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PubMed2006

Selective matrix attachment regions in T helper cell subsets support loop conformation in the Ifng gene.

Cytokine genes undergo progressive changes in chromatin organization when naïve CD4+ T helper (Th) cells differentiate into committed Th1 and Th2 lineages. Here, we analyzed nuclear matrix attachment regions (MARs) in the Ifng gene by DNA array technique in unactivated and activated CD4+ Th cells. This approach was combined with analysis of spatial organization of the Ifng gene by chromosome conformation capture approach to assess the relationship between the gene conformation and matrix attachment organization in functionally different cell subsets. We report that the Ifng gene in unactivated cells displays a linear conformation, but in T-cell receptor-activated cells, it adopts a loop conformation. The selective MARs support the spatial gene organization and characteristically define the Ifng gene in functionally different cell subsets. The pattern of interaction of the Ifng gene with the nuclear matrix dynamically changes in a lineage-specific manner in parallel with the changes in Ifng gene conformation. The data suggest that such structural dynamics provide the means for transcriptional regulation of the Ifng gene in the course of activation and differentiation of CD4+Th cells.

Animals↗
PubMed1998

Partial denaturation of small chromatin fragments: direct evidence for the radial distribution of nucleosomes in folded chromatin fibers.

To examine the internal structure of chromatin fibers, we have developed procedures for partial denaturation of small chromatin fragments (8-30 nucleosomes) from chicken erythrocytes. Electron micrographs of samples prepared under conditions that cause nucleosome dissociation show rods and loops projecting from short compact fibers fixed by glutaraldehyde in 1.7 mM Mg2+. According to previous studies in our laboratory, these images correspond to the top view of partially denatured fibers. Our results indicate that rods and loops consist of extended duplex DNA of different lengths. DNA in loops is nicked, as demonstrated by experiments performed in the presence of high concentrations of ethidium bromide. Length measurements indicate that the radial projections of DNA are produced by unfolding of nucleosomal units. Loops are formed by DNA from denatured nucleosomes in internal positions of the fiber; DNA from denatured nucleosomes in terminal positions form rods. Our micrographs show clearly a radial distribution of DNA loops and rods projecting from fibers. Rods are orthogonal to the surface of the chromatin fragments. Considering that the high ionic strength used in this study (0.8-2.0 M NaCl) neutralizes the electrostatic repulsions between rods and fiber, this observation suggests that rods are extensions of nucleosomes radially organized inside the fiber. The position of the entry points of DNA loops into the fiber could be influenced by constraint on loops, but our results showing that the arc that separates these points in dinucleosome loops is relatively short suggest that consecutive nucleosomes are relatively close to each other in the folded fiber.

Animals↗
PubMed2002

Organization of higher-level chromatin structures (chromomere, chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization.

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.

Animals↗
PubMed2025

An electrostatic repulsion model of centromere organisation.

During cell division, chromosomes reorganise into compact bodies in which centromeres localise precisely at the chromatin surface1-4 to enable kinetochore-microtubule interactions essential for genome segregation5-8. The physical principles guiding this centromere positioning remain unknown. Here, we reveal that human core centromeres are directed to the chromatin surface by repulsion of centromere-associated proteins - independent of condensin-mediated loop extrusion and microtubule engagement. Using cellular perturbations, biochemical reconstitution, and multiscale molecular dynamics simulations, we show that chromatin surface localisation emerges from repulsion between condensed chromatin and both the kinetochore and the highly negatively charged centromere protein, CENP-B. Together, these elements form a centromeric region composed of two domains with opposing affinities, one favouring integration within the mitotic chromosome and the other favouring exposure to the surrounding cytoplasm, thereby driving surface positioning. Tethering synthetic negatively charged proteins to chromatin was sufficient to recapitulate this surface localisation in cells and in vitro, indicating that electrostatic repulsion is a key determinant of surface localisation. These findings demonstrate that centromere layering is not hardwired by chromatin folding patterns but instead emerges from phase separation in chromatin. Our work uncovers electrostatic polarity as a general and programmable mechanism to spatially organise chromatin.

Journal Article↗
PubMed2001

Sequential recruitment of steroid receptor coactivator-1 (SRC-1) and p300 enhances progesterone receptor-dependent initiation and reinitiation of transcription from chromatin.

Employing a cell-free chromatin transcription system that recapitulates progesterone receptor (PR)-mediated transcription in vivo, we have investigated further the coactivator functions of steroid receptor coactivator-1 (SRC-1) in terms of its functional domains as well as cooperation with other coactivators in PR transactivation. By analyzing wild-type and mutant SRC-1 with liganded PR in the chromatin transcription system in vitro, the basic helix-loop-helix/Per-Arnt-Sim domain, the p300-binding domain, and the carboxyl-terminal region (containing the PR-binding site) of SRC-1 were shown to be important for PR transactivation. Although in context of a synthetic promoter its histone acetyltransferase activity was nonessential for PR-mediated transcription, SRC-1 was observed to act synergistically with p300 to enhance PR transactivation from chromatin. Moreover, SRC-1 and p300 were found to function cooperatively to increase the efficiency of productive transcription initiation and reinitiation. Further analysis of synergism between SRC-1 and p300 revealed an obligatory "sequential" recruitment of SRC-1 and p300 to liganded PR. Efficient recruitment of p300 required the presence of SRC-1. In addition, functional analysis of SRC-2 and SRC-3 coactivators indicated that the SRC family modulated PR transactivation from chromatin by a similar mechanism.

