG-14 in chromatin are not stringently regulated, and that a three-fold increase in chromatin-bound protein cause only mild phenotypic changes in the transgenic mice.

Animals↗
PubMed1995

Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

Amino Acid Sequence↗
PubMed1994

The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.

Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.

Alanine↗
PubMed1994

Stimulation of RNA polymerase II elongation by chromosomal protein HMG-14.

The high-mobility group protein 14 (HMG-14) is a non-histone chromosomal protein that is preferentially associated with transcriptionally active chromatin. To assess the effect of HMG-14 on transcription by RNA polymerase II, in vivo-assembled chromatin with elevated amounts of HMG-14 was obtained. Here it is shown that HMG-14 enhanced transcription on chromatin templates but not on DNA templates. This protein stimulated the rate of elongation by RNA polymerase II but not the level of initiation of transcription. These findings suggest that the association of HMG-14 with nucleosomes is part of the cellular process involved in the generation of transcriptionally active chromatin.

Chromatin↗
PubMed1994

The footprint of chromosomal proteins HMG-14 and HMG-17 on chromatin subunits.

The position of chromosomal proteins HMG-14 and HMG-17 in nucleosome cores and in chromatosomes lacking linker histones has been mapped by hydroxyl radical footprinting. Both the nucleosome core and the H1/H5 depleted chromatosome can specifically bind two molecules of HMG-14/-17. The path of HMG-14 on the surface of chromatin subunits is indistinguishable from that of HMG-17. The bound HMGs protect the DNA from hydroxyl radical cleavage 25 base-pairs from the end of the DNA in nucleosome cores and in each of the two major grooves of the DNA flanking the nucleosomal dyad axis. Thus, in both cores and H1/H5-depleted chromatosomes the proteins bridge two adjacent DNA strands on the surface of the particles. The sites occupied by HMG near the end of the chromatosome-length particles are distinct from those occupied by the H1/H5 linker histones. In the region of the dyad axis the binding sites of HMGs overlap those of the linker histones. The placement of HMG-14/-17 near the nucleosomal dyad axis raises the possibility that interactions between histone H1 and HMGs may affect the transcriptional potential of chromatin.

Amino Acid Sequence↗
PubMed1994

Sera from JRA patients contain antibodies against a defined epitope in chromosomal protein HMG-17.

Autoantibodies against the nonhistone nucleosomal protein HMG-17 have been detected in a high percentage of ANA-positive patients with pauciarticular-onset JRA4. Here we report on the epitope mapping of the HMG-17 autoantigen with a set of overlapping and nested synthetic peptides spanning the entire amino acid sequence of the human HMG-17 protein. Competition ELISA experiments defined a proline and lysine rich octapeptide PKPEPKPK as the major epitope recognized by more than 70% of the HMG-17 positive JRA sera. Point mutations introduced in the autoimmune peptide determined the amino acid residues important for autoantibody recognition. Computer based sequence comparison shows close homology between the HMG-17 autoimmune epitope and certain infectious organisms, supporting the possibility that molecular mimicry is an important factor in the etiology of JRA.

Amino Acid Sequence↗
PubMed1993

Aberrant expression of high mobility group chromosomal protein 14 affects cellular differentiation.

High mobility group (HMG) 14 is a ubiquitous chromosomal protein that binds specifically to nucleosomal DNA and may be involved in a process that confers distinct properties to the chromatin structure of transcriptionally active genes. To explore the involvement of this protein in regulation of gene expression, we studied the effect of aberrant expression of HMG-14 protein on cellular differentiation. We produced stably transfected C2C12 mouse myoblasts expressing the human HMG-14 protein under the control of the mouse mammary tumor virus promoter. Transformed colonies retained their potential do differentiate into myotubes. Induction of human HMG-14 expression by dexamethasone inhibited the myogenic process. Revertant colonies, which lost the ability to express human HMG-14, regained the ability to differentiate into myotubes. Inhibition of myoblast differentiation by aberrantly expressed HMG-14 correlated with down-regulation of myogenic determination factors. The results suggest that proper cellular differentiation requires regulated expression of HMG-14 protein and are consistent with the possibility that this protein may be involved in gene regulation.

