Biomedical subjects

P Loidl

Publications and source records attributed to P Loidl.

At least 37 records · Page 2Linked to original sources

PubMed1997

Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein.

The steady state of histone acetylation is established and maintained by multiple histone acetyltransferases and deacetylases, and this steady state affects chromatin structure and function. The identification of a maize complementary DNA encoding the chromatin-bound deacetylase HD2 is reported. This protein was not homologous to the yeast RPD3 transcriptional regulator. It was expressed throughout embryo germination in correlation with the proliferative activity of cells. Antibodies against recombinant HD2-p39 immunoprecipitated the native enzyme complex, which was composed of phosphorylated p39 subunits. Immunofluorescence microscopy and sequence homologies suggested nucleolar localization. HD2 is an acidic nucleolar phosphoprotein that might regulate ribosomal chromatin structure and function.

Acetylation↗

PubMed1997

Cell growth inhibition by the Mad/Max complex through recruitment of histone deacetylase activity.

BACKGROUND: The organization of chromatin is crucial for the regulation of gene expression. In particular, both the positioning and properties of nucleosomes influence promoter-specific transcription. The acetylation of core histones has been suggested to alter the properties of nucleosomes and affect the access of DNA-binding transcriptional regulators to promoters. A recently identified mammalian histone deacetylase (HD1) shows homology to the yeast Rpd3 protein, which together with Sin3 affects the transcription of several genes. Mammalian Sin3 proteins interact with the Mad components of the Myc/Max/Mad network of cell growth regulators. Mad/Max complexes may recruit mammalian Rpd3-like enzymes, therefore, directing histone deacetylase activity to promoters and negatively regulating cell growth. RESULTS: We report the identification of a tetrameric complex composed of Max, Mad1, Sin3B and HD1. This complex has histone deacetylase activity which can be blocked by the histone deacetylase inhibitors trichostatin A and sodium butyrate. The inhibition of cell growth by Mad1 is enhanced by Sin3B and HD1, as measured by colony formation assays. Furthermore, a Mad1-induced block of S-phase progression can be overcome by trichostatin A, as shown in microinjection experiments. CONCLUSIONS: The recruitment of a histone deacetylase by sequence-specific DNA-binding proteins provides a mechanism by which the state of acetylation of histones in nucleosomes and hence the activity of specific promoters can be influenced. The finding that Mad/Max complexes interact with Sin3 and HD1 in vivo suggests a model for the role of Mad proteins in antagonizing the function of Myc proteins.

Animals↗

PubMed1997

Histone acetyltransferases during the cell cycle and differentiation of Physarum polycephalum.

The dynamic state of histone acetylation is maintained by histone acetyltransferases (HATs) and deacetylases. Cellular fractionation of plasmodia of Physarum polycephalum and partial purification of subcellular fractions by chromatography revealed the existence of a cytoplasmic B-type and four nuclear A-type HATs. The cytoplasmic B-enzyme was highly specific for histone H4, causing di-acetylation of H4 in vitro. The nuclear enzymes (HAT-A1 to HAT-A4) accepted all core histones as substrates, but differed by the preference for certain histone species. Enzymes were analyzed during the naturally synchronous cell cycle of macroplasmodia. Each of the enzymes had its individual cell cycle activity pattern, indicating diverse functions in nuclear metabolism. When growing plasmodia were induced to undergo differentiation into dormant sclerotia, an additional enzyme (HAT-AS) appeared at a late stage of sclerotization which correlated with differentiation-specific histone synthesis and acetylation in the absence of DNA replication. When dormant sclerotia were induced to reenter the cell cycle, a further enzyme form (HAT-AG) appeared during a short time period prior to the first post-germination mitosis. This enzyme had a strong preference for H2B, correlating with the overproportional in vivo acetate incorporation in H2B. Both differentiation-associated HATs were undetectable in growing plasmodia. The results demonstrate that different functions of core histone acetylation are based on multiple enzyme forms that are independently regulated during the cell cycle. Transitions from one developmental stage into another are accompanied by specific enzyme forms. With respect to recent data in the literature it may be assumed that these HAT-forms are subunits of a HAT-complex whose composition changes during the cell cycle and differentiation.

Acetyltransferases↗

PubMed1996

Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos.

