Ultrastructural organization of yeast chromatin.

The ultrastructural organization of yeast chromatin was examined in Miller spread preparations of samples prepared from spheroplasts or isolated nuclei of Saccharomyces cerevisiae. Micrographs from preparations dispersed in 1 mM Tris (pH 7.2) illustrate that the basic chromatin fiber in yeast exists in two ultrastructurally distinct conformations. The majority (up to 95%) of the chromatin displays a beaded nucleosomal organization, although adjacent nucleosomes are separated by internucleosomal linkers of variable lengths. Ribonucleoprotein (RNP) fibrils are only occasionally associated with chromatin displaying the conformation. The remaining 5-10% of the chromatin appears to be devoid of discrete nucleosomes and has a smooth contour with a fiber diameter of 30-40 A. Transcriptional units, including putative ribosomal precursor RNA genes, defined by the presence of nascent RNP fibrils are restricted to chromatin displaying this smooth morphology. Chromatin released from nuclei in the presence of 5 mM Mg++ displays higher-order chromatin fibers, 200-300 A in diameter, these fibers appear to be arranged in a manner than reflects the two forms of the basic chromatin fiber.

Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Covalent modifications of histones: expression from chromatin templates.

Recent advances highlight the involvement of histone acetyltransferases in transcriptional activation and histone deacetylases in transcriptional repression. Transcription factors loaded onto regulatory DNA elements may recruit either coactivators with histone acetyltransferase activity or corepressors associated with histone deacetylases. The recruited enzymes may either acetylate or deacetylate proximal nucleosomal histones or nonhistone chromosomal proteins.

Molecular and biochemical basis of intermediate maple syrup urine disease. Occurrence of homozygous G245R and F364C mutations at the E1 alpha locus of Hispanic-Mexican patients.

Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.

5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA.

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.

ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors.

t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.

Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor.

The human estrogen receptor alpha (ER alpha) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER- (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER- but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

Potential role of estrogen receptor alpha (ERalpha) phosphorylated at Serine118 in human breast cancer in vivo.

Post-translational modifications of proteins are known to be important in protein activity and ERalpha is known to be phosphorylated at multiple sites within the protein. The exact function of site-specific phosphorylation in ERalpha is unknown, although several hypotheses have been developed using site-directed mutagenesis and cell culture models. Targeting the ERalpha at the level of such post-translational modification pathways would be a new and exciting approach to endocrine therapy in breast cancer, but adequate knowledge is lacking with regard to the relevance of site-specific phosphorylation in ERalpha in human breast cancer in vivo. Recently, antibodies to P-Serine(118)-ERalpha and P-Serine(167)-ERalpha, two major sites of phosphorylation in ERalpha, have become available and some in vivo data are now available to complement studies in cells in culture. However, the in vivo data are somewhat contradictory and limited by the small cohorts used and the lack of standard well-characterized reagents and protocols.

Signal transduction pathways and the modification of chromatin structure.

Mechanical and chemical signaling pathways are involved in transmitting information from the exterior of a cell to its chromatin. The mechanical signaling pathway consists of a tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and karyoskeleton. The tissue matrix system governs cell and nuclear shape and forms a structural and functional connection between the cell periphery and chromatin. Further, this mechanical signaling pathway has a role in controlling cell cycle progression and gene expression. Chemical signaling pathways such as the Ras/mitogen-activated protein kinase (MAPK) pathway can stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. Activation of the Ras/MAPK pathway results in the alteration of chromatin structure and gene expression. The tissue matrix and chemical signaling pathways are not independent and one signaling pathway can affect the other. In this chapter, we will review chromatin organization, histone variants and modifications, and the impact that signaling pathways have on chromatin structure and function.

Altered nuclear matrix protein profiles in oncogene-transformed mouse fibroblasts exhibiting high metastatic potential.

The nuclear matrix provides the structural support of the nucleus and is involved in various cellular functions of the nucleus. Nuclear matrix proteins, which are both tissue and cell type specific, are altered with transformation and state of differentiation. Here, nuclear matrix protein profiles of oncogene-transformed mouse fibroblasts with various degrees of metastatic activity were analyzed using two-dimensional gel electrophoresis. This study shows that as the metastatic potential increases, similar nuclear matrix protein profiles are associated with each increase regardless of transformation agent.

Nuclear matrix proteins associated with DNA in situ in hormone-dependent and hormone-independent human breast cancer cell lines.