Animals↗
PubMed1993

A signature for the HMG-1 box DNA-binding proteins.

A diverse group of DNA-binding regulatory proteins share a common structural domain which is homologous to the sequence of a highly conserved and abundant chromosomal protein, HMG-1. Proteins containing this HMG-1 box regulate various cellular functions involving DNA binding, suggesting that the target DNA sequences share a common structural element. Members of this protein family exhibit a dual DNA-binding specificity: each recognizes a unique sequence as well as a common DNA conformation. The highly conserved HMG-1/-2 proteins may modulate the binding of other HMG-1 box proteins to bent DNA. We examine the structural and functional relationships between the proteins, identify their signature and describe common features of their target DNA elements.

Amino Acid Sequence↗
PubMed1993

Deposition of chromosomal protein HMG-17 during replication affects the nucleosomal ladder and transcriptional potential of nascent chromatin.

A cell-free system from Xenopus eggs was used to study the role of chromosomal protein HMG-17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG-17 protein to the extracts, which do not contain structural homologs of the HMG-14/-17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG-17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG-17 is incorporated into nucleosomes during chromatin assembly.

Animals↗
PubMed1993

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 17 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.

Animals↗
PubMed1993

Identification and genetic mapping of the murine gene and 20 related sequences encoding chromosomal protein HMG-17.

HMG-17 is an abundant, nonhistone chromosomal protein that binds preferentially to nucleosomal core particles of mammalian chromatin. The human gene for HMG-17 has been localized to Chromosome (Chr) 1p, but the murine gene has not been previously mapped. Here we identify the murine functional gene, Hmg17, from among more than 25 related sequences (probably processed pseudogenes) and show that it is located on mouse Chr 4, in a region known to have conserved linkage relationships with human Chr 1p. We also report the map locations of 20 additional Hmg17-related sequences on mouse Chrs 1, 2, 3, 5, 7, 8, 9, 13, 15, 16, 17, 18, and X. The multiple, dispersed members of the Hmg17 multigene family can be detected efficiently with a single cDNA probe and provide useful markers for genetic mapping studies in mice.

Animals↗
PubMed1993

Evolutionarily conserved motifs and protein binding elements in the 5' region of the chromosomal protein HMG-14 gene.

Although the structure of several genes coding for chromosomal proteins HMG-14 and HMG-17 has been determined, the mechanisms regulating the expression of these genes has not yet been examined. Toward this goal, we have cloned and sequenced a fragment containing the first three exons and 956 bp upstream from the start of translation of the functional mouse HMG-14 gene. Comparison of this sequence to the known sequence of the human HMG-14 gene revealed the presence of five distinct blocks of high sequence identity flanking the start of transcription and the CAAT box. DNase I and mobility-shift analysis identified a DNA region, downstream from the start of transcription, which may be important for the formation of a stable protein-DNA complex. Affinity chromatography on columns containing oligonucleotides corresponding to this sequence indicates that this region is a protein binding site.

Animals↗
PubMed1992

Nucleosome core binding region of chromosomal protein HMG-17 acts as an independent functional domain.

Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date.

Amino Acid Sequence↗
PubMed1992

The transcriptionally-active MMTV promoter is depleted of histone H1.

We have used an ultraviolet light cross-linking and immunoadsorption assay to demonstrate that histones H1 and H2B are bound to the repressed MMTV promoter. Hormone activation results in reduced H1 content with little or no change in H2B. High resolution analysis of the glucocorticoid-inducible DNaseI hypersensitive region demonstrates an NF-1 footprint as well as specific sites of enhanced cleavage on nucleosome B and in the nucleosome B/nucleosome A linker. These results are consistent with a model in which binding of the glucocorticoid receptor to glucocorticoid regulatory elements on the surface of nucleosome B induces a chromatin transition that is necessary for transcription factor (NF-1 and TFIID) recruitment to the MMTV promoter. We hypothesize that association of histone H1 with important cis-elements on the promoter masks these sites, and glucocorticoid-induced displacement of H1 is necessary to expose factor binding sites at the 3' edge of nucleosome B, in the nucleosome B/nucleosome A linker and at the 5' edge of nucleosome A.

