ABSTRACT
The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.
Subject(s)
Autoantigens/metabolism , Cell Cycle/physiology , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Autoantigens/genetics , Base Sequence , Cell Line , Centromere/ultrastructure , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , DNA-Binding Proteins/genetics , Down-Regulation , Histones/metabolism , Humans , Protein BindingABSTRACT
DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.
Subject(s)
Histones/metabolism , Protein Kinases/chemistry , Protein Kinases/physiology , Protein Serine-Threonine Kinases/metabolism , Transcription, Genetic , Adenoviridae/genetics , Animals , Cells, Cultured , Checkpoint Kinase 1 , Culture Media, Serum-Free , DNA Damage , Embryo, Mammalian , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gene Expression Regulation , HCT116 Cells , Histones/genetics , Humans , Mice , Models, Genetic , Phosphorylation/radiation effects , Protein Kinases/analysis , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Substrate Specificity , Ultraviolet RaysABSTRACT
The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly.
Subject(s)
Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Schizosaccharomyces/metabolism , Ubiquitination , Amino Acid Sequence , Histones/chemistry , Methylation , Methyltransferases/metabolism , Mutation/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.
Subject(s)
Activating Transcription Factor 4/metabolism , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-5/metabolism , Peptide Chain Initiation, Translational , Animals , Carcinogenesis/pathology , Cell Line, Tumor , Drosophila melanogaster/metabolism , Eukaryotic Initiation Factor-3 , Fibrosarcoma/pathology , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , Male , Mass Spectrometry , Mice, NudeABSTRACT
Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1's phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α's intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Nucleosomes/metabolism , Casein Kinase II/metabolism , Cell Line , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/chemistry , DNA/metabolism , Histones/metabolism , Humans , Phosphorylation , Protein Binding , Serine/metabolismABSTRACT
Histone H3K4 methylation has been linked to transcriptional activation. KDM5A (also known as RBP2 or JARID1A), a member of the KDM5 protein family, is an H3K4 demethylase, previously implicated in the regulation of transcription and differentiation. Here, we show that KDM5A is physically and functionally associated with two histone deacetylase complexes. Immunoaffinity purification of KDM5A confirmed a previously described association with the SIN3B-containing histone deacetylase complex and revealed an association with the nucleosome remodeling and deacetylase (NuRD) complex. Sucrose density gradient and sequential immunoprecipitation analyses further confirmed the stable association of KDM5A with these two histone deacetylase complexes. KDM5A depletion led to changes in the expression of hundreds of genes, two-thirds of which were also controlled by CHD4, the NuRD catalytic subunit. Gene ontology analysis confirmed that the genes commonly regulated by both KDM5A and CHD4 were categorized as developmentally regulated genes. ChIP analyses suggested that CHD4 modulates H3K4 methylation levels at the promoter and coding regions of target genes. We further demonstrated that the Caenorhabditis elegans homologues of KDM5 and CHD4 function in the same pathway during vulva development. These results suggest that KDM5A and the NuRD complex cooperatively function to control developmentally regulated genes.
Subject(s)
Gene Expression Regulation , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Retinoblastoma-Binding Protein 2/metabolism , Animals , Autoantigens/metabolism , Caenorhabditis elegans/metabolism , Cell Line, Tumor , Chromatin/metabolism , Gene Expression Profiling , HeLa Cells , Histones/metabolism , Humans , MCF-7 Cells , Methylation , Nucleosomes/metabolism , Protein Binding , RNA, Small Interfering/metabolism , Repressor Proteins/metabolism , Transcription, GeneticABSTRACT
Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Protein Methyltransferases/metabolism , Repressor Proteins/metabolism , Animals , Chromatin Assembly Factor-1 , Chromobox Protein Homolog 5 , HeLa Cells , Histone-Lysine N-Methyltransferase , Humans , Methylation , Mice , Models, Biological , Protein Transport , S PhaseABSTRACT
Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer's diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial. Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining "Uncleaved" and "Completely Cleaved" Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Extracellular Matrix Proteins/metabolism , Nerve Tissue Proteins/metabolism , Peptide Hydrolases/metabolism , Serine Endopeptidases/metabolism , Amino Acid Chloromethyl Ketones , Animals , Furin/antagonists & inhibitors , Furin/metabolism , Heparin/chemistry , Humans , Mice , Mice, Inbred ICR , Oligopeptides/pharmacology , Peptide Hydrolases/chemistry , Reelin ProteinABSTRACT
Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that plays a crucial role in heterochromatin-mediated gene silencing. We previously showed that mammalian HP1α is constitutively phosphorylated at its N-terminal serine residues by casein kinase II (CK2), and that this phosphorylation enhances HP1α's binding specificity for nucleosomes containing lysine 9-methylated histone H3 (H3K9me). Although the presence of additional HP1α phosphorylation during mitosis was reported more than a decade ago, its biological significance remains largely elusive. Here we found that mitosis-specific HP1α phosphorylation affected HP1α's ability to bind chromatin. Using biochemical and mutational analyses, we showed that HP1α's mitotic phosphorylation was located in its hinge region and was reversibly regulated by Aurora B kinase and serine/threonine phosphatases. In addition, chromatin fractionation and electrophoretic mobility shift assays revealed that hinge region-phosphorylated HP1α was preferentially dissociated from mitotic chromatin and exhibited a reduced DNA-binding activity. Although HP1's mitotic behaviour was previously linked to H3 serine 10 phosphorylation, which blocks the binding of HP1's chromodomain to H3K9me3, our findings suggest that mitotic phosphorylation in HP1α's hinge region also contributes to changes in HP1α's association with mitotic chromatin.
