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PubMed2003

Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.

Animals↗

PubMed2007

Mouse and rat BDNF gene structure and expression revisited.

Brain-derived neurotrophic factor (BDNF) has important functions in the development of the nervous system and in brain plasticity-related processes such as memory, learning, and drug addiction. Despite the fact that the function and regulation of rodent BDNF gene expression have received close attention during the last decade, knowledge of the structural organization of mouse and rat BDNF gene has remained incomplete. We have identified and characterized several mouse and rat BDNF transcripts containing novel 5' untranslated exons and introduced a new numbering system for mouse and rat BDNF exons. According to our results both mouse and rat BDNF gene consist of eight 5' untranslated exons and one protein coding 3' exon. Transcription of the gene results in BDNF transcripts containing one of the eight 5' exons spliced to the protein coding exon and in a transcript containing only 5' extended protein coding exon. We also report the distinct tissue-specific expression profiles of each of the mouse and rat 5' exon-specific transcripts in different brain regions and nonneural tissues. In addition, we show that kainic acid-induced seizures that lead to changes in cellular Ca(2+) levels as well as inhibition of DNA methylation and histone deacetylation contribute to the differential regulation of the expression of BDNF transcripts. Finally, we confirm that mouse and rat BDNF gene loci do not encode antisense mRNA transcripts, suggesting that mechanisms of regulation for rodent and human BDNF genes differ substantially.

Animals↗

PubMed2003

Transcription elongation factors repress transcription initiation from cryptic sites.

Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Chromatin↗

PubMed1995

Stage-specific distribution of the spermatid-specific histone 2B in the rat testis.

Male germ cells contain a number of histone variants, most of which are synthesized during either the mitotic or the meiotic stages of spermatogenesis. A spermatid-specific H2B (ssH2B) variant has been identified in mouse round spermatids that has an additional 12 amino acids at its carboxyl terminus as compared to somatic H2Bs. Until now, the presence of this protein in other mammals has not been known. Northern blot analysis using an oligonucleotide probe complementary to a unique coding region of the C-terminus of mouse ssH2B showed that mRNA encoding this protein was present in isolated rat round spermatids. Furthermore, immunoblot analysis of basic nuclear proteins from round spermatids probed with an anti-ssH2B antiserum that recognizes the novel peptide sequence indicated that the protein was present in round spermatids but not in pachytene spermatocytes. The ssH2B comigrated with H2A.X by acid-urea (2.5 M) PAGE and migrated slightly more slowly than TH2B by acid-urea-Triton PAGE. When seminiferous tubules were microdissected stage-specifically and basic nuclear proteins were immunoblotted, ssH2B was detected in tubule sections of stages II-VI. It reached a maximum level in stages VII-VIII and then decreased to minimal levels in stages XIII-I. Since ssH2B was not detected in pachytene spermatocytes, the stage-specific levels of ssH2B corresponded to the respective steps of spermatids present in the tubules. While ssH2B constituted a relatively small amount (approximately 2%) of total H2B protein in round spermatids, its presence in both the mouse and the rat suggests that its function may be conserved in mammalian spermatogenic cells.

Animals↗

PubMed2004

Tamoxifen long-term treatment in vitro alters the apoptotic response of MCF-7 breast cancer cells.

