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Results for “actively transcribed chromatin”

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At least 19 recordsLinked to original sources
PubMed1982

TSH stimulates 32P-labeling of thyroid nuclear HMG 14, a protein associated with actively transcribed chromatin.

Thyroid slices were incubated with 32Pi with or without TSH. 32P-labeling of acid-soluble nuclear proteins was then examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. We found that TSH enhanced the labeling of the high mobility group protein HMG 14, a protein that is preferentially associated with actively transcribed chromatin. This observation suggests that changes in HMG 14 phosphorylation may be involved in mediating TSH-induced effects on the structure and function of active chromatin.

Animals↗
PubMed2025

BET family BRD3 initiates DSB-induced chromatin remodeling with TIP60 to promote R-loop-mediated HR.

Mechanisms for genome stability in actively transcribed regions are essential for cellular homeostasis; however, these mechanisms are poorly understood. Herein, we identify the bromodomain and extraterminal domain (BET) family BRD3 as the genome caretaker in actively transcribed chromatin. We identify the protein network between BRD3 and chromatin remodeler TIP60. During transcription, BRD3 localizes to actively transcribed chromatin through its N-terminal bromodomains. Following DNA double-strand breaks (DSBs) at the actively transcribed chromatin, the C-terminal extraterminal (ET) domain of BRD3 recruits CHD4 via its KIKL-like motifs to replace HP1 with the TIP60 (Tat-interactive protein, 60 kDa) complex, promoting H4K16 acetylation and MBTD1 recruitment, which creates chromatin barriers to 53BP1. This process recruits BRCA1 and R-loop-processing factors to promote R-loop-mediated homologous recombination (HR) and suppress 53BP1 and mutagenic non-homologous end-joining. Our study elucidates the mechanism by which BRD3 initiates DSB-induced chromatin remodeling by CHD4 and TIP60 to promote R-loop-mediated HR on actively transcribed chromatin to maintain genome stability.

Humans↗
PubMed1983

Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin.

Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with 32Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of 32P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of 32P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the 32P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, [32P] HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

Animals↗
PubMed1990

Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells.

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.

Animals↗
PubMed1990

Alternative chromatin structure at CpG islands.

Actively transcribed chromatin is structurally different from bulk inactive chromatin. It has been difficult to define the molecular basis of the difference, however, because purified fractions of active chromatin were not available. We have overcome this problem by releasing oligonucleosomes from the nonmethylated CpG-rich islands (CpG islands) of HeLa cell nuclei using restriction endonucleases. Since CpG islands very often include the promoters and 5' transcribed regions of genes, they represent a model for the "active" chromatin structure. CpG island chromatin differs in three respects from bulk chromatin prepared in the same way: histone H1 is present in very low amounts; histones H3 and H4 are highly acetylated; and nucleosome-free regions are present. Except for the latter regions, the average nucleosomal spacing is similar to that of bulk chromatin.

Base Sequence↗
PubMed1984

Association of poly(adenosine diphosphate ribosylated) nucleosomes with transcriptionally active and inactive regions of chromatin.

We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin. Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column [Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin. DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene. Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib). Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes. Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin. Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes. These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins. However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.

Cell Fractionation↗
PubMed1981

Differential phosphorylation of nuclear nonhistone high mobility group proteins HMG 14 and HMG 17 during the cell cycle.

The phosphorylation of the high-mobility group (HMG) proteins at different stages of the cell cycle was studied in synchronized HeLa cells. HMG proteins were extracted and analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Although the molecular weight distribution of HMGs remains unchanged, their total amounts increase by as much as 20-25% in the G1 and S phases when compared with amounts in G2. However, the most significant finding is that there is a 7-fold increase of 32P incorporation into HMG 14 in the G2 phase compared with that in G1, and a 2-fold increase of 32P incorporation into HMG 17 in early S phase relative to the incorporation in the G1 and G2 stages. In contrast, HMG 1 and HMG 2 are not phosphorylated. The clear demonstration of differential phosphorylation of HMG 14 and 17 at specific stages of the cell cycle warrants a serious consideration of their role in tissue-specific maintenance of the altered chromatin structure characteristic of potentially active or actively transcribed chromatin domains.

