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At least 523 records · Page 29Linked to original sources
PubMed2001

Stathmin/Op18 phosphorylation is regulated by microtubule assembly.

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of "mitotic chromatin." Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

Animals↗
PubMed2005

The orphan nuclear receptor Rev-erbalpha recruits the N-CoR/histone deacetylase 3 corepressor to regulate the circadian Bmal1 gene.

Transcriptional regulation plays a fundamental role in controlling circadian oscillation of clock gene expression. The orphan nuclear receptor Rev-erbalpha has recently been implicated as a major regulator of the circadian clock. Expression of Bmal1, the master regulator of circadian rhythm in mammals, is negatively correlated with Rev-erbalpha mRNA level, but the molecular mechanism underlying this regulation is largely unknown. Here we show that Rev-erbalpha dramatically represses the basal activity of the mouse Bmal1 gene promoter via two monomeric binding sites, both of which are required for repression and are conserved between mouse and human. Rev-erbalpha directly binds to the mouse Bmal1 promoter and recruits the endogenous nuclear receptor corepressor (N-CoR)/histone deacetylase 3 (HDAC3) complex, in association with a decrease in histone acetylation. The endogenous N-CoR/HDAC3 complex is also associated with the endogenous Bmal1 promoter in human HepG2 liver cells, where a reduction in cellular HDAC3 level markedly increases the expression of Bmal1 mRNA. These data demonstrate a new function for the N-CoR/HDAC3 complex in regulating the expression of genes involved in circadian rhythm by functioning as corepressor for Rev-erbalpha.

ARNTL Transcription Factors↗
PubMed1997

The transcriptional basis of chromosome pairing.

Pairing between homologous chromosomes is essential for successful meiosis; generally only paired homologs recombine and segregate correctly into haploid germ cells. Homologs also pair in some somatic cells (e.g. in diploid and polytene cells of Drosophila). How homologs find their partners is a mystery. First, I review some explanations of how they might do so; most involve base-pairing (i.e. DNA-DNA) interactions. Then I discuss the remarkable fact that chromosomes only pair when they are transcriptionally active. Finally, I present a general model for pairing based upon the DNA-protein interactions involved in transcription. Each chromosome in the haploid set has a unique array of transcription units strung along its length. Therefore, each chromatin fibre will be folded into a unique array of loops associated with clusters of polymerases and transcription factors; only homologs share similar arrays. As these loops and clusters, or transcription factories, move continually, they make and break contact with others. Correct pairing would be nucleated when a promoter in a loop tethered to one factory binds to a homologous polymerizing site in another factory, before transcription stabilizes the association. This increases the chances that adjacent promoters will bind to their homologs, so that chromosomes eventually become zipped together with their partners. Pairing is then the inevitable consequence of transcription of partially-condensed chromosomes.

Animals↗
PubMed1982

High-resolution scanning electron microscopy of human metaphase chromosomes.

Human metaphase chromosomes, prepared for light microscopy were examined by scanning electron microscopy. Use of an osmium impregnation technique eliminated the need for sputter-coating and allowed high-resolution visualization of uncoated specimens. Chromosomes were of three-dimensional cylindrical profile, with well-defined chromatids and centromeres. Prior to Giemsa-banding a smooth surface morphology was observed. Relaxation of chromosome integrity by Giemsa-banding pretreatment allowed resolution of several orders of chromosome structure not previously demonstrated by scanning electron microscopy. The observed organization of the chromatin fibres allowed parallels to be drawn with the radial loop model of chromosome construction as described by Marsden and Laemmli.

Chromosome Banding↗
PubMed1991

Synaptonemal complex karyotyping in an oligospermic patient with heterochromatin duplication in chromosome n. 9.

Synaptonemal complex karyotyping has been performed in an oligospermia of unknown etiology in a patient carrying a 9qh+ chromosomal polymorphism. The testicular histology showed hypospermatogenesis at the spermatid level and an abnormal pattern of chromatin condensation. Spermatocytes at early pachytene showed a large, asymmetric loop in SC #9, which disappeared at late pachytene, probably because of synaptic adjustment. The loop was formed by a lateral element 7.02% longer than the average normal one. The loop exceeded the centromere towards the short arm, and it is interpreted as a tandem duplication of about 50% of the paracentromeric heterochromatin. The present observation and the previously reported asynaptic loops in carriers of pericentric inversions and showing severe oligospermia suggest that chromosomal variants producing asynaptic loops may be associated with germ cell loss. Further meiotic studies in infertile carriers of such variants are indicated.