In this study, we examined alterations in the apoptotic response of tamoxifen (TAM)-resistant breast cancer cells. We used an in vitro selection approach for TAM resistance by means of long-term culture of MCF-7 breast cancer cells with increasing concentrations of TAM. The apoptotic response to TAM was determined by means of ELISA measurement of apoptotic DNA-histone complexes in cytoplasm and by Annexin-V staining. MCF-7(LT) cells isolated after 5 months of long-term treatment with TAM exhibited a significantly reduced apoptotic response to this drug, even if administered in high concentrations up to 20 microM. This reduced apoptotic response was also observed after treatment with the topoisomerase II inhibitor etoposide, a pro-apoptotic antineoplastic drug. Microarray experiments comparing the transcriptome of MCF-7(LT) and wild-type cells revealed both the down-regulated expression of several genes coding for pro-apoptotic proteins and the up-regulation of genes coding for apoptosis inhibitors. Further experiments to determine expression changes of the receptor tyrosine kinases HER2 and epidermal growth factor receptor did not reveal any alterations in MCF-7(LT) if compared to wild-type cells. Our findings suggest that long-term treatment with TAM in vitro does not necessarily change the expression of receptor tyrosine kinases, but can modulate the expression of apoptotic key genes impairing the apoptotic response of MCF-7 breast cancer cells.

Annexin A5↗

PubMed1983

Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

5-Methylcytosine↗

PubMed2005

Increased life span due to calorie restriction in respiratory-deficient yeast.

A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NAD+-dependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD+ or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a conserved, Sir2-independent mechanism in both PSY316 and BY4742.

Caloric Restriction↗

PubMed2000

Molecular Cloning of MAPK Gene Family Using Degenerate PCR.

MAPK(Mitogen-activated protein kinase) pathways play an important role in morphorgenesis of Candida albicans. According to the conserved amino acid sequence of the known MAPKs, two degenerate primers attaching to subdomain VIB and IX were designed to screen novel MAPKs in C. albicans. The PCR was performed under non-strigent conditons. 100 PCR fragments were sequenced and among 25 novel gene fragments, two novel MAPK gene fragments were obtained. Using these two PCR fragments as probes to screen a Candida albicans genomic DNA library, two novel MAPK genes designated CSK1(Candida albicans sporulation-related MAP kinase1) and CEK2(Candida albicans extracellular signal-regulated kinase 2) were cloned. These two genes share high similarity with three cloned MAPK genes CEK1 and MKC1 and CaHOG1. The CSK1 gene is 1 193 bp in length, containing a 92 bp intron, coding for a 367 aa protein. The CSK1 shares highest similarity with SMK1, with homology 55.3% in nucleotide sequence and 50% in amino acid sequence. SMK1 encodes a MAPK involved in the sporulation pathway in S. cerevisiae. In vitro kinase activity assay showed that the Csk1 kinase exhibited a phosphorylation ability when using MBP as a substrate but not histone H1.

Journal Article↗

PubMed2006

Identification and characterization of SAP25, a novel component of the mSin3 corepressor complex.

The transcriptional corepressor mSin3 is associated with histone deacetylases (HDACs) and is utilized by many DNA-binding transcriptional repressors. We have cloned and characterized a novel mSin3A-binding protein, SAP25. SAP25 binds to the PAH1 domain of mSin3A, associates with the mSin3A-HDAC complex in vivo, and represses transcription when tethered to DNA. SAP25 is required for mSin3A-mediated, but not N-CoR-mediated, repression. SAP25 is a nucleocytoplasmic shuttling protein, actively exported from the nucleus by a CRM1-dependent mechanism. A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. An isotope-coded affinity tag quantitative proteomic analysis of the SAP25 complex revealed that SAP25 is associated with several components of the mSin3 complex, nuclear export machinery, and regulators of transcription and cell cycle. These results suggest that SAP25 is a novel core component of the mSin3 corepressor complex whose subcellular location is regulated by PML.

Active Transport, Cell Nucleus↗

PubMed1990

Flanking sequences of Xenopus 5 S RNA genes determine differential inhibition of transcription by H1 histone in vitro. Mitotic phosphorylation of H1 decreases its inhibitory power.