Cell Cycle↗
PubMed1984

Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome.

Cobalt-60 gamma radiation has been employed as a means of preferentially damaging actively transcribing chromatin within interphase and metaphase Chinese hamster V79-379 lung fibroblasts. The single-strand size distribution and break frequency of bulk 3H-labeled DNA have been compared to those same parameters for active sequences, i.e., sequences complementary to 125I-labeled poly(A+)RNA. The results show that (a) sequences active during interphase are more sensitive than inactive sequences to single-strand break formation by gamma radiation even when the chromatin is condensed in metaphase, (b) repair of strand breaks in the bulk DNA is slower in metaphase than in interphase cells, but (c) during metaphase, repair is faster in active sequences than in the bulk DNA. Furthermore, this study demonstrates that chromatin structure can be probed within intact cells by a method which circumvents isolation of nuclei or chromatin and the use of exogenous nucleases.

Animals↗
PubMed1985

Interaction of triiodothyronine-receptor complexes with simian virus 40 minichromosomes in monkey kidney CV-1 cells.

Thyroid hormone-responsive tissues contain chromatin-localized receptors that bind to DNA and may associate preferentially with actively transcribed chromatin. To study such receptor-chromatin localization, we have used cultured CV-1 cells permissive for simian virus 40 (SV40), in which viral minichromosomes can be separated from the cellular chromatin. CV-1 cells were found to contain intranuclear thyroid hormone-binding sites with an affinity for T3 and T4 and a site concentration similar to those in other thyroid hormone-responsive tissues. When these cells were infected with SV40 or an SV40-human GH gene recombinant, T3 did not affect SV40 replication, early or late gene transcription, or human GH gene expression. However, in both cases, these infections resulted in the association of about 7.5% of the total specific T3-binding activity with the SV40 minichromosome, representing about 1 receptor molecule/65 minichromosomes and a 10-fold enrichment over the cellular chromatin-associated activity (4.3 fmol/micrograms SV40 minichromosomal DNA vs. 0.43 fmol/micrograms chromosomal DNA); 30% of this could be covalently cross-linked to the minichromosome with dissuccinimidyl suberate. The minichromosomes were also found to be transcriptionally active. Thus, thyroid hormone receptors interact preferentially with the SV40 minichromosome, possibly owing to their tendency to associate with transcriptionally active chromatin. This system provides an alternate approach to study the association of thyroid hormone receptors with defined chromosomal segments.

Animals↗
PubMed1978

Fractionation of chromatin by differential solubility in dilute salt.

Chromatin prepared from the livers of rats was fractionated on the basis of solubility in dilute NaCl. Neither of the fractions obtained was enriched in newly synthesized DNA. The salt-soluble fraction had a higher protein content (usually up to 50%) relative to the DNA, and contained 72% or more of the rapidly synthesized RNA. This RNA was found to be complexed with the salt-soluble deoxyribonucleoprotein, not merely co-solubilized with it. Also, polylysine-binding studies showed that about 70% or more of the nucleic acid phosphates were accessible as compared to about 40% in the unfractionated chromatin. These properties suggested that the soluble fraction was enriched in activity transcribed chromatin. In contrast molecular hybridization studies showed that the complexity of the DNA and its homology with cDNA transcribed from rat-liver polysomal mRNA were the same as those of DNA from unfractionated chromatin, or from the salt-insoluble fraction. This suggests that the criteria commonly accepted as distinguishing between euchromatin and heterochromatin in vitro are not invariably valid.

Animals↗
PubMed1978

Separation of transcriptively active and inactive chromatin. Agarose gel chromatography.

