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Results for “histone recycling”

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At least 19 recordsLinked to original sources
PubMed2026

In vitro reconstitution of chromatin replication recapitulates symmetric histone recycling.

Symmetric histone recycling is vital for maintaining epigenetic inheritance upon eukaryotic DNA replication. Recent genome-wide studies have uncovered key determinants of this process, but how these factors collectively support parental histone transfer remains incompletely understood. Here, we successfully reconstitute histone recycling with 24 purified proteins and analyze the products digested by Micrococcal nuclease with Repli-pore-seq, the newly developed pipeline combining nanopore sequencing and deep-learning-based classification. As a result, we identify histones symmetrically recycled as tetrasomes or hexasomes on nucleosome-favorable sequences. We also observe the discordance of the recycled position between lagging and leading strands on the GC-rich DNA sequences. Moreover, removal of Pol δ, Pol32, Dpb3/4, Ctf4, Csm3/Tof1, or Mrc1 disrupts the balance of histone recycling between the two daughter strands, whereas removal of Ctf4, Csm3/Tof1, or Mrc1 additionally alters the positions at which histones were recycled. Furthermore, addition of the lagging-strand maturation factors Fen1 and Cdc9 enhances histone recycling to the lagging strand. These findings provide critical insights into the molecular players and mechanisms underlying symmetric histone recycling.

Histones↗
PubMed2026

Maintenance of nucleosome organization through replication and transcription counteracts aberrant coalescence of active chromatin.

Nucleosomes with their associated modifications organize and regulate the genome. It is unclear how this is integrated with the requirement of replication and transcription to access the DNA template without jeopardizing chromatin function. Here, we reveal a unified requirement for the histone chaperone FACT in mediating nucleosome disruption and reassembly during mammalian replication and transcription. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome wide, and chromatin structure in their wake collapses, with reduced nucleosome occupancy, irregular spacing, and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. Similarly, aberrant compartments form in cells failing to maintain chromatin fiber structure through replication. Nucleosome organization therefore dynamically regulates genome architecture, guarding against spurious chromatin aggregation.

Nucleosomes↗
PubMed2005

Disruption of histone deacetylase gene RPD3 accelerates PHO5 activation kinetics through inappropriate Pho84p recycling.

The histone deacetylase Rpd3p functions as a transcriptional repressor of a diverse set of genes, including PHO5. Here we describe a novel role for RPD3 in the regulation of phosphate transporter Pho84p retention in the cytoplasmic membrane. We show that under repressing conditions (with P(i)), PHO5 expression is increased in a pho4Delta rpd3Delta strain, demonstrating PHO regulatory pathway independence. However, the effect of RPD3 disruption on PHO5 activation kinetics is dependent on the PHO regulatory pathway. Upon switching to activating conditions (without P(i)), PHO5 transcripts accumulated more rapidly in rpd3Delta cells. This more rapid response correlates with a defect in phosphate uptake due to premature recycling of Pho84p, the high-affinity H+/PO4(3-) symporter. Thus, RPD3 also participates in PHO5 regulation through a previously unidentified effect on maintenance of high-affinity phosphate uptake during phosphate starvation. We propose that Rpd3p has a negative role in the regulation of Pho84p endocytosis.

Acid Phosphatase↗
PubMed2006

New histone incorporation marks sites of UV repair in human cells.

Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.

Chromatin↗
PubMed1976

Cooperative alignment of nu bodies during chromosome replication in the presence of cycloheximide.

50% of control DNA is resistant to staphylococcal nuclease after digestion in isolated nuclei, while only 25% of the labeled DNA made in the presence of cycloheximide is resistant to nuclease. Nevertheless, cycloheximide DNA is folded into normal chromosomal subunits as evidence by the observation that it generates nuclease limit-digest DNA fragments that are indistinguishable from controls. These results indicate that cycloheximide chromatin is associated with half the number of normal nu bodies. These nu bodies are probably recycled from the parental chromosome. Partial nuclease digestion of cycloheximide chromatin reveals that a normal pattern of monomer and multimer DNA fragments is generated up to octamers. The data are consistent with the idea that in the presence of cycloheximide, recycled parental histones become cooperatively aligned along the daughter double helices.

Chromatin↗
PubMed2024

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

DNA Polymerase III↗
PubMed1993

Interaction between the beta-amyloid peptide precursor and histones.

The function of the beta-amyloid peptide precursors (beta-APPs) remains undefined. In a search for ligands of beta-APP we found that the most prominent beta-APP-binding proteins were histones. To measure the specificity of binding the secreted or extracellular form of beta-APP(751), beta-APP-S, was purified from recombinant baculovirus-infected Sf-9 cells and was labeled with iodine-125. The labeled beta-APP-S bound to each of the histones, which had previously been adsorbed to nitrocellulose membranes. Differences in their apparent affinities were small. beta-APP-S did not bind to histones incorporated into chromatin. beta-APP-S bound relatively weakly to fibronectin, laminin, collagen type I, or collagen type IV. It did not bind to other basic proteins tested, myelin basic protein, or cytochrome c. The beta-APP-S-histone complex adhered to most filters and gel media, which precluded a direct determination of its stability. The apparent dissociation constant of beta-APP-S bound to histone-4-Sepharose was about 30 nM. Although beta-APP normally does not contact histones, it may bind those that are released from damaged cells. A function of beta-APP may be to bind and recycle substances such as histones, proteases, and matrix proteins from the extracellular medium.