The nuclear matrix is a dynamic RNA-protein complex that organizes chromatin and regulates nuclear DNA metabolism. Nuclear matrix proteins informative in the diagnosis of cancer have been identified. Here, the nuclear matrix breast cancer proteins (NMBCs) cross-linked to nuclear DNA in situ with cisplatin in human breast cancer cell lines were analyzed by two-dimensional gel electrophoresis. We identified NMBCs that were differentially associated with nuclear DNA of hormone-dependent and -independent breast cancer cell lines. Three DNA cross-linked NMBCs were found to be exclusive to estrogen receptor-positive, hormone-dependent breast cancer cells, whereas two NMBCs were observed only in estrogen receptor-negative, hormone-independent breast cancer cells. Changes in these NMBCs were observed when hormone-dependent breast cancer cells became hormone independent. Furthermore, we show that the intermediate filament protein vimentin is associated with the nuclear DNA of MDA-MB-231 breast cancer cells, an estrogen receptor-negative, hormone-independent breast cancer cell line with high metastatic potential. These nuclear matrix DNA-binding proteins may play important roles in breast tumorigenesis.

Altered profiles in nuclear matrix proteins associated with DNA in situ during progression of breast cancer cells.

Nuclear matrix proteins (NMPs) show promise as informative biomarkers in following the pathogenesis of breast cancer. The nuclear matrix is a dynamic RNA-protein network involved in the organization and expression of chromatin. Cisplatin, which preferentially cross-links nuclear matrix proteins to DNA in situ, may be used to identify NMPs that organize and/or regulate the processing of DNA. In this study, we analyzed the nuclear matrix proteins from an estrogen receptor-positive breast cancer cell line panel consisting of MCF-7, MIII, LCC1, and LCC2 cell lines. This cell line panel reflects the stages of malignant progression in breast cancer. Proteins isolated from nuclear matrices and proteins cross-linked to nuclear DNA in situ with cisplatin were analyzed by two-dimensional gel electrophoresis. Specific changes in nuclear matrix proteins bound to nuclear DNA were identified. In concordance with estrogen independence and antiestrogen insensitivity, a loss in cisplatin cross-linking of specific NMPs to nuclear DNA was observed. Our results suggest that progression of breast cancer is accompanied by a reorganization of chromosomal domains, which may lead to alterations in gene expression.

Gene-specific differences in the aflatoxin B1 adduction of chicken erythrocyte chromatin.

Mature and immature chicken erythrocyte nuclei were treated with activated aflatoxin B1 (2,3-dichloroaflatoxin B1), producing covalently bound DNA adducts. This reaction produces alkali-labile sites in the DNA which can be identified by using a variation of the Maxam-Gilbert sequencing procedure. We determined the aflatoxin B1 accessibility of defined regions of the erythroid genome by using different specific probes and monitoring the disappearance of similar-sized fragments generated by restriction enzyme digestion. The genes studied were the erythroid-specific beta-globin and histone H5 genes, which are potentially active in mature erythroid nuclei and transcriptionally active in immature erythrocytes, and the vitellogenin and ovalbumin genes, which are both transcriptionally inactive in these cells. The beta-globin and histone H5 genes were more accessible than the repressed vitellogenin and ovalbumin genes to aflatoxin B1 modification in mature and immature erythroid chromatin. Micrococcal nuclease was used to probe the nucleosomal organization of active (beta-globin and histone H5) and repressed (vitellogenin and ovalbumin) genes in chicken erythrocytes. The vitellogenin and ovalbumin genes show a canonical nucleosome repeat pattern in mature and immature chicken erythrocyte nuclei. In contrast, the beta-globin and histone H5 genes lack a distinct nucleosomal repeat pattern in these cells. These results support the hypothesis that transcriptionally active genes are preferentially accessible to carcinogen modification because of their disrupted chromatin structure.

Reduced levels of histones H1o and H1b, and unaltered content of methylated DNA in rainbow trout hepatocellular carcinoma chromatin.

The levels of histone subtypes and DNA methylation of aflatoxin-induced rainbow trout hepatocellular carcinoma and adult liver nuclei were compared. The hepatocellular carcinoma nuclei were enriched in the ubiquitinated species of histone H2A and depleted in histones H1o and H1b. The 5-methylcytosine content and methylation patterns of the vitellogenin genes and the transcriptionally inactive TPG-3 protamine gene were not altered in the trout hepatocellular carcinoma DNA. Thus, undermethylation of DNA is not a general feature of chemically induced tumors in vivo.

In situ cross-linking by cisplatin of nuclear matrix-bound transcription factors to nuclear DNA of human breast cancer cells.