Base Sequence↗
PubMed1992

Autoantibodies to the chromosomal protein HMG-17 in juvenile rheumatoid arthritis.

OBJECTIVE: To determine the antibody profiles in sera from patients with juvenile rheumatoid arthritis (JRA). METHODS: Immunoblotting using nuclear extracts and recombinant high-mobility group (HMG) nonhistone chromosomal proteins. RESULTS: Antibodies directed against HMG-17 were found in 47% of antinuclear antibody (ANA)-positive patients with pauciarticular-onset JRA and in 16% of ANA-positive patients with polyarticular-onset JRA. HMG-17 values of 6% and 8%, respectively, were detected in ANA-negative patients with JRA and in those with nonrheumatic diseases. CONCLUSION: There is evidence for a high prevalence of anti-HMG-17 antibodies in sera of patients with pauciarticular-onset JRA.

Adolescent↗
PubMed1992

Genetic mapping of the murine gene and 14 related sequences encoding chromosomal protein HMG-14.

The high-mobility-group chromosomal protein HMG-14 preferentially binds to nucleosomal core particles of mammalian chromatin and may modulate the chromatin configuration of transcriptionally active genes. The human gene for HMG-14 has been localized to the Down syndrome region of Chromosome (Chr) 21 and may be involved in the etiology of this syndrome. Here we show, by means of genetic linkage analysis of interspecific and intersubspecific backcross mice, that the murine functional gene, Hmg14, is located on the distal end of mouse Chr 16, a region known to have conserved synteny with human Chr 21. In addition to the functional gene for HMG-14, both human and mouse genomes contain many related sequences that are probably processed pseudogenes. Here we map the locations of 14 Hmg14-related sequences in two mouse genomes. The 14 mapped loci are widely dispersed on ten chromosomes (Chrs 3, 5, 7, 9, 11, 12, 16, 17, 19, and X) and can be detected efficiently with a single cDNA probe. Thus, the Hmg14 multigene family is well suited to serve as genetic markers for other linkage studies in mice.

Animals↗
PubMed1992

Developmental regulation of chromatin composition during mouse embryogenesis: somatic histone H1 is first detectable at the 4-cell stage.

We have examined the distribution of histone H1 in oocytes and preimplantation embryos of the mouse, using a polyclonal antibody raised against the histone H1 subtypes present in somatic cells. Immunofluorescence and immunoblotting analyses failed to detect somatic histone H1 in germinal vesicle (GV)-stage oocytes. In contrast, somatic histone H1 was detectable by immunofluorescence in the nuclei of GV oocytes previously injected with histone H1 as well as the nuclei of ovarian granulosa cells, and by immunoblotting in 8-cell embryos. 1- and 2-cell embryos examined by immunofluorescence did not contain detectable somatic histone H1. At the early 4-cell stage (54-56 hours post-hCG), 5 of 52 embryos contained somatic histone H1 in one or more nuclei. By the late 4-cell stage (66-68 hours post-hCG), however, 58 of 62 embryos contained somatic histone H1. In 8-cell embryos, morulae and blastocysts, all nuclei contained somatic histone H1 in every case. When embryos were exposed to the transcriptional inhibitor, alpha-amanitin, beginning at the late 2-cell stage, they cleaved to the 4-cell stage but fewer than 10% developed histone H1 immunoreactivity. When treatment began at the early 4-cell stage, the embryos that remained at the 4-cell stage in the presence of the drug developed histone H1 immunoreactivity in half of the cases. Embryos that reached the 5- to 8-cell stage in the presence of the drug developed histone H1 immunoreactivity in every case.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