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At least 775 records · Page 43Linked to original sources
PubMed1999

Nucleosome dynamics. II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles.

H2A-H2B exchange with the intranuclear histone pool upon chromatin transcription in vivo is generally viewed as being triggered by the DNA positive supercoiling wave pushed by the elongating polymerase. This notion was tested here by investigating a potential release of H2A-H2B by ethidium bromide-induced positive supercoiling in the loop of mononucleosomes reconstituted on DNA minicircles. The results of gel electrophoresis, fluorescence titration and electron microscopy showed that such a positive supercoiling was not able to release H2A-H2B, nor to unfold the nucleosome to any detectable extent. The reason appeared to be the ease with which the loop could undergo a positive crossing, a surprising observation in view of the DNA left-handed wrapping around the octamer. Moreover, the influence of histone acetylation suggested that such loop flexibility to positive crossing is mediated by histone N-terminal tails which, by interacting with entering and exiting DNAs, reduce their electrostatic repulsion. These conclusions are confirmed and extended in the accompanying article through relaxation with topoisomerase I.

Animals↗
PubMed2001

EM studies of female meiosis in wood lemmings with different sex chromosome constitutions.

The chromosomes were studied throughout meiotic prophase by electron microscopy of surface-spread oocytes from one XX, four X*X, and three X*Y female wood lemmings, Myopus schisticolor. The X* chromosome had originated from X by a deletion and an inversion in the short arm. The deletion was confirmed in pachytene cells from X*X females; a D-loop was present in the sex bivalent in 16.8% of the cells, and asynapsis of unequal ends was seen in 9.1% of other cells. At late pachytene the D-loop underwent synaptic adjustment. The breakpoints of the deletion are in G-light bands. No inversion loop was seen, which also is in agreement with Ashley's ('88) hypothesis; at least one of the presumed breakpoints of the inversion is in G-dark chromatin. Various types of synaptic abnormalities, such as nonhomologous pairing (triple pairing, interchange, self-synapsis), univalents, foldbacks, and broken lateral elements, were encountered in all types of female. X*Y females showed a high frequency of abnormal oocytes (70.7%), which significantly exceeded that of X*X (23.1%) and XX (8.1%). Univalents were particularly common in the X*Y females. J. Exp. Zool. 290:504-516, 2001.

Animals↗
PubMed2024

The HIV-1 Transcriptional Program: From Initiation to Elongation Control.

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

HIV-1↗
PubMed1999

Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1.

BACKGROUND: A central question in vertebrate transcriptional regulation is how cis-regulatory modules, including enhancers, silencers and promoters, communicate with each other over long distances to mandate proper gene expression. In order to address this question we analysed protein/DNA interactions in the human beta-globin locus control region (LCR). One of the many proteins that are potentially implicated in LCR function is Bach1. Bach1 possesses a basic leucine zipper (bZip) domain, as well as a BTB/POZ domain that has been shown to be involved in the regulation of chromatin structure. Bach1 forms heterodimers with small Maf proteins through its leucine zipper and binds to Maf recognition elements (MARE). RESULTS: Using atomic force microscopy we visualized large looped DNA structures between MAREs located in different regulatory elements within the human beta-globin LCR that were mediated by Bach1/MafK heterodimers. The formation of these DNA loops required the Bach1 BTB/POZ protein interaction domain. Furthermore, in transfection studies we found that Bach1 repressed the enhancer activity of the LCR in a BTB/POZ domain-dependent manner. CONCLUSION: Our results suggest that Bach1 and other BTB/POZ transcription factors may represent a class of nuclear architectural proteins that mediate long range interactions between cis-regulatory elements in order to regulate gene expression.

Basic-Leucine Zipper Transcription Factors↗
PubMed2002

TRAP/SMCC/mediator-dependent transcriptional activation from DNA and chromatin templates by orphan nuclear receptor hepatocyte nuclear factor 4.

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.

Acetyltransferases↗
PubMed2003

Cha, a basic helix-loop-helix transcription factor involved in the regulation of upstream stimulatory factor activity.

