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Results for “chromatin loop formation”

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PubMed1999

Formation of the 3' end of histone mRNA.

All metazoan messenger RNAs, with the exception of the replication-dependent histone mRNAs, terminate at the 3' end with a poly(A) tail. Replication-dependent histone mRNAs end instead in a conserved 26-nucleotide sequence that contains a 16-nucleotide stem-loop. Formation of the 3' end of histone mRNA occurs by endonucleolytic cleavage of pre-mRNA releasing the mature mRNA from the chromatin template. Cleavage requires several trans-acting factors, including a protein, the stem-loop binding protein (SLBP), which binds the 26-nucleotide sequence; and a small nuclear RNP, U7 snRNP. There are probably additional factors also required for cleavage. One of the functions of the SLBP is to stabilize binding of the U7 snRNP to the histone pre-mRNA. In the nucleus, both U7 snRNP and SLBP are present in coiled bodies, structures that are associated with histone genes and may play a direct role in histone pre-mRNA processing in vivo. One of the major regulatory events in the cell cycle is regulation of histone pre-mRNA processing, which is at least partially mediated by cell-cycle regulation of the levels of the SLBP protein.

3' Untranslated Regions↗
PubMed2006

Tpk3 and Snf1 protein kinases regulate Rgt1 association with Saccharomyces cerevisiae HXK2 promoter.

Hexokinase 2 is an essential factor for signalling repression through the Saccharomyces cerevisiae high-glucose sensing pathway. The main regulatory mechanism that controls the HXK2 gene expression in yeast is mediated by the Rgt1 and Med8 transcription factors, which repress HXK2 expression in low-glucose containing media. In this study, we show that the repression activity of Rgt1 is regulated by Snf1 and Tpk3 protein kinases. Binding of Rgt1 to the HXK2 promoter requires Rgt1 phosphorylation by Snf1 or by an Snf1-dependent protein kinase. Conversely, Rgt1 hyperphosphorylation by the Tpk3 or by a Tpk3-dependent protein kinase dissociates Rgt1 from the repressor complex. Two-hybrid and chromatin immunoprecipitation experiments indicate that an Snf1-dependent interaction between Rgt1 and Med8 in the repressor complex is also essential for Rgt1 repression. The repression of HXK2 transcription by Rgt1 likely occurs through the formation of a DNA loop in the HXK2 locus, spanning the promoter and coding regions. These results suggest that a novel silent-chromatin loop is responsible for Rgt1-dependent transcriptional regulation of the HXK2 gene.

Binding Sites↗
PubMed1996

Spermatogenesis in Drosophila.

A short summary on the present knowledge on spermatogenesis in Drosophila is given which also points out particular questions of interest in the context of this morphogenetic process. Such points of interest are the formation of lampbrush loops in primary spermatocytes, the chromosomal events during meiosis, the occurrence of chromatin rearrangements and the regulation of gene activities at the posttranscriptional level. The activities and some major conclusions from my laboratory are subsequently described. They include studies of the expression of histone variants, the structure and function of lampbrush loops and the expression of genes participating in sperm morphogenesis.

Animals↗
PubMed2005

cAMP-response elements in Aplysia creb1, creb2, and Ap-uch promoters: implications for feedback loops modulating long term memory.