The dynamic state of core histone acetylation is maintained by histone acetyltransferases and deacetylases. In germinating maize embryos, four nuclear histone deacetylases can be distinguished. From a chromatin fraction prepared at 72 h after start of embryo germination, we have purified the nuclear histone deacetylase HD2 to homogeneity. Using a sequence of chromatographic steps, we achieved the purification of an enzymatically active high molecular weight protein complex with an apparent molecular mass of 400 kDa, as determined by gel filtration chromatography. The purified enzyme was characterized in terms of enzymatic and kinetic properties, and sensitivity to several histone deacetylase inhibitors. In SDS-polyacrylamide gels, HD2 split into three polypeptides of 45, 42, and 39 kDa, suggesting that the native enzyme is a multimer-protein complex. Electrophoresis under nondenaturing conditions in combination with second dimension SDS-gel electrophoresis indicated that all three protein components of the HD2 complex were enzymatically active. Polyclonal antibodies against each of the three polypeptides were raised in rabbits. Each antiserum reacted with all three polypeptides on Western blots, suggesting that p45, p42, and p39 are highly homologous. This homology was confirmed by amino acid sequencing of peptides generated from each of the three HD2 components.

Blotting, Western↗

PubMed1996

Purification of histone deacetylase HD1-A of germinating maize embryos.

We have purified the soluble nuclear histone deacetylase HD1-A of germinating maize embryos. By a combination of 6 chromatographic steps we achieved a 77,000-fold purification of an enzymatically active protein. Gel filtration chromatography revealed a molecular weight of 45 kDa of the native enzyme and electrophoretic analysis of the purified enzyme by SDS-PAGE resulted in a single band at a molecular weight of 48 kDa, indicating that the enzyme is a monomer protein. When fractions with enzyme activity of different stages of chromatographic purification were subjected to isoelectric focusing, enzyme activity focused at a pH of around 6.4 as measured in an activity gel assay; second dimension SDS-PAGE again revealed a protein spot at a molecular weight of 48 kDa.

Cell Nucleus↗

PubMed1996

A comparative study of histone deacetylases of plant, fungal and vertebrate cells.

The enzymatic equilibrium of reversible core histone acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase (HD). These enzyme activities exist as multiple enzyme forms. The present report describes methods to extract different HD-forms from three organisms, germinating maize embryos, the myxomycete Physarum polycephalum, and chicken red blood cells; it provides data on the chromatographic separation and partial purification of HD-forms. In germinating maize embryos three HDs (HD1-A, HD1-B, HD2) can be discriminated; HD1-A, HD1-B, and HD2 were characterized in terms of their dependence on pH, temperature and various ions, as well as kinetic parameters (Km for core histones) and inhibition by various compounds. The same parameters were investigated for the corresponding enzymes of Physarum polycephalum, and mature and immature chicken erythrocytes. Based on these results, optimum assay conditions were established for the different enzyme forms. The kinetic data revealed that the maize histone deacetylase HD1-B peak after partial purification by Q-Sepharose chromatography was heterogeneous and consisted of two histone binding sites that differed significantly in their affinity for purified core histones. Optimized affinity chromatography on poly-Lysine Agarose indeed showed that the former defined deacetylase HD1-B can be separated clearly into two individual HD enzyme forms. The high multiplicity of histone deacetylases underlines the importance of these enzymes for the complex regulation of core histone acetylation.

Acetylation↗

PubMed1996

Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos.

From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.

Acetylation↗

PubMed1996

Oncogene- and tumor-suppressor gene-related proteins in plants and fungi.

Protooncogene- and tumor-suppressor gene proteins serve essential functions in the regulation of proliferation and differentiation of cells. Abnormal regulation or mutation of these genes, or transformation with retroviral homologs, may lead to tumor development in animals. In contrast to vertebrates, only few data on these genes exist in plants and fungi. Plant nuclear protooncogene homologs, such as myb and myc have multiple regulatory functions in metabolic pathways not existing in mammalian cells; they are involved in the complex regulation of anthocyanin (purple pigment) and phlobaphene (red pigment) biosynthesis, lignin production, trichome differentiation, dehydration stress gene expression and seed development. Apart from these well-characterized roles in plant-specific pathways, few experimental data have been reported on a functional significance in growth and development. A screening for nuclear protooncogene- and tumor-suppressor gene-related proteins in the myxomycete Physarum polycephalum revealed the existence of homologs of vertebrate c-myc, c-fos, c-jun, p53, and retinoblastoma proteins during the synchronous cell cycle or sclerotization. The p53 homologs of Physarum and Zea mays were shown to be specific for quiescent stages of their life cycles. Plants and lower eukaryotes, such as fungi, may be useful experimental systems to elucidate novel functions of protooncogene- and tumor-suppressor proteins in cell cycle regulation and development, or to reveal target genes that might be difficult to identify in complex mammalian systems. Recent data indicate that oncogenes and tumor suppressors in animals have more cellular targets than originally proposed; some of these might be as unexpected as in plant secondary metabolism.

