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Results for “Chromatin Assembly and Disassembly”

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At least 19 recordsLinked to original sources
PubMed1993

The role of nucleoplasmin in chromatin assembly and disassembly.

Nucleoplasmin is the most abundant nuclear protein in Xenopus oocytes and eggs. The term 'molecular chaperone' was coined to describe its role in the assembly of the nucleosome subunits of chromatin. Although histones and DNA can self-assemble into nucleosomes, nucleoplasmin can facilitate this process in vitro by competing against non-specific charge interactions. In vivo nucleoplasmin binds histones H2A and H2B and transfers them to DNA. Another acidic nuclear protein, N1, binds and transfers histones H3 and H4. Nucleoplasmin has at least one other role in modulating chromatin structure in Xenopus eggs. It is required for the first stage of sperm chromatin decondensation. It binds and removes sperm basic proteins and replaces them by histones H2A and H2B, again forming nucleosomes, and resulting in decondensation of the compacted sperm chromatin. In addition we propose that the properties of the nuclear localization signal of nucleoplasmin can be explained by a model in which heat shock cognate protein hsc70 has a chaperone role in signal presentation during nuclear transport.

Amino Acid Sequence↗
PubMed2026

Insights into FACT in Cancers with Targeted Therapeutic Implications.

Facilitates chromatin transcription (FACT) is an evolutionarily conserved chromatin remodeling factor. It controls chromatin states in an ATP-independent manner via the regulation of chromatin assembly and disassembly. Through such regulation, FACT is involved in controlling transcription and other DNA-transacting processes such as replication and repair. However, it is surprisingly found to be upregulated in various cancers, and upregulated FACT induces oncogenesis and supports cancer cell survival, aggressiveness and metastasis, thus implying it to be a prognostic marker for cancer with an attractive targeted therapeutic potential. Here, we describe the involvement of FACT in various cancers with mechanistic insights and potential targeted therapeutic implications.

Humans↗
PubMed2025

Nucleosome stability safeguards cell identity, stress resilience and healthy aging.

Nucleosomes are the minimal repeating units of chromatin. Their dynamic assembly and disassembly underpins chromatin organization and genome regulation. However, it remains unclear how intrinsic nucleosome stability contributes to higher-level yet fundamental cellular and organismal properties-such as preservation of cell identity, lineage specification, stress resilience and ultimately healthy aging. To address this, we tested the impact of decreased intrinsic nucleosome stability across multiple cell, tissue and organismal models by introducing histone mutants that weaken histone-histone interactions. While nucleosome instability did not broadly alter global chromatin accessibility, DNA damage, cell proliferation or viability, it impaired lineage-specific gene expression programs, altered lineage specification and activated intrinsic inflammatory and stress pathways in a manner reminiscent of aging in mouse tissues and human cells. Consistently, nucleosome instability accelerated the onset of age-associated transcriptional alterations and functional decline in Caenorhabditis elegans and Drosophila melanogaster, and reduced cellular resilience to exogenous perturbations-including environmental, epigenetic and mitotic stress-in human cells and Saccharomyces cerevisiae. These cross-species findings identify nucleosome stability as an evolutionarily conserved epigenetic safeguard that preserves cell identity and stress resilience and supports organismal function and healthy aging.

Journal Article↗
PubMed2005

Histone dynamics on the interleukin-2 gene in response to T-cell activation.

Several models have been proposed for the mechanism of chromatin remodelling across the promoters of inducible genes in mammalian cells. The most commonly held model is one of cooccupation where histone proteins are modified by acetylation or phosphorylation and nucleosomes are remodelled, allowing the assembly of transcription factor complexes. Using chromatin immunoprecipitation, we observed an apparent decrease of histone acetylation and phosphorylation signals at the proximal promoter region of the inducible interleukin-2 and granulocyte-macrophage colony-stimulating factor genes in response to T-cell activation. We showed that this apparent decrease was due to a loss of histone H3 and H4 proteins corresponding to a decrease in nucleosome occupation of the promoter. This histone loss is reversible; it is dependent on the continual presence of appropriate activating signals and transcription factors and is not dependent on the acetylation status of the histone proteins. These data show for the first time that histone proteins are lost from a mammalian promoter upon activation of transcription and support a model of activation-dependent disassembly and reassembly of nucleosomes.