Adult↗
PubMed1993

Atomic force microscopy of mammalian sperm chromatin.

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, ellipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.

Animals↗
PubMed1989

Evolution of chromosome bands: molecular ecology of noncoding DNA.

Giemsa dark bands, G-bands, are a derived chromatin character that evolved along the chromosomes of early chordates. They are facultative heterochromatin reflecting acquisition of a late replication mechanism to repress tissue-specific genes. Subsequently, R-bands, the primitive chromatin state, became directionally GC rich as evidenced by Q-banding of mammalian and avian chromosomes. Contrary to predictions from the neutral mutation theory, noncoding DNA is positionally constrained along the banding pattern with short interspersed repeats in R-bands and long interspersed repeats in G-bands. Chromosomes seem dynamically stable: the banding pattern and gene arrangement along several human and murine autosomes has remained constant for 100 million years, whereas much of the noncoding DNA, especially retroposons, has changed. Several coding sequence attributes and probably mutation rates are determined more by where a gene lives than by what it does. R-band exons in homeotherms but not G-band exons have directionally acquired GC-rich wobble bases and the corresponding codon usage: CpG islands in mammals are specific to R-band exons, exons not facultatively heterochromatinized, and are independent of the tissue expression pattern of the gene. The dynamic organization of noncoding DNA suggests a feedback loop that could influence codon usage and stabilize the chromosome's chromatin pattern: DNA sequences determine affinities of----proteins that together form----a chromatin that modulates----rate constants for DNA modification that determine----DNA sequences. Theories of hierarchical selection and molecular ecology show how selection can act on Darwinian units of noncoding DNA at the genome level thus creating positionally constrained DNA and contributing minimal genetic load at the individual level.

Base Sequence↗
PubMed1990

From gene to chromosome: organization levels defined by the interplay of transcription and replication in vertebrates.

In higher eukaryotes, gene activation is accompanied by an increased sensitivity to DNaseI over a domain that extends beyond the limits of the gene itself, or of the gene cluster to which it belongs. This increased sensitivity probably reflects both the partial decondensation of chromatin and an increased communication with the outside of the nucleus. In addition, gene activation usually causes a coreplication domain that extends much beyond the decondensation domain to switch to an early replication time in S phase. This switch is produced, at least in some cases, by an early firing of origins of replication situated in flanking condensed chromatin. Some of the recently identified DNA domains that tether chromosomal loops to the nuclear matrix do represent the borders of decondensation domains. They may also constitute pausing sites for replication forks. The different replication times of successive 200- to 400-kb regions along the genome may have been the basis for the observed long-term differentiation of very large genomes in domains of different overall sequence composition (G:C content and distribution of short repeated motifs). Chromosomal bands represent a low resolution picture of this pattern. Just like gene methylation, differential replication timing and the consequent compositional differentiation of the genome have probably contributed to making the management of very large genomes workable.

Animals↗
PubMed1994

Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail and found that the interaction is saturable, of high affinity, and evolutionarily conserved. Competition studies revealed the existence of two different types of interaction related to different structural features of MARs: one involving the minor groove of double-stranded MAR DNA and one involving single-stranded regions. We obtained similar results for the interaction of MARs with intact nuclear matrices from rat liver. A model in which the interaction of nuclear matrix proteins with single-stranded MAR regions serves to stabilize the transcriptionally active state of chromatin is discussed.

Animals↗
PubMed1995

A novel cis-acting centromeric DNA element affects S. pombe centromeric chromatin structure at a distance.

The chromatin structure of the central core region of Schizosaccharomyces pombe centromeric DNA is unusual. This distinctive chromatin structure is associated only with central core sequences in a functional context and is modulated by a novel cis-acting DNA element (centromere enhancer) within the functionally critical K centromeric repeat, which is found in multiple copies in all three S. pombe centromeres. The centromere enhancer alters central core chromatin structure from a distance and in an orientation-independent manner without altering the nucleosomal packaging of sequences between the enhancer and the central core. These findings suggest a functionally relevant structural interaction between the enhancer and the centromeric central core brought about by DNA looping.