Cisplatin is an antitumor drug that is used to treat several types of cancers. In this study, we analyzed the proteins that were cross-linked to DNA in situ in MCF-7 human breast cancer cells incubated with cisplatin. We show that cisplatin cross-links nuclear matrix proteins to DNA. In immunoblotting experiments, we found that nuclear matrix-associated transcription factors and cofactors (estrogen receptor, HET/SAF-B, hnRNP K, and histone deacetylase 1) were cross-linked to nuclear DNA. These transcription factors and cofactors have essential roles in the regulation of genes involved in the proliferation of breast cancer cells and in the organization and structure of chromatin. We applied a novel protocol to demonstrate that the nuclear matrix-bound transcription factors/cofactors were cross-linked to DNA fragments attached to the nuclear matrix. These results suggest that the cross-linking of nuclear matrix-associated transcription factors and cofactors to DNA may be one of the mechanisms by which cisplatin inhibits transcription and replication processes.

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions.

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.

The protamine gene chromatin in developing trout testis exists in an altered state.

Micrococcal nuclease was used to probe the nucleosomal organization of the rainbow trout germ-line-specific protamine multi-gene family in testis and erythrocytes. In erythrocyte chromatin, the repressed protamine genes show a distinct nucleosomal repeat pattern. However, in early-stage testis chromatin, where the protamine genes are expressed, they lack a distinct nucleosomal repeat pattern, indicating that the disrupted chromatin structure is related to their transcriptional activity. Micrococcal nuclease-digested testis and erythrocyte chromatin was separated into soluble and insoluble fractions. Transcriptionally active/competent genes of testis that had been labeled by nuclear nick-translation were enriched in the low-salt eluted, micrococcal nuclease-sensitive chromatin fraction. This fraction was not enriched in protamine DNA sequences. In testis, but not erythrocytes, protamine DNA sequences were slightly enriched in chromatin that fractionated with insoluble nuclear material, suggesting that transcriptionally active protamine gene chromatin has an insoluble character. Since the different protamine genes may not be simultaneously expressed, our results show the distribution of both transcriptionally active and inactive protamine genes. However, our observations indicate that the active germ-line-specific protamine gene chromatin shares several, but not all, of the features associated with other active tissue-specific genes.

Nuclear distribution of histone deacetylase: a marker enzyme for the internal nuclear matrix.

Nuclear matrins are proteins that localize to the internal nuclear matrix. In a previous study, we reported that histone deacetylase is a component of the internal matrix, suggesting that histone deacetylase is a nuclear matrin. Here, we demonstrate that the majority of the histone deacetylase activity is associated with the internal nuclear matrices of chicken and trout liver. Thus, the association of the histone deacetylase with the internal nuclear matrix is neither tissue- nor species-specific. Using histone deacetylase as a marker enzyme for the partitioning of the internal nuclear matrix during nuclear fractionations, we show that in contrast to the internal nuclear matrices of trout liver, trout hepatocellular carcinoma and chicken liver, the stability of the chicken erythrocyte internal nuclear matrix is temperature-dependent. Our results support a model that has the histone deacetylase mediating transient interactions between the internal nuclear matrix and chromatin regions undergoing dynamic acetylation, for example transcriptionally active chromatin regions.

Differential compaction of transcriptionally competent and repressed chromatin reconstituted with histone H1 subtypes.

Chromatin fragments stripped of H1 histones regain the ability to form higher order structures and aggregates in 0.15 M NaCl following reconstitution with histone H1. However, transcriptionally competent chromatin fragments are resistant to chicken erythrocyte H1/H5 histone-induced 0.15 M NaCl aggregation/precipitation. In this study, we investigated the ability of stripped chromatin fragments reconstituted with one of four histone H1 subtypes (chicken erythrocyte H1, H5, trout liver H1a, H1b) at various stoichiometries to form salt precipitable higher order structures. Our results provide evidence that chicken erythrocyte histone H1 was more effective than histone H5 and trout liver histone H1b better than H1a in forming higher order structures. None of the histone H1 subtypes could render transcriptionally competent chromatin fragments insoluble in 0.15 M NaCl. These results are consistent with the ideas that the histone H1 subtypes differ in their capacities to compact chromatin fiber, and that the alterations in the structure of transcriptionally competent nucleosomes interfere with the capacity of all H1 subtypes to form higher order structures.

Inducible upregulation of oestrogen receptor-beta1 affects oestrogen and tamoxifen responsiveness in MCF7 human breast cancer cells.

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.