Subject(s)
Cell Cycle , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Mitosis , Aurora Kinase B/metabolism , Chromobox Protein Homolog 5 , DNA/metabolism , HEK293 Cells , HeLa Cells , Histones/metabolism , Humans , Phosphorylation , Protein Binding , Protein Phosphatase 2/metabolism , Protein Phosphatase 2C/metabolismABSTRACT
The human T-cell lymphotropic virus type 1 (HTLV-1) infects and transforms CD4+ lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. Here, we demonstrate that the HTLV-1 pX splice-variant p30II markedly enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter, dependent upon its conserved Myc-responsive E-box enhancer elements, which are associated with increased S-phase entry and multinucleation. Enhancement of c-Myc transforming activity by HTLV-1 p30II is dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and it requires TIP60 histone acetyltransferase (HAT) activity and correlates with the stabilization of HTLV-1 p30II/Myc-TIP60 chromatin-remodeling complexes. The p30II oncoprotein colocalizes and coimmunoprecipitates with Myc-TIP60 complexes in cultured HTLV-1-infected ATLL patient lymphocytes. Amino acid residues 99 to 154 within HTLV-1 p30II interact with the TIP60 HAT, and p30II transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner, as determined by microarray gene expression analyses. Importantly, these results suggest that p30II functions as a novel retroviral modulator of Myc-TIP60-transforming interactions that may contribute to adult T-cell leukemogenesis.
Subject(s)
Acetyltransferases/metabolism , Cyclins/genetics , E-Box Elements/genetics , Leukemia-Lymphoma, Adult T-Cell/virology , Proto-Oncogene Proteins c-myc/metabolism , Retroviridae Proteins/metabolism , Transcriptional Activation , Acetyltransferases/analysis , Alternative Splicing , CD4-Positive T-Lymphocytes/chemistry , CD4-Positive T-Lymphocytes/virology , Cell Transformation, Neoplastic , Chromatin Assembly and Disassembly , Cyclin D2 , Gene Expression Profiling , Histone Acetyltransferases , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/physiology , Humans , Leukemia-Lymphoma, Adult T-Cell/genetics , Lysine Acetyltransferase 5 , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Retroviridae Proteins/analysis , Retroviridae Proteins/genetics , Transcription, GeneticABSTRACT
Identification of the interaction partners of a protein is one of useful straightforward methods to gain insight into the molecular functions of the protein in cells. The pre-deposited forms of histones are associated with the specific histone chaperones to assemble into chromatin. Here, I describe an affinity purification method using the FLAG/HA double epitope-tagging technique and its application to purify particular histone variant-interacting chaperone complexes from soluble fraction to study dynamic chromatin functions. The purification is performed under low salt condition to obtain native histone variant complexes, and it would be useful to identify the specific chaperone proteins involved in the specific chromatin functions via histone variants.
Subject(s)
Histone Chaperones/isolation & purification , Histones/isolation & purification , Molecular Biology/methods , Chromatography, Affinity , HeLa Cells , Humans , Protein Binding , Protein Isoforms/isolation & purification , SolubilityABSTRACT
Chromobox 2 (CBX2), a component of polycomb repressive complex 1 (PRC1), binds lysine 27-methylated histone H3 (H3K27me3) via its chromodomain (CD) and plays a critical role in repressing developmentally regulated genes. The phosphorylation of CBX2 has been described in several studies, but the biological implications of this modification remain largely elusive. Here, we show that CBX2's phosphorylation plays an important role in its nucleosome binding. CBX2 is stably phosphorylated in vivo, and domain analysis showed that residues in CBX2's serine-rich (SR) region are the predominant phosphorylation sites. The serine residues in an SR region followed by an acidic-residue (AR) cluster coincide with the consensus target of casein kinase II (CK2), and CK2 efficiently phosphorylated the SR region in vitro. A nucleosome pull-down assay revealed that CK2-phosphorylated CBX2 had a high specificity for H3K27me3-modified nucleosomes. An electrophoretic mobility-shift assay showed that CK2-mediated phosphorylation diminished CBX2's AT-hook-associated DNA-binding activity. Mutant CBX2 lacking the SR region or its neighboring AR cluster failed to repress the transcription of p21, a gene targeted by PRC1. These results suggest that CBX2's phosphorylation is critical for its transcriptional repression of target genes.