We report here the characterization of Cha, a transcription factor of the basic helix-loop-helix (bHLH) family. The basic region of Cha shares DNA-interacting amino acids with members of class C bHLH transcription factors. In addition, the HLH region of Cha presents a Myc-type dimerization domain signature required for heterodimer formation between members of this class. Cha protein and mRNA were ubiquitously expressed in many human tissues. Electrophoretic mobility shift assays showed that Cha and upstream stimulatory factor (USF)-1 formed a complex that specifically bound to E-box DNA elements. Moreover, pull-down and co-immunoprecipitation experiments showed an interaction between Cha and USF-1. Cha did not bind to E-box DNA elements and required USF-1 for protein-DNA complex formation. Moreover, Cha inhibited USF-1-stimulated transcription of CD2 (a USF-1-dependent gene) and E-box promoter reporter plasmids. Chromatin immunoprecipitation assays showed that Cha occupied the CD2 promoter in resting, but not in mitogen-stimulated, T cells. Finally, Cha mRNA and protein expression were high in resting T cells and absent in mitogen-activated T cells and inversely correlated with CD2 expression. Contrarily, overexpression of Cha in T cells significantly reduced CD2 expression. In summary, our results indicated that Cha is a new bHLH transcription factor that negatively regulates USF-dependent transcription.

Amino Acid Sequence↗
PubMed2006

Nuclear localisation of endogenous SUMO-1-modified PDGF-C in human thyroid tissue and cell lines.

We investigated post-translational modification and subcellular localisation of endogenous platelet-derived growth factor-C (PDGF-C) in human thyroid papillary carcinomas (PTC), non-neoplastic thyroid tissues, and a selection of cultured cell lines. PDGF-C expressed nuclear localisation in 95% of all tested cell types in culture and in 10% of the thyrocytes from both PTC and non-neoplastic tissue. The cell lines expressed two forms of full-length PDGF-C, approximately 39 and approximately 55 kDa, in cell membrane and cytosol, while the approximately 55 kDa form dominated in the nucleus where it was partly chromatin-associated. The approximately 55 kDa form was post-translationally modified by SUMO-1. The putative PDGF-C SUMOylation site is the surface exposed (314)lysine part of a positively charged loop ((312)RPKTGVRGLHK(322)) with characteristics of a nuclear localisation signal. The tissue thyrocytes expressed a non-SUMOylated approximately 43 kDa and the 55 kDa PDGF-C. The SUMO-1 modified approximately 55 kDa PDGF-C expression was low in PTC where the approximately 43 kDa PDGF-C dominated. This is in contrast to non-neoplastic tissue and cultured cells where the SUMOylated approximately 55 kDa PDGF-C was strongly expressed. Our data provide novel evidence for nuclear localisation of PDGF-C, post-translational modification by SUMOylation and the expression of a novel form of PDGF-C in human papillary thyroid carcinomas.

Amino Acid Sequence↗
PubMed1994

Nuclear structure and the microdistribution of radiation damage in DNA.

Evidence for the roles of proteins and metal ions in the microheterogeneity of DNA damage is reviewed. Decondensation of chromatin in hypotonic buffers markedly sensitizes the DNA to radiation, while treatment of nuclei with hypertonic buffers strips the DNA of histones and other nuclear proteins and enhances the radiosensitivity of the DNA with respect to double-strand break (dsb) formation. Addition of the radical scavenger DMSO reduces the yield of strand breaks, but dehistonized chromatin remains approximately 2.5 times more sensitive to radiation than does native chromatin at 0.1 M DMSO. DNA-protein crosslink (DPC) formation is relatively unaffected by the removal of the majority of histones from chromatin. Most DPC form at or near the nuclear matrix, and matrix is stabilized and radiosensitized by Cu++. To elucidate the role of Cu++, the induction of dsb and DPC by gamma-radiation has been compared with that by hydroxyl radical from Fe(++)-EDTA, or Cu++ catalysed Fenton reactions. Data comparing the size of DNA fragments produced, the effect of expanding or dehistonizing chromatin, and the effects of radical scavengers suggest that gamma-radiation and Fe(++)-EDTA produce dsb at open chromatin sites, whereas Cu(++)-generated dsb are similar to radiation-induced DPC in their location at the nuclear matrix. Both metal ions appeared to produce damage by site-specific generation of hydroxyl radicals. The nuclear matrix, the proteinaceous skeleton which anchors chromosomal loops and provides sites for DNA replication and transcription, binds metal ions and matrix-attachment DNA regions (MARs) consisting of 300 + bp of AT-rich DNA. The interaction of cloned MARs with isolated nuclear matrices has been found to be hypersensitive to crosslinking upon gamma-irradiation, in comparison with associations formed by similarly sized DNA fragments lacking MAR sequences. Thus, the non-random distribution of radiation damage is partially explained by the protection of DNA afforded by histones and chromatin structure and partially by the hypersensitivity of DNA-nuclear matrix associations.

