Search PubMed⌕ Search

Biomedical subjects

M Bustin

Publications and source records attributed to M Bustin.

At least 37 records · Page 2Linked to original sources
PubMed1998

Dynamic relocation of chromosomal protein HMG-17 in the nucleus is dependent on transcriptional activity.

Chromosomal proteins HMG-14/-17 are nucleosomal binding proteins, which alter the structure of the chromatin fiber and enhance transcription, but only from chromatin templates. Here we show that in tissue culture cells, HMG-17 protein colocalizes with sites of active transcription. Incubation of permeabilized cells with a peptide corresponding to the nucleosomal binding domains of HMG-14/-17 specifically arrested polymerase II-dependent transcription. In these cells the peptide displaces HMG-17 from chromatin and reduces the cellular content of the protein. These results suggest that the presence of HMG-14/-17 in chromatin is required for efficient polymerase II transcription. In non-permeabilized, actively transcribing cells, the protein is dispersed in a punctate pattern, throughout the nucleus. Upon transcriptional inhibition by alpha-amanitin or actinomycin D, the protein gradually redistributes until it localizes fully to interchromatin granule clusters, together with the splicing factor SC35. The results suggest that the association of HMG-17 with chromatin is dynamic rather than static, and that in the absence of transcription, HMG-17 is released from chromatin and accumulates in interchromatin granule clusters. Thus, the intranuclear distribution of chromosomal proteins which act as architectural elements of chromatin structure may be dynamic and functionally related to the transcriptional activity of the cell.

Binding Sites↗
PubMed1998

DNA damage activates p53 through a phosphorylation-acetylation cascade.

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

Acetylation↗
PubMed1998

The chromatin unfolding domain of chromosomal protein HMG-14 targets the N-terminal tail of histone H3 in nucleosomes.

Nonhistone chromosomal protein HMG-14 is a nucleosomal binding protein that unfolds the higher-order chromatin structure and enhances the transcriptional potential of chromatin, but not that of DNA. Both the transcriptional enhancement and the chromatin unfolding activities of HMG-14 are mediated through the C-terminal region of the protein. Here we study the molecular interactions of both this region and the N-terminal region of HMG-14 with nucleosome cores. By protein photocrosslinking we demonstrate that the N-terminal domain of HMG-14 targets a restricted region in histone H2B, whereas the C-terminal chromatin unfolding domain of HMG-14 targets a restricted region in the N terminus of histone H3. The N-terminal regions of the core histones are involved in the folding of oligonucleosomes and are the target of various activities associated with chromatin unfolding and transcriptional activation. We suggest that specific interactions between the C-terminal domain of HMG-14 and the N-terminal tail of histone H3 reduce the compaction of chromatin. These findings provide insights into the molecular mechanism whereby HMG-14/-17 proteins reduce the repressive effect of chromatin, and they also broaden the scope of the molecular interactions involving the N termini of the core histones in nucleosomes.

Binding Sites↗
PubMed1998

Stimulation of replication efficiency of a chromatin template by chromosomal protein HMG-17.

The effect of chromosomal protein HMG-17 on the replication of a chromatin template was studied with minichromosomes containing the SV40 origin of replication. The minichromosomes were assembled from M13 DNA in Xenopus egg extracts in either the absence or presence of HMG-17. Structural data show that HMG-17 was efficiently incorporated into the chromatin and induced an extended chromatin structure. Using an in vitro SV40 replication system, we find that minichromosomes containing HMG-17 replicate with higher efficiency than minichromosomes deficient of HMG-17. The replicational potential of chromatin was enhanced only when HMG-17 was incorporated into the template during, but not after, chromatin assembly. HMG-17 stimulated replication only from a chromatin template, but not from protein-free DNA. Thus, HMG-17 protein enhances the rate of replication of a chromatin template by unfolding the higher order chromatin structure and increasing the accessibility of target sequences to components of the replication machinery.

Animals↗
PubMed1998

High mobility group protein-1 (HMG-1) is a unique activator of p53.

The binding of p53 protein to DNA is stimulated by its interaction with covalent as well as noncovalent modifiers. We report the identification of a factor from HeLa nuclear extracts that activates p53 DNA binding. This factor was purified to homogeneity and identified as the high mobility group protein, HMG-1. HMG-1 belongs to a family of highly conserved chromatin-associated nucleoproteins that bend DNA and facilitate the binding of various transcription factors to their cognate DNA sequences. We demonstrate that recombinant His-tagged HMG-1 enhances p53 DNA binding in vitro and also that HMG-1 and p53 can interact directly in vitro. Unexpectedly, HMG-1 also stimulates DNA binding by p53Delta30, a carboxy-terminally deleted form of the protein that is considered to be constitutively active, suggesting that HMG-1 stimulates p53 by a mechanism that is distinct from other known activators of p53. Finally, using transient transfection assays we show that HMG-1 can increase p53 and p53Delta30-mediated transactivation in vivo. HMG-1 promotes the assembly of higher order p53 nucleoprotein structures, and these data, along with the fact that HMG-1 is capable of bending DNA, suggest that HMG-1 may activate p53 DNA binding by a novel mechanism involving a structural change in the target DNA.