Animals↗

PubMed1995

Nuclear matrix and the cell cycle.

The facts that the nuclear matrix represents a structural framework of the cell nucleus and that nuclear events, such as DNA replication, transcription, and DNA repair, are associated with this skeletal structure suggest that its components are subject to cell cycle-regulatory mechanisms. Cell cycle regulation has been shown for nuclear lamina assembly and disassembly during mitosis and chromatin reorganization. Little attention has so far been paid to internal nuclear matrix proteins and matrix-associated proteins with respect to the cell cycle. This survey attempts to summarize available data and presents experimental evidence that important metabolic functions of the nucleus are regulated by the transient, cell cycle-dependent attachment of enzymes and regulatory proteins to the nuclear matrix. Results on thymidine kinase and RNA polymerase during the synchronous cell cycle of Physarum polycephalum demonstrate that reversible binding to the nuclear matrix represents an additional level of regulation for nuclear processes.

Animals↗

PubMed1995

Inhibition of maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum.

HC toxin, the host-selective toxin of the maize pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 microM. Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did not affect histone acetyltransferases. After partial purification of histone deacetylases HD1-A, HD1-B, and HD2 from germinating maize embryos, we demonstrated that the different enzymes were similarly inhibited by the toxins. Inhibitory activities were reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the uncompetitive type and reversible. HC toxin, in which the epoxide group had been hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had been reduced to the corresponding alcohol, the modified toxin was less active than native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and the myxomycete Physarum polycephalum were also inhibited, indicating that the host selectivity of HC toxin is not determined by its inhibitory effect on HD. Consistent with these results, we propose a model in which HC toxin promotes the establishment of pathogenic compatibility between C. carbonum and maize by interfering with reversible histone acetylation, which is implicated in the control of fundamental cellular processes, such as chromatin structure, cell cycle progression, and gene expression.

Acetyltransferases↗

PubMed1994

Subcellular location of enzymes involved in core histone acetylation.

Multiple enzyme forms of histone deacetylase and histone acetyltransferase exist in germinating maize embryos. We analyzed the association of the different enzymes to chromatin by ion exchange chromatography of subcellular fractions from different time points of embryo germination. The vast majority of histone deacetylase HD-1A was not bound to chromatin, since it was solubilized during chromatin isolation, regardless of its phosphorylation state and the phase of embryo germination. In contrast, HD-2 was chromatin bound during the entire germination pathway. Histone deacetylase HD-1B was present in a chromatin-bound and a soluble form; the ratio between these two forms changed during germination. Both nuclear histone acetyltransferases, HAT-A1 and HAT-A2, were tightly chromatin-bound and could only be released from chromatin by salt extraction. To test whether histone acetyltransferases or deacetylases are associated with the nuclear matrix, we analyzed nuclear matrix preparations from yeast, Physarum, and maize step by step for both enzyme activities. This analysis confirmed that part of the activity is chromatin bound, but no significant enzyme activity could be found in the final nuclear matrix, regardless of the preparation protocol. This result was further substantiated by detailed analysis of histone deacetylases and acetyltransferases during cellular fractionation and nuclear matrix preparation of chicken erythrocytes. Altogether our results suggest that the participation of these enzymes in different nuclear processes may partly be regulated by a distinct location to intranuclear components.

Acetylation↗

PubMed1994

Histone acetylation: facts and questions.

The DNA of eukaryotic cells is organized in a complex with proteins, either as interphase chromatin or mitotic chromosomes. Nucleosomes, the structural subunits of chromatin, have long been considered as static structures, incompatible with processes occurring in chromatin. During the past few years it has become evident that the histone part of the nucleosome has important regulatory functions. Some of these functions are mediated by the N-terminal core histone domains which contain sites for posttranslational modifications, among them lysine residues for reversible acetylation. Recent results indicate that acetylation and deacetylation of N-terminal lysines of nucleosomal core histones represent a means of molecular communication between chromatin and the cellular signal transduction network, resulting in heritable epigenetic information. Data on enzymes involved in acetylation and the pattern of acetylated lysine sites on chromosomes, as well as genetic data on yeast transcriptional repression, suggest that acetylation may lead to structural transitions as well as specific signalling within distinct chromatin domains.

Acetylation↗

PubMed1993

Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC-PK1 cell line. A comprehensive investigation of different preparation procedures.