The Aplysia genes encoding for cAMP-response element-binding protein 1 (CREB1), CREB2, and ubiquitin C-terminal hydrolase (Ap-uch) have been implicated in the formation of long term memory. However, nothing is known about the promoter regions of these genes or the transcription factors that regulate them. We cloned the promoter regions of creb1, creb2, and Ap-uch and identified a canonical cAMP-response element (CRE) in the promoter region of creb1. Variants of the canonical CRE were identified in all three promoters. TATA boxes and C/EBP-binding motifs are also present in the promoter regions of these genes. Promoter immunoprecipitation assays and chromatin immunoprecipitation assays indicated that CREB1 and CREB2 bind to the promoter regions of creb1 and creb2, suggesting that feedback loops modulate the formation of long term memory. In a positive feedback loop, phosphorylated CREB1 might induce its own gene via CREs. In support of this suggestion, treatment with serotonin enhanced binding of CREB1 to its promoter region and increased mRNA levels of creb1. Levels of Ap-uch mRNA also increased in response to serotonin; however, binding of CREB1 or CREB2 to the promoter region of Ap-uch was not detected. The finding that the promoter region of creb2 has a CRE raises the intriguing possibility that its expression is regulated by CREB1 and/or CREB2. CREB2 may repress its own gene, forming a negative feedback loop, and CREB2 up-regulation via CREB1 may limit the activity of the CREB1-mediated positive feedback loop.

Animals↗
PubMed1980

Folding up genes and chromosomes.

The basic chromatin fiber is composed of a tandem array of nucleosomes, adjacent nucleosomes being separated by a variable amount of DNA. In vivo, this fiber is folded to form a higher-order fiber with a diameter of 200 to 300 A. The distribution of nucleosomes within the higher-order fiber has been analyzed in negatively stained preparations of chromatin fibers released from metaphase cells or interphase nuclei by mechanical disruption. The fiber appears to be composed to discrete packing domains in each of which there is a high density of closely apposed nucleosomes; within these regions several discrete packing patterns are observed. Adjacent domains are connected by regions of the fiber which are thinner and contain a lower density of nucleosomes. From these observations we suggest that the 200 to 300 A fiber is mosaic and that domains differ from each other in composition so as to confer sequence specificity upon the structure. In addition, compositional differences may have relevance to the functional state of the underlying genes; thus, higher order structure of a region is related to its functional state. Above this level of organization, studies on the early stages of chromatin folding in meiotic prophase suggest that 200 to 300 A fibers are organized as loops which emanate from the longitudinal axis of the chromosome, in a manner consistent with a model for mitotic chromosome folding by means of radial loop formation. Active ribosomal RNA genes are identifiable in these preparations and appear to be organized in a similar fashion; each active gene appears to comprise a loop of chromatin.

Animals↗
PubMed2000

HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology.

The nature of nuclear structures that are required to confer transcriptional regulation by distal enhancers is unknown. We show that long-range enhancer-dependent beta-globin transcription is achieved in vitro upon addition of the DNA architectural protein HMG I/Y to affinity-enriched holo RNA polymerase II complexes. In this system, HMG I/Y represses promoter activity in the absence of an associated enhancer and strongly activates transcription in the presence of a distal enhancer. Importantly, nucleosome formation is neither necessary for long-range enhancer regulation in vitro nor sufficient without the addition of HMG I/Y. Thus, the modulation of DNA structure by HMG I/Y is a critical regulator of long-range enhancer function on both DNA and chromatin-assembled genes. Electron microscopic analysis reveals that HMG I/Y binds cooperatively to preferred DNA sites to generate distinct looped structures in the presence or absence of the beta-globin enhancer. The formation of DNA topologies that enable distal enhancers to strongly regulate gene expression is an intrinsic property of HMG I/Y and naked DNA.

Base Sequence↗
PubMed2006

Loop formation by the transgene WAP:6xHishGH in transgenic rabbit fibroblasts, revealed by fluorescence in situ hybridization to nuclear halos.

Using fluorescence in situ hybridization (FISH) to somatic nuclear halos from transgenic rabbits WAP:6xHishGH, we present evidence for stability of transgenesis at the chromatin level. FISH performed on fibroblasts from a homozygous individual showed 2 independent loops from both chromosomes of pair 7. On a heterozygous individual, FISH detected a single loop. According to the concept of chromatin loops and their influence on gene expression, this shows that the human growth hormone transgene, which was actively expressed in mammary gland under the influence of the tissue-specific promoter, was inactive in examined skin fibroblasts.