DNA↗
PubMed1997

Clusters of nucleosomes containing chromosomal protein HMG-17 in chromatin.

Chromosomal proteins HMG-14 and HMG-17 are nucleosome binding proteins which can function as architectural elements to alter the structure of the chromatin fiber and enhance transcription from chromatin templates. Here we study the spatial organization of these HMG proteins in the nucleus and the distribution of nucleosomes containing HMG-17 in the chromatin fiber. By confocal immunofluorescence microscopy we find that HMG-14/17 proteins are clustered into foci containing either HMG-14 or HMG-17. These results suggest that HMG-14/17 proteins segregate into distinct nuclear domains. Indeed, immunofractionation of defined length oligonucleosomes, with affinity pure antibodies to HMG-17, indicates that oligonucleosomes containing HMG-17 are devoid of HMG-14. Quantitative analysis indicates that in cellular chromatin nucleosomes containing HMG-17 are clustered. The average size of the cluster is six contiguous HMG-17-containing nucleosomes. The nucleosomes in this cluster contain either two or zero molecules of HMG-17 and a complete set of four core histones. We suggest that HMG-14/17 proteins modify the nucleosomal organization of the 30 nm chromatin fiber, to unfold the higher order chromatin structure and facilitate access to the underlying DNA sequence. Clustering of architectural elements, such as HMG proteins and linker histone subtypes into distinct domains, may lead to structural and functional heterogeneity along the chromatin fiber.

Animals↗
PubMed1997

The histone acetyltransferase activity of human GCN5 and PCAF is stabilized by coenzymes.

Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases.

Acetyl Coenzyme A↗
PubMed1997

V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins.

Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.

DNA↗
PubMed1997

Enhancement of the transcription potential of nascent chromatin by chromosomal proteins HMG-14/-17 is coupled to nucleosome assembly and not DNA synthesis.

We have previously demonstrated that in Xenopus egg extracts, which support DNA strand synthesis and chromatin assembly, incorporation of chromosomal proteins HMG-14/-17 into nascent nucleosomes increases the transcriptional potential of a chromatin template carrying the Xenopus 5S RNA gene. Here we use the single-stranded and double-stranded forms of a plasmid carrying a 5S RNA maxigene, to test whether the effect of HMG-14/-17 on transcription requires DNA synthesis and whether these proteins will affect transcription through a region containing nucleosomes. We find that most of the transcripts were about 350 nucleotides long, suggesting that HMG-14/-17 enhance transcription through a region that could contain nucleosomes. HMG-14/-17 enhance transcription of chromatin templates assembled onto double-stranded DNA, in the absence of DNA synthesis. Single-round transcription assays suggest that HMG-14/-17 increase transcription from templates assembled onto both single- and double-stranded DNA by increasing the specific activity, and not the number, of transcriptionally active templates. We conclude that the effect of HMG-14/-17 on the transcriptional potential of chromatin is linked to nucleosome assembly and is not linked to DNA synthesis.

Animals↗
PubMed1997

Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14.

Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes.

Amino Acid Sequence↗
PubMed1997

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Animals↗
PubMed1997

Chromatin modifications during oogenesis in the mouse: removal of somatic subtypes of histone H1 from oocyte chromatin occurs post-natally through a post-transcriptional mechanism.

We examined the distribution of the somatic subtypes of histone H1 and the variant subtype, H1(0), and their encoding mRNAs during oogenesis and early embryogenesis in the mouse. As detected using immunocytochemistry, somatic H1 was present in the nuclei of oocytes of 18-day embryos. Following birth, however, somatic H1 became less abundant in both growing and non-growing oocytes, beginning as early as 4 days of age in the growing oocytes, and was scarcely detectable by 19 days. Together with previous results, this defines a period of time when somatic H1 is depleted in oocytes, namely, from shortly after birth when the oocytes are at prophase I until the 4-cell stage following fertilization. At the stages when somatic H1 was undetectable, oocyte nuclei could be stained using an antibody raised against histone H1(0), which suggests that this may be a major linker histone in these cells. In contrast to the post-natal loss of somatic H1 protein, mRNAs encoding four (H1a, H1b, H1d, H1e) of the five somatic subtypes were present, as detected using RT-PCR in growing oocytes of 9-day pups, and all five subtypes including H1c were present in fully grown oocytes of adults. All five subtypes were also present in embryos, both before and after activation of the embryonic genome. mRNA encoding H1(0) was also detected in oocytes and early embryos. Whole-mount in situ hybridization using cloned H1c and H1e cDNAs revealed that the mRNAs were present in the cytoplasm of oocytes and 1-cell embryos, in contrast to the sea urchin early embryo where they are sequestered in the cell nucleus. We suggest that, as in many somatic cell types, the chromatin of mouse oocytes becomes depleted of somatic H1 and relatively enriched in histone H1(0) postnatally, and that somatic H1 is reassembled onto chromatin in cleavage-stage embryos. The post-natal loss of somatic H1 appears to be regulated post-transcriptionally by a mechanism not involving nuclear localization.