Sheared chromatin fractionated by currently accepted methods of agarose gel exclusion chromatography, undergoes a limited and non-specific aggregation resulting from the high ionic strength and divalent cation concentration of the column elution buffer. Such aggregation causes the artifactual appearance of radioactively labeled, newly synthesized RNA within the column exclusion volume, erroneously suggesting an enrichment for actively transcribed chromatin. Claims for the efficacy of agarose gel exclusion as a method for separating template-active and -inactive chromatin are based largely on assays for active chromatin which rely on localization of specific molecular complexes of chromatin and nascent RNA. Under the conditions employed, the present studies invalidate this assay and thus cast considerable doubt on the agarose gel exclusion method itself.

Animals↗
PubMed1990

Interaction of HMG14 with chromatin.

Neutron scattering has been used to study the interaction of HMG14 with chromatin. Chromatin depleted of H1/H5 was reconstituted separately with histones H1 and H5, and complexed with HMG14. We have also studied the conformation of complexes formed by the binding of HMG14 to nucleosome dimers without linker DNA. Our data on the binding of HMG14 to linkerless nucleosome dimers argue against a significant change in the exit and entry angles of nucleosomal core DNA. Data on the condensation of chromatin into a higher-order structure suggest that there is no dramatic difference between the roles of H1 and H5 in their influence on HMG14 complex formation. However, there is a decrease of about 25% in the mass per unit length of chromatin fibers on HMG14 binding, which is not accompanied by a change in the fiber repeat distance. This is evidence that there are fewer nucleosomes per repeat in HMG14 containing chromatin fibers than in normal chromatin. Alteration of chromatin structure in this manner may be part of the role of HMG14 in actively transcribed chromatin.

Animals↗
PubMed1987

Ca2+/calmodulin regulation of prolactin gene expression.

Despite the extensive literature on the biological actions of Ca2+ and calmodulin, very little is known about their involvement in nuclear functions, e.g., regulation of specific gene expression. To date, the only genes other than prolactin and growth hormone shown to be regulated by perturbations in cell Ca2+ are those coding for two glucose-regulated proteins. However, there is a growing body of indirect evidence for nuclear functions of Ca2+ and calmodulin, and we suspect that other examples of Ca2+-regulated genes will emerge. We have described in this chapter several different experimental approaches which we have employed to examine first whether prolactin gene expression is regulated by changes in cell Ca2+ content, and then to begin searching for the components of the mechanism by which Ca2+ exerts its effects on the prolactin gene. The tentative identification of 56-kDa nuclear matrix protein as both a calmodulin-binding protein and a substrate of a Ca2+-calmodulin-dependent protein kinase suggests that NMP 56 may be a subunit of a multifunctional Ca2+-calmodulin-protein kinase. This enzyme was recently detected in the nuclear matrix fraction of neuronal nuclei, and was shown to phosphorylate a chromatin protein similar to high mobility group protein 17 (HMG 17). Since HMG 17 is associated with actively transcribed chromatin, its phosphorylation in GH3 cells might play a role in the Ca2+-calmodulin-dependent regulation of prolactin gene expression by hormones and growth factors.

Animals↗
PubMed1990

[A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture].

A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.

Amino Acids↗
PubMed1990

Islands of acetylated histone H4 in polytene chromosomes and their relationship to chromatin packaging and transcriptional activity.