Acetylation↗
PubMed2006

The histone chaperone anti-silencing function 1 stimulates the acetylation of newly synthesized histone H3 in S-phase.

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. We have found that budding yeast lacking Asf1 has greatly reduced levels of histone H3 acetylated at lysine 9. Lysine 9 is acetylated on newly synthesized budding yeast histone H3 prior to its assembly onto newly replicated DNA. Accordingly, we found that the vast majority of H3 Lys-9 acetylation peaked in S-phase, and this S-phase peak of H3 lysine 9 acetylation was absent in yeast lacking Asf1. By contrast, deletion of ASF1 has no effect on the S-phase specific peak of H4 lysine 12 acetylation; another modification carried by newly synthesized histones prior to chromatin assembly. We show that Gcn5 is the histone acetyltransferase responsible for the S-phase-specific peak of H3 lysine 9 acetylation. Strikingly, overexpression of Asf1 leads to greatly increased levels of H3 on acetylation on lysine 56 and Gcn5-dependent acetylation on lysine 9. Analysis of a panel of Asf1 mutations that modulate the ability of Asf1 to bind to histones H3/H4 demonstrates that the histone binding activity of Asf1 is required for the acetylation of Lys-9 and Lys-56 on newly synthesized H3. These results demonstrate that Asf1 does not affect the stability of the newly synthesized histones per se, but instead histone binding by Asf1 promotes the efficient acetylation of specific residues of newly synthesized histone H3.

Acetylation↗
PubMed2007

Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene.

The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (-6,244/-194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (-448/+1,204). The presence of nucleosomes was observed approximately from position (-20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons.

Acetylation↗
PubMed2005

Biochemical observation of the rapid mobility of nuclear HMGB1.

Formaldehyde-crosslinked and sonicated chromatin fragments were obtained from 15-day chicken embryo erythrocytes and purified on caesium chloride gradients. Polyclonal antibodies raised against chicken HMGB1 were used to immuno-precipitate fragments carrying HMGB1 in two protocols: (1) affinity purified antibodies covalently coupled to agarose beads and (2) diluted antiserum. The DNA of the antibody-bound chromatin was quantified and its sequence content assessed by quantitative real-time PCR to give values of the absolute enrichments generated. Amplicons were monitored within the active beta-globin locus, in the adjacent heterochromatin, in the lysozyme locus (containing an active housekeeping gene and the inactive lysozyme gene) and at the promoter of the inactive ovalbumin gene. For all amplicons the Bound/Input ratio was close to unity, implying no preferential location of HMGB1 on the chromatin. This initially unexpected result can now be understood in the light of the exceptional mobility of HMGB1 revealed by FLIP experiments showing that only 1-2 s are needed for HMGB1 to cross the nucleus: crosslinking times of 1 min were used in the present experiments.

Active Transport, Cell Nucleus↗
PubMed2004

Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair.

The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8. Strains lacking INO80, ARP5, or ARP8 have defects in transcription. Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease. The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations. Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates. Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129. Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage. These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

Adenosine Triphosphate↗
PubMed2004

INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

Adenosine Triphosphate↗
PubMed2006

Structural basis for the histone chaperone activity of Asf1.

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Angstroms shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-beta sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a beta strand to the Asf1 beta sheet sandwich. Interactions with both H3 and H4 were required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a "strand-capture" mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones.

Amino Acid Substitution↗
PubMed2005

The PHD finger/bromodomain of NoRC interacts with acetylated histone H4K16 and is sufficient for rDNA silencing.