Animals↗
PubMed1996

Chromosomal proteins HMG-14 and HMG-17 are synthesized throughout the S-phase in Burkitt's lymphoma.

Here we test whether the targeting of chromosomal proteins HMG-14 and HMG-17 to transcriptionally active regions is due to coincidence of the synthesis of these proteins with the replication and assembly of active chromatin. Cells from the Burkitt's lymphoma line CA46 were synchronized with mimosine. The mRNA levels and the protein synthesis rates of HMG-14/-17 were determined at various stages during the S phase. The expression pattern of HMG-14/-17 was essentially constant throughout this phase of the cell cycle. The half-life for HMG-14/-17 protein corresponded to the doubling time of the cells. These results indicate that HMG-14/-17 are synthesized throughout S-phase and persist through the cell cycle. Targeting of HMGs to active genes is not simply due to the timing of their synthesis during S phase.

Antineoplastic Agents↗
PubMed1995

Assembly of somatic histone H1 onto chromatin during bovine early embryogenesis.

We have examined the distribution of somatic histone H1 in bovine oocytes and preimplantation embryos, using an antibody that recognizes histone H1 subtypes present in somatic cells. Immunoreactive H1 was not detectable on the chromosomes of metaphase II of meiosis nor in the nuclei of early cleavage-stage embryos. In most embryos, immunoreactive H1 was assembled onto embryonic chromatin during the fourth to sixth cell cycle after fertilization. No immunoreactive somatic histone H1 was detected, however, when embryos were incubated in the presence of alpha-amanitin beginning early during the fourth cell cycle. These results indicate that somatic subtypes of histone H1 are assembled onto embryonic chromatin in a developmentally regulated manner that requires embryonic transcription. Aphidicolin, an inhibitor of DNA replication, also inhibited the assembly of somatic histone H1 onto chromatin when present at early stages of the 4th cell cycle. It is suggested that, because the bulk of histone gene expression in proliferating cells occurs during DNA replication, expression of genes encoding immunoreactive H1 is inhibited in embryos blocked before or soon after entering the S-phase. These findings on the control of somatic histone H1 assembly onto chromatin in cattle show a remarkable similarity to those found in the mouse. Such evolutionary conservation suggests that the somatic histone H1 complement of chromatin may regulate critical aspects of chromatin activity during mammalian oogenesis or early embryogenesis.

Animals↗
PubMed1995

Homodimers of chromosomal proteins HMG-14 and HMG-17 in nucleosome cores.

In this work, we report that nucleosome core particles interact with an equimolar mixture of the chromosomal proteins HMG-14 and HMG-17 to form, exclusively, complexes containing two molecules of either HMG-14 or HMG-17 (homodimers). Analysis of the binding of various mixtures of wild-type proteins and their deletion mutants indicates that homodimer formation is not dependent on contacts between the nucleosome-bound HMG-14/-17 proteins themselves. We suggest that HMG-14/-17 proteins in nucleosomes cross-talk by inducing specific allosteric transitions in the chromatin subunit.

Allosteric Regulation↗
PubMed1995

Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcription complexes.

The role of chromosomal proteins HMG-14 and HMG-17 in the generation of transcriptionally active chromatin was studied in a Xenopus laevis egg extract which supports complementary DNA strand synthesis and chromatin assembly. Chromosomal proteins HMG-14/HMG-17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. The transcriptional potential of chromatin was enhanced only when these proteins were incorporated into the template during, but not after, chromatin assembly. HMG-14 and HMG-17 stimulate transcription by increasing the activity, and not the number, of transcribed templates. They unfold the chromatin template without affecting the nucleosomal repeat or decreasing the content of histone B4. We suggest that HMG-14/HMG-17 enhance transcription by inducing an extended conformation in the chromatin fiber, perhaps due to interactions with histone tails in nucleosomes. By disrupting the higher order chromatin structure HMG-14/HMG-17 increase the accessibility of target sequences to components of the transcriptional apparatus.

Amino Acid Sequence↗
PubMed1995

The HMG-14/-17 chromosomal protein family: architectural elements that enhance transcription from chromatin templates.

Chromosomal proteins HMG-14 and HMG-17 enhance the transcriptional potential of chromatin when incorporated into nucleosomes during, but not after, chromatin assembly on replicating DNA. Two molecules of either HMG-14 or HMG-17 can bind to nucleosome cores, independently of the underlying DNA sequence, in a cooperative fashion to limit nucleosome mobility and stabilize the structure of the nucleosome core without stabilizing the higher order chromatin structure. By modifying the structure of nucleosomes, the proteins affect the local structure of the chromatin fiber leading to an increase in the rate of transcriptional elongation but not initiation. We suggest that HMG-14/-17 are architectural elements which assist in the assembly of an unfolded chromatin fiber thereby decreasing the repressive activity of histones and facilitating transcriptional processes.

Animals↗