The four histones of the nucleosome core particle are all subject to enzyme-catalysed, post-translational acetylation at defined lysine residues in their amino-terminal domains. Much circumstantial evidence suggests a role for this process in modifying chromatin structure and function, but detailed mechanisms have not been defined. To facilitate studies on the functional significance of histone acetylation, we have prepared antibodies specific for the acetylated isoforms of histone H4. Because of the extreme evolutionary conservation of H4, these antisera can be applied to a wide variety of organisms and experimental systems. In the present study we have used polytene chromosomes from the salivary glands of larvae of the midge Chironomus to examine the distribution of acetylated H4 in interphase chromatin. By indirect immunofluorescence, antisera to acetylated H4 labeled the four Chironomus chromosomes with reproducible patterns of sharply defined, fluorescent bands. An antiserum to non-acetylated H4 gave a completely different, more-diffuse labelling pattern. Thus, there are defined regions, or islands, in the interphase genome that are enriched in acetylated H4. Double-labelling experiments with two antisera specific for H4 molecules acetylated at different sites, showed that each antiserum gave the same banding pattern. Immunolabelling patterns were not dependent on the pattern of phase-dense bands characteristic of these chromosomes; strongly labelled regions could correspond to phase-dense bands (i.e. condensed chromatin), to interbands or, frequently, to band-interband junctions. Immunogold electron microscopy confirmed the immunofluorescence results and showed further that regions of relatively high labelling could be either transcriptionally active or quiescent, as judged by the presence or absence of ribonucleoprotein particles. Two rapidly transcribed genes on chromosome 4 of Chironomus form characteristic 'puffs', the Balbiani rings BRb and BRc. The antiserum to non-acetylated H4 gave diffuse labelling throughout these puffs, demonstrating the continued presence of this histone in these transcriptionally active regions. Antisera to acetylated H4 strongly labelled the boundaries of BRb and BRc, and revealed clearly defined islands of increased H4 acetylation just within the expanded chromatin of the puffs. Labelling within the central region of each puff was much less intense. A similar pattern was observed in puffs on other chromosomes. Thus, increased H4 acetylation is not found throughout actively transcribed chromatin but occurs only at defined sites, possibly in the non-transcribed flanking regions. H4 acetylation is clearly not required for the passage of RNA polymerase through the nucleosome and we speculate that its role may be to facilitate the binding to DNA of polymerases and other proteins prior to the onset of transcription and possibly replication.

Acetylation↗
PubMed1984

High-mobility group chromosomal proteins of wheat.

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.

Amino Acids↗
PubMed1985

Hormonal control of the phosphorylation of histones, HMG proteins and other nuclear proteins.

Hormone-dependent phosphorylation and dephosphorylation of nuclear proteins may play an important part in regulating nuclear function and specific gene expression. Some progress has been made in identifying specific nuclear proteins whose phosphorylation is affected by specific hormones; however, relatively little is known about the regulatory mechanisms involved, or about the molecular consequences of increased or decreased phosphorylation. It is suspected--but not yet proved--that cAMP-dependent effects on transcription are mediated at least partly by increases in nuclear cAMP-dependent protein kinase (A-kinase) activity, and consequent increases in the phosphorylation of specific chromatin proteins. In several instances, increased phosphorylation has been found to precede or correlate with cAMP-mediated induction of specific gene products. Several chromatin proteins are susceptible to cAMP-dependent phosphorylation in vivo, including histones H1 and H3, the high mobility group protein HMG 14 (which is preferentially associated with actively transcribed chromatin), and at least three other basic nonhistone proteins. The A-kinase phosphorylation sites of the majority of H1, H3 and HMG 14 molecules in chromatin appear to be inaccessible to A-kinase in vivo; nothing is known about the factors determining their accessibility, which may be tightly regulated and may vary significantly from cell to cell and tissue to tissue. Many hormone-induced changes in nuclear protein phosphorylation may be cAMP-independent. cAMP-independent mechanisms could involve a variety of nuclear enzymes including, for example, cGMP-dependent, Ca2+/calmodulin-dependent, Ca2+/phospholipid-dependent and polyamine-dependent protein kinases. So far, however, there is little solid evidence in support of a role for any specific cAMP-independent protein kinase in mediating hormonally induced increases in the phosphorylation of specific, identified nuclear proteins.

Animals↗
PubMed1984

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes.

An antibody was raised against "high mobility group" nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. - Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. - It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.

Animals↗