Binding Sites↗
PubMed2006

Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation.

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.

Amino Acid Sequence↗
PubMedDate not supplied

Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test.

The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium-sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA-nuclear damage. The varicocele patients showed 32.4% +/- 22.3% of spermatozoa with fragmented DNA, significantly different from the group of fertile subjects (12.6% +/- 5.0%). Nevertheless, this was not different from that of normozoospermic patients (31.3% +/- 16.6%) (P = .83) and with abnormal semen parameters (36.6% +/- 15.5%) (P = .31). No significant differences were found between the normozoospermic patients and the patients with abnormal semen parameters. Strikingly, the proportion of the degraded cells in the total of sperm cells with fragmented DNA was 1 out of 4.2 (23.9% +/- 12.9%) in the case of varicocele patients, whereas it was 1 out of 8.2 to 9.7 in the normozoospermic patients (11.1% +/- 9.9%) in the patients with abnormal sperm parameters (12.2% +/- 8.3%) and in the fertile group (10.3% +/- 7.2%). Thus, whereas no differences in the percentage of sperm cells with fragmented DNA were evident with respect to other infertile patients, individuals with varicocele exhibit a higher yield of sperm cells with the greatest nuclear DNA damage level in the population with fragmented DNA. This finding illustrates the value of assessing different patterns of DNA-nuclear damage within each sperm cell and the particular ability of the Halosperm kit to reveal them.

Adolescent↗
PubMed1983

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes.

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic "pulverized" appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.

Cell Cycle↗
PubMed2003

A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins.

RecG protein differs from other helicases analysed to atomic resolution in that it mediates strand separation via translocation on double-stranded (ds) rather than single-stranded (ss) DNA. We describe a highly conserved helical hairpin motif in RecG and show it to be important for helicase activity. It places two arginines (R609 and R630) in opposing positions within the component helices where they are stabilized by a network of hydrogen bonds involving a glutamate from helicase motif VI. We suggest that disruption of this feature, triggered by ATP hydrolysis, moves an adjacent loop structure in the dsDNA-binding channel and that a swinging arm motion of this loop drives translocation. Substitutions that reverse the charge at R609 or R630 reduce DNA unwinding and ATPase activities, and increase dsDNA binding, but do not affect branched DNA binding. Sequences forming the helical hairpin and loop structures are highly conserved in Mfd protein, a transcription-coupled DNA repair factor that also translocates on dsDNA. The possibility of type I restriction enzymes and chromatin-remodelling factors using similar structures to drive translocation on dsDNA is discussed.

Adenosine Triphosphate↗
PubMed1977

Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin.

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.

Animals↗
PubMed2004

Nuclear architecture and genome functioning in plants and animals: what can we learn from both?

In this short review, we combine information about large-scale chromosome and chromatin structure in the interphase nucleus from animals and plants in relation to transcription. We propose a rosette-like structure for interphase chromosomes in higher eukaryotes in which condensed heterochromatin domains act as organizing centers from which euchromatic loops sprout that contain the transcription units available to that particular cell. In the same context, we address the apparent discrepancy between the spatial distribution of transcriptionally active sites in the nucleus and our understanding of the linear organization of genes on the linear genome. It is likely that this paradox can be solved by taking into account the probably large number of genes for nonprotein-coding RNAs.

Animals↗
PubMed1982

A ? developmental characteristic of the ultrastructure of childhood leukaemic lymphoblasts.

Areas of chromatin demarcated from the rest of the nucleus ("separations") were found frequently in acute lymphoblastic leukaemias of childhood, in some patients in apparently all leukaemic cells, but rarely in non-lymphocytic leukaemias. In normal lymphocytes, separations were most common in fetal thymus, and less frequent in fetal lymph node and bone marrow, and child thymus, lymph node and marrow. Separations may be a developmental marker, and fetal lymphoid tissues, especially thymus, may have significance in the origin of lymphoblastic leukaemias of childhood. Nuclear loops enclosing cytoplasmic material were associated with, and may have similar significance to, separations.