In Xenopus laevis chromatin histone H1 selectively inhibits the transcription of oocyte 5 S RNA genes while not affecting the transcription of somatic 5 S RNA genes (Schlissel, M. S., and Brown, D. D. (1984) Cell 37, 903-913; Wolffe, A. P. (1989) EMBO J. 8, 527-537). To explore possible mechanisms of this specific action of H1 we analyzed the in vitro transcription of H1.DNA complexes. We found that the selective inhibitory effect of H1 in this system depends entirely on the flanking sequences of 5 S RNA genes and not on the coding sequence itself. At an H1:DNA ratio above approximately 0.4, H1 strongly inhibited the transcription of the gene surrounded by the A + T-rich flanks characteristic of oocyte 5 S RNA genes, whereas it did not prevent transcription of the genes surrounded by G + C-rich somatic-type flanks. This was reflected by strongly preferential binding of H1 to isolated 5 S RNA genes contained within A + T-rich flanks. We also showed that superphosphorylation of H1 with growth-associated (mitotic) H1 kinase invariably decreased H1's ability to inhibit transcription in an in vitro system.

Animals↗

PubMed1999

Molecular cloning and nuclear localization of a histone deacetylase homologue in Plasmodium falciparum.

Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression and developmental events. The acetylation state of histones is controlled by a dynamic equilibrium between activities of histone acetylase and deacetylase enzymes. Histone deacetylase (HDAC) was recently suggested to be the target of a fungus-derived antiprotozoal agent exhibiting structural similarity to known HDAC inhibitors. We have initiated a study of HDAC of human malaria parasite, Plasmodium falciparum, to evaluate its potential as the target for novel antimalarials and its role in parasite development. We have isolated HDAC1 gene from the P. falciparum genomic and cDNA libraries. The nucleotide sequence contains no intervening sequence and its open reading frame (ORF) codes for a protein of 449 amino acid residues. We have named the protein, PfHDAC1, as the sequence shows significant homology to yeast, human and other eukaryotic HDACs. Northern blot analysis of the total RNA from different asexual and sexual stages of the parasite reveals the presence of single mRNA transcript, which is predominantly expressed in mature asexual blood stages and in gametocytes. Antiserum raised against a carboxyl terminal peptide immunoprecipitated an in vitro translated P. falciparum HDAC gene product and recognized an approximately 50 kDa protein in the Triton X-100 insoluble fraction of parasites. Immunoelectron microscopy analysis showed majority of the protein localized in the nucleus of P. falciparum. To our knowledge, this is the first HDAC gene isolated from the malaria parasite.

Amino Acid Sequence↗

PubMed2006

Structural characterization of Set1 RNA recognition motifs and their role in histone H3 lysine 4 methylation.

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation.

Amino Acid Sequence↗

PubMed1992

TSH is able to induce cell cycle-related gene expression in rat thyroid cell.

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18-20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c-jun and a gene coding for a zinc-finger protein Xrox 20/Egr2, both having a pattern of expression similar to the c-fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP/ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.

Animals↗

PubMed1980

Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell.

We describe the first example of a predominantly nuclear protein which during mitosis becomes part of the mitotic apparatus. This protein has been termed the nuclear-mitotic apparatus (NuMA) protein. In interphase cells NuMA protein is restricted to the nucleus and is a constituent of isolated nuclear matrices, but in mitotic cells it is observed by indirect immunofluorescence microscopy to be concentrated at the polar regions of the mitotic apparatus. This mitotic localization is dependent on the integrity of the spindle, since treatments which disrupt the spindle result in dispersion of NuMA protein throughout the cell. Comparison to the subcellar distribution of tubulin at different stages of the cell cycle indicates that NuMA protein is distinct from the previously identified components of the mitotic spindle. Its association with the nuclear matrix and its localization during mitosis to the site of nuclear reassembly suggest the interesting possibility that NuMA protein could be representative of a class of proteins involved in the early events of nuclear reassembly. NuMA is present in the nuclei and mitotic spindle of all types of human cells that have been examined, but proteins of similar molecular weight (300,000 daltons in dissociating solvents) or immunological specificity are not detected in cells of other species (including monkey). However, the NuMA protein is synthesized in a human/Chinese hamster hybrid cell containing a reduced number of human chromosomes. Immunofluorescence studies of this hybrid cell showed that the distribution of NuMA protein is equivalent to that in human cells. These results suggest that the human gene coding for NuMA protein, unlike other genes coding for human specific nuclear proteins, can be expressed in human/hamster hybrid cells and that the cell hybrids will be useful in further characterization of NuMA protein.