Animals↗
PubMed2023

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Child↗
PubMed1996

Localization of mouse Rad51 and Lim15 proteins on meiotic chromosomes at late stages of prophase 1.

BACKGROUND: In meiosis, eukaryotic chromosomes show a series of morphological changes, during which chromosomes synapse and recombine. To understand the mechanisms of the morphological changes and recombination of chromosomes, we examined stage-specific localization of the Rad51 and Lim15 proteins on the chromosomes in meiotic prophase 1. These proteins are homologous with the RecA protein and have general properties of searching and pairing of homologous DNA sequences. We used mouse chromosomes whose small sizes allow us to identify the locations of these proteins on the entire structures of the chromosomes. RESULTS: In the leptotene and zygotene stages, the Rad51 protein was present on chromatin loops of mouse testis chromosomes then the protein left the loops. In the pachytene stage, the Rad51 protein was present almost exclusively along the core of the synaptonemal complexes (SC). When the stage proceeded to diplotene, the protein was present in the synaptic regions of chromosomes, in particular, in the chiasma regions. The protein was not present on separated homologous SC cores. On the other hand, the Lim15 protein that was found on chromatin loops in early prophase 1, was present almost exclusively at both ends of the SC cores throughout the late stages of prophase 1. CONCLUSION: The Rad51 and Lim15 proteins are present in chromatin loops when chromosomes form SC. The proteins may promote pairing of homologous DNA sequences that would lead formation of SC. The Rad51 protein in the SC cores may be involved in chiasma formation in late stages. The Lim15 protein, instead, may be involved in recombination in the telomeric region or in cohesion of sister chromatids for segregation.

Adenosine Triphosphatases↗
PubMed2004

Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes.

Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.

Binding Sites↗
PubMed1992

Chromosome structures.

Chromosomes are large subcellular structures, visible in the light microscope, that are found in the nuclei of most eukaryotic cells. Each chromosome consists of a single very long DNA molecule that has been compacted approximately 10,000-fold by interactions with proteins, such that the resulting chromosome structure fits within a typical eukaryotic nucleus of only 10 microns in diameter. Several levels of structural organisation are involved in the formation of chromosomes. Most chromosomal DNA is wrapped in left-handed superhelical turns around protein 'spools', called histone octamers, to form nucleosomes. Arrays of these nucleosomes, or 'beads on a string', are further compacted into solenoidal structures, called 30 nm chromatin fibres. The chromatin fibres are, in turn, compacted approximately 250-fold to form topologically independent 'looped' DNA domains, each loop containing about 20,000-100,000 nucleotide pairs of DNA extending from a proteinaceous central scaffold. Some chromosomes, such as lampbrush and polytene chromosomes, can be seen in certain specialised cells during interphase. Metaphase chromosomes, which can be stained to reveal characteristic banding patterns, are formed in most eukaryotic cells during mitosis. Formation of chromosome structures and the nuclei that envelop them involves discrete steps of nucleosome assembly, scaffold assembly, and nuclear envelope assembly, and can be carried out in cell-free extracts of animal eggs. Centromeres, the regions that mediate attachment of a chromosome to a meiotic or mitotic spindle, and telomeres, the natural ends of chromosomes, are structures that ensure that the correct number of full length chromosomes are maintained during the cell cycle. Most chromosome structures (nucleosomes, chromatin fibres, and scaffold loop domains) form from virtually any DNA sequence, but centromeres and telomeres are both composed of specific DNA sequences complexed with specific binding proteins. Recently, complete DNA sequences of entire chromosomes have been determined, and our rapidly emerging knowledge of chromosome structures is beginning to provide insights into the molecular basis of human disease.

Cell Cycle↗
PubMed2006

The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression.