Amyloid beta-Protein Precursor↗
PubMed1999

The movement of coiled bodies visualized in living plant cells by the green fluorescent protein.

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.

Arabidopsis↗
PubMed1984

The distribution of H1 histone is nonuniform in chromatin and correlates with different degrees of condensation.

Salt induces aggregation of large chromatin fragments maximally at 150-200 mM NaCl. The soluble fragments are depleted of H1 histones while the aggregated fragments are enriched. H1 histones did not equilibrate between the soluble and insoluble chromatin fractions when they were recycled through the process of salt-induced aggregation. The chromatin fragments that resisted aggregation retained more H1c subtype than they did H1 ab, correlating with previous results which showed complexes of H1c with DNA resisted salt-induced aggregation much more than complexes of DNA with other subtypes. The chromatin that was soluble at physiological concentrations of NaCl was DNase I sensitive and enriched in acetylated core histones. We conclude that H1 histone is nonuniformly distributed in chromatin in a stable pattern that probably correlates with the different degrees of condensation known to exist in vivo.

Acetylation↗
PubMed2004

Identification of alternatively spliced human biotinidase mRNAs and putative localization of endogenous biotinidase.

Biotinidase is essential for recycling the vitamin biotin and for transferring biotin to proteins, such as histones, suggesting that the enzyme localizes to various cellular and extracellular sites. To better understand the functions of the enzyme, we examined its gene structure and subcellular localization. Using RACE-PCR and a BLAST search, we extended the 5' sequence of the biotinidase gene. Three novel, alternatively spliced variants of biotinidase, 1a, 1b, and 1c, were identified in multiple human tissues. Exon 1c is present only in testes. The sequence of the 5' splice variants, 1a and 1b, suggest that biotinidase localizes to the mitochondria and/or ER, respectively. Using indirect immunofluorescence studies, biotinidase localizes to organelles in the cytoplasm, but not nucleus, of human fibroblasts and Hep G2 cells. Endogenous expression was examined by isopycnic gradient centrifugation of rat liver organelles, which identified an 85kDa biotinidase protein with biotinyl-hydrolase and transferase activities in microsomes and possibly lysosomes. A 48kDa protein, which also reacts with anti-biotinidase, localizes to mitochondria. The 48kDa protein is not N-glycosylated but is biotinylated, is in the inner mitochondrial matrix, but has no biotinyl-hydrolase or transferase activities. The function and validation of the mitochondrial species remains to be determined. The 5' splice variants and organelle fractionation studies indicate that biotinidase is directed to the secretory pathway and perhaps mitochondria.

5' Flanking Region↗
PubMed2004

Transcriptional activation by nuclear receptors.

Transcriptional activation by nuclear receptors (NRs) involves the recruitment of distinct classes of co-activators and other transcription-related factors to target promoters in the chromatin environment of the nucleus. Chromatin has a general repressive effect on transcription, but also provides opportunities for NRs to regulate transcription by directing specific patterns of chromatin remodelling and histone modification. Ultimately, the transcription of hormone-regulated genes by NRs is critically dependent on co-ordinated physical and functional interactions among the receptors, chromatin, co-activators with chromatin-, histone- and factor-modifying activities, and the RNA polymerase II transcriptional machinery. In addition, several mechanisms exist to terminate or attenuate NR-dependent signalling, including modification, recycling, subcellular redistribution and degradation of the receptors or their associated cofactors. The complexity of NR-dependent transcription provides multiple targets for regulatory inputs, thus allowing each hormone-responsive cell to direct its transcriptional output in a physiologically appropriate manner.

Animals↗
PubMed1981

Nucleosome dissociation at physiological ionic strengths.

Monomer nucleosomes purified on isokinetic sucrose gradients are shown to dissociate into component DNA and histones at physiological ionic strength upon dilution to a DNA concentration below 20 microgram/ml. The starting material is 11S, contains 145-190 BP DNA, and equimolar amounts of the four core histones with slightly less H1. Dilution of monomers in the presence of 0.14 M NaCl results in the rapid conversion of 10-40% of the 3H thymidine labeled material from 11S to 5S (5S is coincident with the S value of monomer length DNA). The proportion of nucleosomes which dissociate increases with increasing NaCl concentration between 0.15 M and 0.35 M and decreases with increasing DNA concentration above 1 microgram/ml. Recycling 11S monomers, which remain after dissociation, through a second dilution in salt generates an equivalent proportion of 5S material as seen after the initial dilution. Thus, the dissociation does not result from special properties of a subset of nucleosomes. An equilibrium between intact monomer and free DNA and histones appears to be rapidly established under the conditions described and the dissociated DNA will reassociate with histones to form 11S monomers if conditions of high DNA concentration and low ionic strength are established.