Bone Marrow Cells↗
PubMed2006

Comparative genomics modeling of the NRSF/REST repressor network: from single conserved sites to genome-wide repertoire.

We constructed and applied an open source informatic framework called Cistematic in an effort to predict the target gene repertoire for transcription factors with large binding sites. Cistematic uses two different evolutionary conservation-filtering algorithms in conjunction with several analysis modules. Beginning with a single conserved and biologically tested site for the neuronal repressor NRSF/REST, Cistematic generated a refined PSFM (position specific frequency matrix) based on conserved site occurrences in mouse, human, and dog genomes. Predictions from this model were validated by chromatin immunoprecipitation (ChIP) followed by quantitative PCR. The combination of transfection assays and ChIP enrichment data provided an objective basis for setting a threshold for membership and rank-ordering a final gene cohort model consisting of 842 high-confidence sites in the human genome associated with 733 genes. Statistically significant enrichment of NRSE-associated genes was found for neuron-specific Gene Ontology (GO) terms and neuronal mRNA expression profiles. A more extensive evolutionary survey showed that NRSE sites matching the PSFM model exist in roughly similar numbers in all fully sequenced vertebrate genomes but are notably absent from invertebrate and protochordate genomes, as is NRSF itself. Some NRSF/REST sites reside in repeats, which suggests a mechanism for both ancient and modern dispersal of NRSEs through vertebrate genomes. Multiple predicted sites are located near neuronal microRNA and splicing-factor genes, and these tested positive for NRSF/REST occupancy in vivo. The resulting network model integrates post-transcriptional and translational controllers, including candidate feedback loops on NRSF and its corepressor, CoREST.

Animals↗
PubMed2006

The homeoprotein Alx3 expressed in pancreatic beta-cells regulates insulin gene transcription by interacting with the basic helix-loop-helix protein E47.

The regulation of insulin gene expression in pancreatic beta-cells is the result of the coordinate activity of specific combinations of transcription factors assembled on different promoter elements. We investigated the involvement of the aristaless-related homeoprotein Alx3 in this process. We found that Alx3 is coexpressed with insulin in pancreatic islets, as well as in the beta-cell line MIN6, and it is also present in glucagon- and somatostatin-expressing cells. Chromatin immunoprecipitation assays indicated that Alx3 present in MIN6 cells and in mouse pancreatic islets occupies the promoter of the mouse insulin genes. EMSAs indicated that Alx3 present in MIN6 cells binds to the A3/4 regulatory element of the insulin I promoter. We found that Alx3 transactivates the insulin promoter by acting on the E2A3/4 enhancer in conjunction with the basic helix-loop-helix transcription factors E47/Pan1 and Beta2/NeuroD, and that Alx3 physically interacts via the homeodomain with E47/Pan1 but not with Beta2/NeuroD. Alx3 binds to the A3/4 element as a dimer, and the homeodomain is sufficient to recruit E47/Pan1 to the insulin promoter. Deletion studies in transfected HeLa cells indicated that proline-rich regions located at either side of the Alx3 homeodomain work together with E47/Pan1, and that this requires the integrity of the amino-terminal activation domain to transactivate. Thus, these studies support the notion that Alx3 participates in the regulation of insulin gene expression in pancreatic beta-cells.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Differential functions for the transcription factor E2A in positive and negative gene regulation in pre-B lymphocytes.

The transcription factors encoded by the E2A gene have been shown to play essential roles in the initiation and progression of lymphocyte development. However, there is still a lack of comprehensive understanding of E2A downstream genes in B-cell development. We previously developed a gene tagging-based chromatin immunoprecipitation (ChIP) system to directly evaluate E2A target genes in B-cell development. Here, we have improved this ChIP strategy and used it in conjunction with microarray analysis on E2A-deficient pre-B-cell lines to determine E2A target genes in lymphocyte development. Both microarray data and ChIP studies confirmed that E2A directly controls IgH gene expression. The microarray assay also revealed genes that were significantly up-regulated after E2A disruption. ChIP analysis showed that E2A was most likely to be directly involved in repression of some of these target genes such as Nfil3 and FGFR2. An inducible E2A reconstitution system further demonstrated that E2A-mediated repression of Nfil3 and FGFR2 was reversible. Collectively, these findings indicate that E2A is a positive regulator for one set of genes and a negative regulator for another set of genes in developing B lymphocytes.

Animals↗