Blotting, Southern↗
PubMed2003

Dosage compensation: an intertwined world of RNA and chromatin remodelling.

Dosage compensation mechanisms in flies and mammals provide an exquisite example of chromatin associated RNAs in chromosome-wide transcription regulation. Recent progress shows that chromatin modifications are also closely linked to these processes. Concerted action of the RNA/chromatin-modifying enzymes may play a crucial role in determining transcriptional output. Furthermore, non-coding RNAs appear to play a dual role, being targeting modules as well as encoding for target sites for complex recognition.

Animals↗
PubMedDate not supplied

DNA unpacking in guinea pig sperm chromatin by heparin and reduced glutathione.

The kinetics of nuclear decondensation and DNA unpacking induced by the action of a physiological concentration of heparin and glutathione of guinea pig spermatozoa was studied. Sperm (acrosomeless) suspensions were incubated at several different temperatures (37, 40, 43, and 46 degrees C), with a constant concentration of either heparin (50 microM) or reduced glutathione (12.5 mM) and increasing concentrations of the other reagent. Nuclei spermatozoa remained highly condensed when incubated in the medium alone or in either GSH or heparin alone for up to 72 h. Swelling of nuclei spermatozoa was initially observed during the first 20 min of incubation. The sperm nuclei initiate decompaction at the central part of the nuclear structure while at the periphery there remain numerous residues of densely packed chromatin. The swollen chromatin pattern presents the characteristic organization into "hub-like" nuclear bodies that measured 10-100 nm diameter joined by a network of chromatin fibers. At full nuclei decondensation chromatin end fibers are loose, probably meaning that DNA is not organized into loop domains. DNA presence was verified by the use of ethidium bromide and acridine orange.

Animals↗
PubMed1998

Mutations synthetically lethal with cep1 target S. cerevisiae kinetochore components.

CP1 (encoded by CEP1) is a Saccharomyces cerevisiae chromatin protein that binds a DNA element conserved in centromeres and in the 5'-flanking DNA of methionine biosynthetic (MET) genes. Strains lacking CP1 are defective in chromosome segregation and MET gene transcription, leading to the hypothesis that CP1 plays a general role in assembling higher order chromatin structures at genomic sites where it is bound. A screen for mutations synthetically lethal with a cep1 null allele yielded five recessive csl (cep1 synthetic lethal) mutations, each defining a unique complementation group. Four of the five mutations synergistically increased the loss rate of marker chromosomes carrying a centromere lacking the CP1 binding site, suggesting that the cep1 synthetic lethality was due to chromosome segregation defects. Three of these four CSL genes were subsequently found to be known or imputed kinetochore genes: CEP3, NDC10, and CSE4. The fourth, CSL4, corresponded to ORF YNL232w on chromosome XIV, and was found to be essential. A human cDNA was identified that encoded a protein homologous to Csl4 and that complemented the csl4-1 mutation. The results are consistent with the view that the major cellular role of CP1 is to safeguard the biochemical integrity of the kinetochore.

Amino Acid Sequence↗
PubMed2006

INSM1 functions as a transcriptional repressor of the neuroD/beta2 gene through the recruitment of cyclin D1 and histone deacetylases.

INSM1/IA-1 (insulinoma-associated 1) is a developmentally regulated zinc-finger transcription factor, exclusively expressed in the foetal pancreas and nervous system, and in tumours of neuroendocrine origin. We have identified an INSM1 binding site in the neuroD/beta2 promoter and demonstrated transcriptional repressor activity of INSM1 by transient transfection assay. A chromatin immunoprecipitation assay confirmed that in vivo INSM1 is situated on the promoter region of the neuroD/beta2 gene. In an attempt to elucidate the molecular mechanism of transcriptional repression by the INSM1 gene, cyclin D1 was identified as an interacting protein by using a 45-day-old human foetal brain cDNA library and a yeast two-hybrid screen. The physical association between INSM1 and cyclin D1 was confirmed by in vitro and in vivo pull-down assay. Cyclin D1 co-operates with INSM1 and suppresses neuroD/beta2 promoter activity. Co-immunoprecipitation of INSM1, cyclin D1 and HDACs (histone deacetylases) in mammalian cells revealed that INSM1 interacts with HDAC-1 and -3 and that this interaction is mediated through cyclin D1. Overexpression of cyclin D1 and HDAC-3 significantly enhanced the transcriptional repression activity of INSM1 on the neuroD/beta2 promoter. A further chromatin immunoprecipitation assay confirmed that HDAC-3 occupies this same region of the neuroD/beta2 promoter, by forming a transcription complex with INSM1. Thus we conclude that INSM1 recruits cyclin D1 and HDACs, which confer transcriptional repressor activity.