Animals↗
PubMed2006

Birth of a chimeric primate gene by capture of the transposase gene from a mobile element.

The emergence of new genes and functions is of central importance to the evolution of species. The contribution of various types of duplications to genetic innovation has been extensively investigated. Less understood is the creation of new genes by recycling of coding material from selfish mobile genetic elements. To investigate this process, we reconstructed the evolutionary history of SETMAR, a new primate chimeric gene resulting from fusion of a SET histone methyltransferase gene to the transposase gene of a mobile element. We show that the transposase gene was recruited as part of SETMAR 40-58 million years ago, after the insertion of an Hsmar1 transposon downstream of a preexisting SET gene, followed by the de novo exonization of previously noncoding sequence and the creation of a new intron. The original structure of the fusion gene is conserved in all anthropoid lineages, but only the N-terminal half of the transposase is evolving under strong purifying selection. In vitro assays show that this region contains a DNA-binding domain that has preserved its ancestral binding specificity for a 19-bp motif located within the terminal-inverted repeats of Hsmar1 transposons and their derivatives. The presence of these transposons in the human genome constitutes a potential reservoir of approximately 1,500 perfect or nearly perfect SETMAR-binding sites. Our results not only provide insight into the conditions required for a successful gene fusion, but they also suggest a mechanism by which the circuitry underlying complex regulatory networks may be rapidly established.

Amino Acid Sequence↗
PubMed1989

Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle of Physarum polycephalum.

Monoclonal antibodies raised against the 34-kD nucleolar protein, B-36, from the slime mold Physarum polycephalum have been used to examine the electron microscopic localization of B-36 during the cell cycle in Physarum. During interphase, B-36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B-36, fibrillarin. During mitosis, B-36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA-specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that in Physarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the "NOR" equivalents do not separate during mitosis as in mammalian cells. In addition, the B-36-enriched nucleolar remnants appear to be recycled from one cell cycle to the next.

Antibodies, Monoclonal↗
PubMed2007

Drosophila photoreceptors express cysteine peptidase tan.

The Drosophila mutant tan (t) shows reciprocal pigmentation defects compared with the ebony (e) mutant. Visual phenotypes, however, are similar in both flies: Electroretinogram (ERG) recordings lack "on" and "off" transients, an indication of impaired synaptic transmission to postsynaptic cells L1 and L2. Cloning of tan revealed transcription of the gene in the retina, apparently in photoreceptor cells. We expressed Tan in Escherichia coli and confirmed by Western blotting and mass spectroscopic analyses that Tan is expressed as preprotein, followed by proteolytic cleavage into two subunits at a conserved --Gly--Cys-- motif like its fungal ortholog isopenicillin-N N-acyltransferase (IAT). Tan thus belongs to the large family of cysteine peptidases. To discriminate expression of Tan and Ebony in retina and optic neuropils, we raised antisera against specific Tan peptides. Testing for colocalization with GMR-driven n-Syb-GFP labeling revealed that Tan expression is confined to the photoreceptor cells R1-R8. A close proximity of Tan and Ebony expression is evident in lamina cartridges, where three epithelial glia cells envelop the six photoreceptor terminals R1-R6. In the medulla, R7/R8 axonal terminals appeared lined up side by side with glial extensions. This local proximity supports a model for Drosophila visual synaptic transmission in which Tan and Ebony interact biochemically in a putative histamine inactivation and recycling pathway in Drosophila.

Animals↗
PubMedDate not supplied

Polycation-cell surface interactions and plasma membrane compartments in mammals. Interference of oligocation with polycationic condensation.

At lower concentrations, polyarginine, polylysine, protamine, histones H1, H2A, H2B and H3 cause lysis of human erythrocytes, whereas at higher concentrations inner histones are not hemolytic but induce only surface condensation and alterations in the cell-shape. Antibody coated erythrocytes treated with polyarginine result in ghost-like spheres having globular bodies 1 micron in diameter on the surface. Human fibroblasts and lymphocytes, and Ehrlich ascites cells treated with polyarginine also form surface globular bodies similar in size. Nucleate cell-polyarginine mixtures with lower polycation doses result in cytolysis, while higher polycation doses produce pyknosis of the cell surface accompanied by reorganization of a membrane-like structure. Changes in spectrofluorometric values result from 1,6-diphenyl-1,3, 5-hexatrien binding to cell lipids, match the plasma membrane alterations. Reciprocal shake incubation amplifies and/or conditions these polycation-induced alterations. The homogeneity of pyknotic surface bodies and the apparent polycationic membrane reorganization requiring oscillatory friction forces suggest the preexistence of a multizonal glycocalyx distribution corresponding to plasma membrane compartments. The possible role of this compartmentalization in receptor and membrane recycling, as well as the involvement of reversible catalytic-like polycation condensation in macromolecular changes are discussed.

Animals↗
PubMed2006

CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25.

SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.

Animals↗