FarUpStream Element (FUSE) Binding Protein (FBP) binds the human c-myc FUSE in vitro only in single-stranded or supercoiled DNA. Because transcriptionally generated torsion melts FUSE in vitro even in linear DNA, and FBP/FBP Interacting Repressor (FIR) regulates transcription through TFIIH, these components have been speculated to be the mechanosensor (FUSE) and effectors (FBP/FIR) of a real-time mechanism controlling c-myc transcription. To ascertain whether the FUSE/FBP/FIR system operates according to this hypothesis in vivo, the flux of activators, repressors and chromatin remodeling complexes on the c-myc promoter was monitored throughout the serum-induced pulse of transcription. After transcription was switched on by conventional factors and chromatin regulators, FBP and FIR were recruited and established a dynamically remodeled loop with TFIIH at the P2 promoter. In XPB cells carrying mutant TFIIH, loop formation failed and the serum response was abnormal; RNAi depletion of FIR similarly disabled c-myc regulation. Engineering FUSE into episomal vectors predictably re-programmed metallothionein-promoter-driven reporter expression. The in vitro recruitment of FBP and FIR to dynamically stressed c-myc DNA paralleled the in vivo process.

Cell Line↗
PubMed2005

Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer.

It is well established that gene expression in eukaryotes is controlled by sequence-dependent binding of trans-acting proteins to regulatory elements like promoters, enhancers or silencers. A less well understood level of gene regulation is governed by the various structural and functional states of chromatin, which have been ascribed to changes in covalent modification of core histone proteins. And, much on how topological domains in the genome take part in establishing and maintaining distinct gene expression patterns is still unknown. Here we present a set of regulatory proteins that allow to reversibly alter the DNA structure in vivo and in vitro by adding low molecular weight effectors that control their oligomerization and DNA binding. Using this approach, we completely regulate the activity of an SV40 enhancer in HeLa cells by reversible loop formation to topologically separate it from the promoter. This result establishes a new mechanism for DNA-structure-dependent gene regulation in vivo and provides evidence supporting the structural model of insulator function.

Base Sequence↗
PubMed2004

Multiple EBNA1-binding sites within oriPI are required for EBNA1-dependent transactivation of the Epstein-Barr virus C promoter.

The transactivating function of the oriPI-EBNA1 complex is essential for activation of the Epstein-Barr virus (EBV) C promoter (Cp) in lymphoblastoid cell lines expressing the viral growth programme. Furthermore, the oriPI-EBNA1 complex is believed to play an important role during promoter switching upon primary infection of B-lymphocytes and establishment of latent infection in vivo. Previously, it was shown that six EBNA1-binding sites within oriPI were required for transactivation of the heterologous thymidine kinase promoter. Here, we define the number of EBNA1-binding sites within oriPI necessary for its biological function as EBNA1-dependent Cp enhancer. We show that four EBNA1-binding sites within oriPI lead to significant upregulation of Cp in response to EBNA1 and eight or more to full activation. Thus, multiple EBNA1 homodimers at oriPI are required for the formation of a transcriptionally active Cp complex, a process that involves EBNA1-induced changes in the chromatin structure including DNA looping and nucleosome destabilization.

Binding Sites↗
PubMed2005

Nick-forming sequences may be involved in the organization of eukaryotic chromatin into approximately 50 kbp loops.

Phenomena involving the disassembly of chromosomes to approximately 50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is approximately 100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.

Apoptosis↗
PubMed1987

[The structure of eukaryotic chromosomes].

Recent models for the organization of the chromosomes of eukaryotes in mitosis and interphase are discussed with respect to their condensation hierarchy. Possible functional interpretations are indicated. At present, an alternating coiling and loop formation, induced by histones and nonhistone proteins, respectively, appears to be the most probable structure. The relationship of DNA properties to chromatin structure and function is also briefly discussed.

Animals↗
PubMed1980

Chromatin organization during meiotic prophase of Bombyx mori.

Chromatin organization during the early stages of male meiotic prophase in Bombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200-300 A in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5-25 mu of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.

Animals↗