Subject(s)
Gene Expression Regulation/physiology , Nucleosomes/physiology , Polycomb Repressive Complex 1/metabolism , Chromatin , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Silencing , HEK293 Cells , Histones , Humans , Mutation , Phosphorylation , Polycomb Repressive Complex 1/genetics , Protein Binding , Protein DomainsABSTRACT
Protein 6b, encoded by T-DNA from the pathogen Agrobacterium tumefaciens, stimulates the plant hormone-independent division of cells in culture in vitro and induces aberrant cell growth and the ectopic expression of various genes, including genes related to cell division and meristem-related class 1 KNOX homeobox genes, in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b is found in nuclei and binds to several plant nuclear proteins. Here, we report that 6b binds specifically to histone H3 in vitro but not to other core histones. Analysis by bimolecular fluorescence complementation revealed an interaction in vivo between 6b and histone H3. We recovered 6b from a chromatin fraction from 6b-expressing plant cells. A supercoiling assay and digestion with micrococcal nuclease indicated that 6b acts as a histone chaperone with the ability to mediate formation of nucleosomes in vitro. Mutant 6b, lacking the C-terminal region that is required for cell division-stimulating activity and interaction with histone H3, was deficient in histone chaperone activity. Our results suggest a relationship between alterations in nucleosome structure and the expression of growth-regulating genes on the one hand and the induction of aberrant cell proliferation on the other.
Subject(s)
Chromatin/metabolism , Histones/metabolism , Molecular Chaperones/metabolism , Oncogene Proteins/metabolism , Rhizobium/metabolism , Arabidopsis/genetics , Genes, Plant , Mitogens , Molecular Sequence Data , Plant Epidermis/cytology , Plant Leaves/cytology , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Nicotiana/cytologyABSTRACT
It has been believed that protein tagging caused by consecutive rare codons involves tmRNA action at the internal mRNA site. We demonstrated previously that ribosome stalling either at sense or stop codons caused by certain arrest sequences could induce mRNA cleavage near the arrest site, resulting in nonstop mRNAs that are recognized by tmRNA. These findings prompted us to re-examine the mechanism of tmRNA tagging at a run of rare codons. We report here that either AGG or CGA but not AGA arginine rare-codon clusters inserted into a model crp mRNA encoding cAMP receptor protein (CRP) could cause an efficient protein tagging. We demonstrate that more than three consecutive AGG codons are needed to induce an efficient ribosome stalling therefore tmRNA tagging in our system. The tmRNA tagging was eliminated by overproduction of tRNAs corresponding to rare codons, indicating that a scarcity of the corresponding tRNA caused by the rare-codon cluster is an important factor for tmRNA tagging. Mass spectrometry analyses of proteins generated in cells lacking or possessing tmRNA encoding a protease-resistant tag sequence indicated that the truncation and tmRNA tagging occur within the cluster of rare codons. Northern and S1 analyses demonstrated that nonstop mRNAs truncated within the rare-codon clusters are detected in cells lacking tmRNA but not in cells expressing tmRNA. We conclude that a ribosome stalled by the rare codon induces mRNA cleavage, resulting in nonstop mRNAs that are recognized by tmRNA.
Subject(s)
Codon , Escherichia coli Proteins/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , Receptors, Cell Surface/genetics , Ribosomes/genetics , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Cyclic AMP Receptor Protein , Escherichia coli Proteins/metabolism , Molecular Sequence Data , RNA, Bacterial/genetics , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Receptors, Cell Surface/metabolism , Transcription Factors/metabolismABSTRACT
Deposition of the major histone H3 (H3.1) is coupled to DNA synthesis during DNA replication and possibly DNA repair, whereas histone variant H3.3 serves as the replacement variant for the DNA-synthesis-independent deposition pathway. To address how histones H3.1 and H3.3 are deposited into chromatin through distinct pathways, we have purified deposition machineries for these histones. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that we show are necessary to mediate DNA-synthesis-dependent and -independent nucleosome assembly, respectively. Notably, these complexes possess one molecule each of H3.1/H3.3 and H4, suggesting that histones H3 and H4 exist as dimeric units that are important intermediates in nucleosome formation. This finding provides new insights into possible mechanisms for maintenance of epigenetic information after chromatin duplication.
Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , DNA Replication/genetics , DNA/biosynthesis , Histones/metabolism , Nucleosomes/metabolism , Chromatin Assembly Factor-1 , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Epigenesis, Genetic/genetics , HeLa Cells , Histone Chaperones , Histones/genetics , Humans , Macromolecular Substances , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/physiology , Repressor Proteins/physiology , Animals , Binding Sites , CCCTC-Binding Factor , Chickens , Chromatin/chemistry , Chromatin/metabolism , DNA, Complementary/metabolism , DNA-Binding Proteins/metabolism , Dimerization , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , K562 Cells , Mass Spectrometry , Models, Biological , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleophosmin , Precipitin Tests , Protein Binding , Repressor Proteins/metabolism , Silver Staining , Species SpecificityABSTRACT
A mutational block in the early stages of the glycolytic pathway facilitates the degradation of the ptsG mRNA encoding the major glucose transporter IICBGlc in Escherichia coli. The degradation is RNase E dependent and is correlated with the accumulation of either glucose-6-P or fructose-6-P (Kimata et al., 2001, EMBO J 20: 3587-3595; Morita et al., 2003, J Biol Chem 278: 15608-15614). In this paper, we investigate additional physiological effects resulting from the accumulation of glucose-6-P caused by a mutation in pgi encoding phosphoglucose isomerase, focusing on changes in gene expression. The addition of glucose to the pgi strain caused significant growth inhibition, in particular in the mlc background. Cell growth then gradually resumed as the level of IICBGlc decreased. We found that the transcription of the cps operon, encoding a series of proteins responsible for the synthesis of colanic acid, was markedly but transiently induced under this metabolic stress. Both genetic and biochemical studies revealed that the metabolic stress induces cps transcription by activating the RcsC/YojN/RcsB signal transduction system. Overexpression of glucose-6-P dehydrogenase eliminated both growth inhibition and cps induction by reducing the glucose-6-P level. Mutations in genes responsible for the synthesis of glucose-1-P and/or dTDP-glucose eliminated the activation of the Rcs system by the metabolic stress. Taken together, we conclude that an increased synthesis of dTDP-glucose activates the Rcs phosphorelay system, presumably by affecting the synthesis of oligosaccharides for enterobacterial common antigen and O-antigen.
Subject(s)
Bacterial Proteins/physiology , Escherichia coli Proteins/physiology , Escherichia coli/metabolism , Glucose/analogs & derivatives , Glucose/biosynthesis , Glycolysis , Multienzyme Complexes/physiology , Phosphoprotein Phosphatases/physiology , Phosphotransferases/physiology , Protein Kinases/physiology , Thymine Nucleotides/biosynthesis , Transcription Factors , Escherichia coli/genetics , Escherichia coli/growth & development , Gene Expression Regulation, Bacterial , Gene Silencing , Genes, Bacterial , Glucose/metabolism , Glucose-6-Phosphate/metabolism , Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/physiology , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/metabolism , Glycolysis/genetics , Mutation , Polysaccharides/biosynthesis , RNA, Messenger/analysis , Repressor Proteins/genetics , Repressor Proteins/physiology , Signal Transduction , Transcription, Genetic/genetics , Transcription, Genetic/physiologyABSTRACT
Recently, it has been found that ribosome pausing at stop codons caused by certain nascent peptides induces cleavage of mRNA in Escherichia coli cells (1, 2). The question we addressed in the present study is whether mRNA cleavage occurs when translation elongation is prevented. We focused on a specific peptide sequence (AS17), derived from SecM, that is known to cause elongation arrest. When the crp-crr fusion gene encoding CRP-AS17-IIA(Glc) was expressed, cAMP receptor protein (CRP) proteins truncated around the arrest sequence were efficiently produced, and they were tagged by the transfer-messenger RNA (tmRNA) system. Northern blot analysis revealed that both truncated upstream crp and downstream crr mRNAs were generated along with reduced amounts of the full-length crp-crr mRNA. The truncated crp mRNA dramatically decreased in the presence of tmRNA due to rapid degradation. The 3' ends of truncated crp mRNA correspond well to the C termini of the truncated CRP proteins. We conclude that ribosome stalling by the arrest sequence induces mRNA cleavage near the arrest point, resulting in nonstop mRNAs that are recognized by tmRNA. We propose that the mRNA cleavage induced by ribosome stalling acts in concert with the tmRNA system as a way to ensure quality control of protein synthesis and possibly to regulate the expression of certain genes.
Subject(s)
Escherichia coli/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomes/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Blotting, Northern , Blotting, Western , Cell Division , Codon , Codon, Terminator , Cyclic AMP/metabolism , Mass Spectrometry , Membrane Transport Proteins/metabolism , Models, Biological , Models, Genetic , Molecular Sequence Data , Peptides/chemistry , Plasmids/metabolism , Protein Structure, Tertiary , RNA/metabolism , Recombinant Fusion Proteins/metabolism , Ribosomes/chemistry , SEC Translocation Channels , SecA ProteinsABSTRACT
To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.