Animals↗

PubMed1987

Isolation of a cDNA clone for transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids.

We have isolated a cDNA clone for rat transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. The clone was identified initially by hybrid selection of TP1 mRNA. The sequence of the 251-nucleotide cDNA includes the entire coding region for the protein, thereby confirming the identity of the clone as well as predicting two changes in the published amino acid sequence.

Amino Acid Sequence↗

PubMed2006

Genome-wide analysis of Polycomb targets in Drosophila melanogaster.

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. Here, we determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the D. melanogaster genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways.

Animals↗

PubMed2001

Ssn6-Tup1 regulates RNR3 by positioning nucleosomes and affecting the chromatin structure at the upstream repression sequence.

The DNA damage inducible gene ribonucleotide reductase (RNR3) is regulated by a transcriptional repression mechanism by the recruitment of the Ssn6-Tup1 corepressor complex to its promoter by the sequence-specific DNA-binding protein Crt1. Ssn6-Tup1 is reported to represses transcription by interfering with transcription factors, recruiting histone deacetylases, and positioning nucleosomes at the promoter of its target genes. Two of the three mechanisms involve effects on chromatin structure, and therefore, we have delineated the nucleosomal structure of RNR3 in the repressed and derepressed state using multiple nuclease mapping strategies. A regular array of positioned nucleosomes is detected over the repressed RNR3 promoter that extends into the coding sequence. Treating cells with DNA damaging agents or deleting CRT1, SSN6, or TUP1 derepresses RNR3 transcription, and causes a dramatic disruption of nucleosome positioning over its promoter. Furthermore, derepression of RNR3 correlated with changes in nuclease sensitivity within the upstream repression sequence (URS) region. Specifically, the loss of a MNase-hypersensitive site, and the appearance of strong DNase I hypersensitivity, was observed over the URS. Interestingly, we find that the binding of Crt1 to the promoter in the absence of Ssn6 or Tup1 is insufficient for nucleosome positioning or regulating chromatin structure at the URS; thus, these two functions are strictly dependent upon Ssn6-Tup1. We propose that RNR3 is regulated by changes in nucleosome positioning and chromatin structure that are mediated by Ssn6, Tup1, and Crt1.

Blotting, Northern↗

PubMed1988

Analysis and in vivo disruption of the gene coding for adenylate kinase (ADK1) in the yeast Saccharomyces cerevisiae.

The gene (designated ADK1) encoding the so-called cytosolic adenylate kinase of the yeast Saccharomyces cerevisiae was isolated using a single mixed oligonucleotide hybridization probe designed from the published amino acid sequence. ADK1 was found to be identical to an adenylate kinase gene recently isolated by an approach entirely different from ours (Magdolen, V., Oechsner, U., and Bandlow, W. (1987) Curr. Genet. 12, 405-411). The gene resides on yeast chromosome IV adjacent to the histone gene H2A-1. Southern blot analysis revealed only one copy of the gene, and no other related yeast DNA sequences were detected. By gene disruption it is shown that the ADK1 gene is needed for normal cell proliferation but is not essential for cell viability. Immunological studies confirmed the absence of the ADK1 gene product in mutant cells; in extracts of total cellular protein, however, there were still about 10% of the wild-type enzymatic activity present. This indicates the existence of two or more adenylate kinase isozymes in yeast. From preliminary 31P NMR measurements on suspensions of yeast cells, a significant decrease in the level of nucleoside triphosphates was found in the mutant strain carrying the disrupted and partially deleted ADK1 locus.

Adenylate Kinase↗