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC-PK1 cells extend predominantly over 40-80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC-PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.

Animals↗

PubMed1993

Histone deacetylase. A key enzyme for the binding of regulatory proteins to chromatin.

Core histones can be modified by reversible, posttranslational acetylation of specific lysine residues within the N-terminal protein domains. The dynamic equilibrium of acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase. Recent data on histone deacetylases and on anionic motifs in chromatin- or DNA-binding regulatory proteins (e.g. transcription factors, nuclear proto-oncogenes) are summarized and united into a hypothesis which attributes a key function to histone deacetylation for the binding of regulatory proteins to chromatin by a transient, specific local increase of the positive charge in the N-terminal domains of nucleosomal core histones. According to our model, the rapid deacetylation of distinct lysines in especially H2A and H2B would facilitate the association of anionic protein domains of regulatory proteins to specific nucleosomes. Therefore histone deacetylation (histone deacetylases) may represent a unique regulatory mechanism in the early steps of gene activation, in contrast to the more structural role of histone acetylation (histone acetyltransferases) for nucleosomal transitions during the actual transcription process.

Animals↗

PubMed1993

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum.

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

Acetylation↗

PubMed1993

Identification of proteins immunologically related to vertebrate lamins in the nuclear matrix of the myxomycete Physarum polycephalum.

Lamin-like proteins have been identified as components of the nuclear matrix of the myxomycete Physarum polycephalum. A 67 kDa homologue was detected by immunoblotting of nuclear matrix proteins with affinity-purified anti-lamin antibodies of human autoimmune sera. A 65 kDa lamin B homologue was identified with polyclonal antibodies against turkey erythrocyte lamin B in nuclei and nuclear matrix of Physarum amoebae and plasmodia. Indirect immunofluorescence microscopy revealed that the 65 and 67 kDa proteins are localized in the nuclear lamina of plasmodial nuclei.

Animals↗

PubMed1992

Specificity of Zea mays histone deacetylase is regulated by phosphorylation.

Mono Q ion exchange high performance liquid chromatography (HPLC) reveals that the main histone deacetylase activity (HD1) of germinating Zea mays embryos consists of multiple enzyme forms. Chromatography of HD1 after treatment with alkaline phosphatase yields two distinct histone deacetylase forms (HD1-A, HD1-B). The same is true for chromatography after phosphatase treatment of a total cell extract. One of these enzyme forms (HD1-A) is subject to phosphorylation, which causes a change in the substrate specificity of the enzyme, as shown with HPLC-purified individual core histone species; the substrate specificity for H2A increases more than 2-fold after phosphorylation, whereas the specificity for H3 decreases to about 60%. The total histone deacetylase activity is quantitatively released from isolated nuclei after extraction with moderate ionic strength buffers; no significant residual enzyme activity could be detected in the nuclear matrix.

Animals↗

PubMed1992

Intranuclear compartmentalization of transcribed and nontranscribed c-myc sequences in Namalva-S cells.

This investigation is centered on the intranuclear localization of transcribed and nontranscribed c-myc sequences in human Namalva-S cells bearing t(8;14) translocation. Southern hybridization showed that the breakpoint in the truncated allele of c-myc lies outside the characteristic 12.8-kbp EcoRI fragment: as Northern analysis indicated, this reorganization induces a high level of c-myc transcription. Following high-salt treatment, EcoRI digestion and centrifugation, isolated nuclei from the same cells are separated into two residual fractions: a heavier P fraction including nuclear matrix structures and a light S fraction representing dehistonized chromatin fibres. Comparative hybridization experiments revealed that the above procedure separates the c-myc sequences between the two fractions. To locate the site of intranuclear c-myc transcription, we performed run-on experiments with two fractions, topologically analogous to the residual P and S fractions but maintaining the original chromatin organization. These experiments indicated that chromatin P fraction harbours actively transcribed c-myc sequences while chromatin S harbours nontranscribed ones. Further experiments have clarified that the transcribed c-myc sequences are firmly bound to the matrix by multiple attachment sites, arranged throughout the entire gene locus. It was found, moreover, that at the site of attachment the interaction between DNA and the matrix components is realized via proteins. Controls with the beta-globin gene, which is constitutively nonexpressed in Namalva-S cells but upon induction is highly expressed in murine erythroleukemia cells, completely confirmed the conclusion we had made for the intranuclear localization of c-myc. Thus the experiments presented here support the more common idea that the transcribed and nontranscribed sequences are precisely compartmentalized.

Animals↗