The SNF2h-containing chromatin-remodeling complex NoRC is responsible for silencing a fraction of mammalian rRNA genes (rDNA). NoRC silences transcription by establishing heterochromatic features-including DNA methylation, hypoacetylation of histone H4, and methylation of H3K9-at the rDNA promoter []. We have investigated the mechanism of NoRC-mediated rDNA silencing and show that binding of the bromodomain of TIP5, the large subunit of NoRC, to acetylated nucleosomes is a prerequisite for NoRC function. A point mutation within the bromodomain impairs the association of NoRC with chromatin, prevents heterochromatin formation, and abolishes transcriptional repression. Moreover, the association of NoRC with chromatin requires acetylation of histone H4 at lysine 16 (acH4K16), and binding to acH4K16 is required for subsequent deacetylation of H4K5, H4K8, and H4K12, indicating that acetylation of H4K16 plays an active role in NoRC-mediated heterochromatin formation. The bromodomain cooperates with an adjacent PHD finger to recruit HDAC1, DNMT1, DNMT3, and SNF2h to rDNA. If specifically targeted to the rDNA promoter, the PHD finger/bromodomain is capable of establishing heterochromatic features and rDNA silencing. Thus, the PHD finger/bromodomain represents an autonomous unit that binds to acH4K16 and coordinates the chain of events that establish the repressed state of rDNA.

Acetylation↗
PubMed2006

Polycomb group complexes self-regulate imprinting of the Polycomb group gene MEDEA in Arabidopsis.

Fertilization in flowering plants initiates the development of the embryo and endosperm, which nurtures the embryo. A few genes subjected to imprinting are expressed in endosperm from their maternal allele, while their paternal allele remains silenced. Imprinting of the FWA gene involves DNA methylation. Mechanisms controlling imprinting of the Polycomb group (Pc-G) gene MEDEA (MEA) are not yet fully understood. Here we report that MEA imprinting is regulated by histone methylation. This epigenetic chromatin modification is mediated by several Pc-G activities during the entire plant life cycle. We show that Pc-G complexes maintain MEA transcription silenced throughout vegetative life and male gametogenesis. In endosperm, the maternal allele of MEA encodes an essential component of a Pc-G complex, which maintains silencing of the paternal MEA allele. Hence, we conclude that a feedback loop controls MEA imprinting. This feedback loop ensures a complete maternal control of MEA expression from both parental alleles and might have provided a template for evolution of imprinting in plants.

Arabidopsis↗
PubMed2005

Spatio-temporal activation of chromatin on the human CYP24 gene promoter in the presence of 1alpha,25-Dihydroxyvitamin D3.

The vitamin D3 24-hydroxylase gene (CYP24) is one of the most strongly induced genes known. Despite this, its induction by the hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3) has been characterized only partially. Therefore, we monitored the spatio-temporal, 1alpha,25OH2D3-dependent chromatin acetylation status of the human CYP24 promoter by performing chromatin immunoprecipitation (ChIP) assays with antibodies against acetylated histone 4. This was achieved by performing PCR on 25 contiguous genomic regions spanning the first 7.7 kb of the promoter. ChIP assays using antibodies against the 1alpha,25OH2D3 receptor (VDR) revealed that, in addition to the proximal promoter, three novel regions further upstream associated with VDR. Combined in silico/in vitro screening identified in three of the four promoter regions sequences resembling known VDREs and reporter gene assays confirmed the inducibility of these regions by 1alpha,25OH2D3)=. In contrast, the fourth VDR-associated promoter region did not contain any recognizable classical VDRE that could account for the presence of the protein on this region. However, re-ChIP assays monitored on all four promoter regions simultaneous association of VDR with retinoid X receptor, coactivator, mediator and RNA polymerase II proteins. These proteins showed a promoter region-specific association pattern demonstrating the complex choreography of the CYP24 gene promoter activation over 300 minutes. Thus, this study reveals new information concerning the regulation of the CYP24 gene by 1alpha,25OH2D3, and is a demonstration of the simultaneous participation of multiple, structurally diverse response elements in promoter activation in a living cell.