Adenoviridae↗
PubMed1993

The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene.

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2006

E2A and IRF-4/Pip promote chromatin modification and transcription of the immunoglobulin kappa locus in pre-B cells.

The immunoglobulin kappa light chain (Igkappa) locus is regulated in a lineage- and stage-specific manner during B-cell development. The highly restricted timing of V to J gene recombination at the pre-B-cell stage is under the control of two enhancers, the intronic enhancer (kappaEi) and the 3' enhancer (kappaE3'), flanking the constant exon. E2A transcription factors have been indicated to be directly involved in the regulation of Igkappa locus activation. In this study, we utilize E2A-deficient pre-B cells to directly investigate the mechanism of E2A-mediated Igkappa activation. We demonstrate that Igkappa germ line transcription is severely impaired and recombination is blocked in the absence of E2A. Reconstitution of E2A-/- pre-B cells with inducible human E2A (E47R) is sufficient to promote chromatin modification of Igkappa and rescue Igkappa germ line transcription and Jkappa gene recombinase accessibility. Furthermore, we show that increased E2A recruitment to kappaEi and kappaE3' correlates with activation of Igkappa in pre-B cells and that recruitment of E2A to kappaE3' is in part dependent on the transcription factor IRF-4. Inhibition of IRF-4 expression in pre-B cells leads to a significant reduction of Igkappa germ line transcription and enhancer acetylation. In the absence of E2A, increased IRF-4 expression is not sufficient to promote Igkappa enhancer chromatin modification or transcription, suggesting that the sequential involvement of IRF-4 and E2A is necessary for the activation of the Igkappa locus. Finally, we provide genetic evidence in the mouse that E2A gene dosage can influence the development of pre-B cells during the phase of Igkappa gene activation.

Acetylation↗
PubMed1983

Rosette-like structures from nuclei with condensed (chromomeric) chromatin but not from nuclei with diffuse (nucleomeric or nucleosomic) chromatin.

The structure of chromatin of rat hepatocyte nuclei has been studied. At low ionic strength (20-50) chromatin in isolated nuclei, depending on the concentration of MgCl2 in the solution (0-2 and 4-5 mM), may be present in two states, respectively, diffuse and condensed. The major structural component of the nuclei with condensed chromatin is globular structures 100 nm in diameter, i.e. chromomers. By treating chromomer-containing nuclei with heparin and dextransulfate (polyanions/DNA equal 1), one can isolate rosette-like structures having an electron-dense core and numerous loops (the number loops in the rosette, 15-30, total length of all the loops, 15-20 micrometers, core diameter, 30-60 nm). The action of endogeneous nuclease on the nuclei and DNase I (but not RNase) on the rosette results in the break-down of the loops. Pronase or higher concentrations of polyanions (polyanions/DNA equal 4) induces partial or total decondensation of the rosette core and unfolding of the loops into a continuous linear structure. Rosette structures are not isolated from nuclei with diffuse chromatin. Rosette structures are discussed in terms of the known levels of the organization of chromatin.

Animals↗
PubMed1996

The unique, complex organization of the transcriptionally silent sperm chromatin.

The sperm nucleus contains one haploid copy of the genome that is completely transcriptionally silent and is not being replicated. Recent evidence has revealed that this "silent" chromatin nevertheless contains a complex organization at all levels. This includes DNA loop domain formation by the sperm nuclear matrix that is gene specific and highly ordered folding patterns of the chromosomes, particularly with respect to centromere and telomere positioning. Such specificity in the sperm DNA organization suggests functional requirements for their existence. As these begin to emerge, the sperm nucleus is becoming an important model for the study of the eukaryotic genome.

Animals↗