Acetylation↗
PubMed2026

Genome assembly of Astatotilapia latifasciata uncovers B chromosome-linked chromatin reorganization.

B chromosomes (Bs) are supernumerary genomic elements found in many eukaryotes, yet their full sequence composition, functional potential, and regulatory impact on the host genome remain unclear. Here, we present a chromosome-level genome assembly of the cichlid fish Astatotilapia latifasciata, integrating PacBio long reads, Illumina short reads, and Hi-C chromatin contact maps to resolve both A and B chromosomes. The 0.93 Gb assembly (N50 = 36.2 Mb) includes a 34 Mb B chromosome containing 789 predicted protein-coding genes and a markedly higher density of transposable elements (TEs), especially long terminal repeats (LTR) retrotransposons. Transcriptome profiling revealed that B-linked genes are predominantly transcriptionally repressed relative to their A chromosome paralogs. Hi-C-based chromatin modeling uncovered distinct 3D structural configurations associated with the B chromosome, including fewer topologically associating domains (TADs), reduced loop formation, and altered compartmentalization. These changes are linked to long-range chromatin interactions and genomic rearrangements, suggesting that the B chromosome reshapes the nuclear architecture of the host genome. Our study proposes a potential regulatory role of Bs in genome and provides a genomic resource for investigating chromosome evolution in cichlids.

Animals↗
PubMed2004

Spt3 and Mot1 cooperate in nucleosome remodeling independently of TBP recruitment.

We have investigated the requirements for nucleosome remodeling upon transcriptional induction of the GAL1 promoter. We found that remodeling was dependent on two SAGA complex components, Gcn5 and Spt3. The involvement of the latter was surprising as its function has been suggested to be directly involved in TATA-binding protein (TBP) recruitment. We demonstrated that this novel function was in fact independent of TBP recruitment and this was further validated using a Gal4-driven synthetic promoter. Most importantly, we showed that the involvement of Spt3 in chromatin remodeling was independent of transcription, as it was also observed for a nonpromoter nucleosome located next to an activator-binding site. In an effort to explore how the Spt3 function was elicited, we found that Mot1, an ATPase of the Snf2 family that genetically interacts with Spt3, was also required for nucleosome remodeling independently of TBP recruitment. Interestingly enough, Spt3 and Mot1 were recruited on the GAL1 promoter as well as on the nonpromoter site in an interdependent manner. These findings show that the two proteins cooperate in nucleosomal transactions.

Adenosine Triphosphatases↗
PubMed2005

Cell cycle regulation of chromatin at an origin of DNA replication.

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Adenosine Triphosphatases↗
PubMed2005

Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division.

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.

Animals↗
PubMed2006

Physical interaction of apoptosis-inducing factor with DNA and RNA.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which upon apoptosis induction translocates to the nucleus where it interacts with DNA by virtue of positive charges clustered on the AIF surface. Here we show that the AIF interactome, as determined by mass spectroscopy, contains a large panel of ribonucleoproteins, which apparently bind to AIF through the RNA moiety. However, AIF is devoid of any detectable RNAse activity both in vitro and in vivo. Recombinant AIF can directly bind to DNA as well as to RNA. This binding can be visualized by electron microscopy, revealing that AIF can condense DNA, showing a preferential binding to single-stranded over double-stranded DNA. AIF also binds and aggregates single-stranded and structured RNA in vitro. Single-stranded poly A, poly G and poly C, as well double-stranded A/T and G/C RNA competed with DNA for AIF binding with a similar efficiency, thus corroborating a computer-calculated molecular model in which the binding site within AIF is the same for distinct nucleic acid species, without a clear sequence specificity. Among the preferred electron donors and acceptors of AIF, nicotine adenine dinucleotide phosphate (NADP) was particularly efficient in enhancing the generation of higher-order AIF/DNA and AIF/RNA complexes. Altogether, these data support a model in which a direct interaction of AIF contributes to the compaction of nucleic acids within apoptotic cells.

Amino Acid Sequence↗