Acetyltransferases↗
PubMed1993

Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa.

In the four decades since the discovery of the basic structure of the DNA double helix, researchers have been investigating the more dynamic tertiary structures that DNA assumes in the various forms of chromatin. The tertiary structure of DNA is important because it is directly related to the function of the genome: for the cell to access the information that is present in the genome accurately and efficiently, the DNA must be in an organized form. This paper reviews the recent work on one particular enigmatic structural form of eukaryotic DNA, that of the highly condensed spermatozoa. Based on the literature and on recently completed experiments in the field, a new model for DNA packaging in sperm nuclei is presented. In this model, each individual DNA loop domain in the sperm chromatin is condensed into a toroid-shaped structure termed the DNA loop doughnut.

Animals↗
PubMed1995

Microvascular pattern in the metaphysis during bone growth.

BACKGROUND: Little is known about the three-dimensional micromorphology of vessels in the growth zone of long bones, where significant vasculogenesis occurs. Therefore, we examined the microvascular pattern of the femoral metaphysis. METHODS: Six-week-old normal rats of either sex were used. We cast the femurs of 14 rats with Mercox for scanning electron microscopy (SEM), and in 10 rats we prepared tissue sections of femurs for light (LM) and transmission electron microscopy (TEM). RESULTS: In the LM, calcified cartilage was found to define cylindrical compartments beneath the last row of hypertrophied chondrocytes of the metaphyseal growth plate. These compartments ran in the bone's longitudinal axis and contained a single capillary profile. Endothelial cells of these capillaries often showed increased cytoplasmic volume and loose texture of nuclear chromatin. Cast metaphyses by SEM showed numerous parallel vascular loops with nodular protrusions 10-12 microns in diameter at their tips. The loops had ascending and descending limbs with a luminal diameter of 10-14 microns. Small projections 4-5 microns in diameter and delicate crests were sometimes found on the tip of the larger nodes. In a 100 x 100 microns area, there were 14-17 large nodes. By TEM, capillary sprouts were identified at the level beneath the last row of hypertrophied chondrocytes. These capillaries had voluminous endothelial cells rich in free ribosomes and rough endoplasmic reticulum. Endothelial cell nuclei were rounded and showed loose chromatin texture. Endothelial cells were connected by intermediate junctions and there was no basal lamina. Deeper into the metaphysis, arterioles and sinusoids were present. CONCLUSIONS: We conclude that the metaphyseal plate of the growing rat offers an optimal model to study vasculogenesis. Capillary sprouts can be readily identified, measured, and counted because they are located within a plane bordering against avascular cartilage. In addition, by using microvascular corrosion casting in SEM not only capillary sprouting per se but also different stages of neovascularization, indicated by differently sized nodular projections at the tip of vascular loops, can be studied in the growing long bone.

Animals↗
PubMed1997

Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization.

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60-80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Animals↗
PubMedDate not supplied

Sperm chromatin.

Available data on dry and hydrated nuclear volume of mammalian spermatozoa indicate that available volume is clearly insufficient to contain sperm chromatin packed in nucleosome-like structures. Therefore, sperm DNA-protein complexes must be packed differently than somatic DNA-protein complexes. Packing of DNA in fixed, dehydrated mammalian sperm approaches the physical limits of molecular compaction, making mammalian sperm chromatin the most condensed eukaryotic DNA known. The fundamental packaging unit of sperm chromatin is a toroid approximately 900-A outer diameter. 200-A thickness, and 150-A diameter hole. Each toroid contains 60 kilobases of DNA and is linked to other toroids by uncoiled DNA stretches. The factors that contribute to mammalian chromatin structuration are still under study. The role of protamines in sperm chromatin condensation and nuclear shaping has been overstressed to the exclusion of other possible factors. Chromatin organization in sperm nuclei is maintained during sperm condensation by tight interactions with the nuclear matrix at fixed sites, inducing the formation of individual toroid-shaped DNA loop stuctures. Observations that abnormal manchettes affect sperm head shape and chromatin organization inducing sterility speak about manchette importance during chromatin organization. The presence in sperm chromatin of regions packaged in specific ways with several types of protamines or even with histones, indicates that nuclear shaping and chromatin organization must be under DNA control. The structural properties that distinguish sperm DNA from somatic DNA may play the most important role in chromatin organization.

Animals↗
PubMed2002

Sperm nuclear halos can transform into normal chromosomes after injection into oocytes.

Mouse sperm nuclei extracted with an ionic detergent and 2 M NaCl retain their overall morphology, but upon subsequent reduction of the protamine disulfides they lose all elements of chromatin structure except the organization of DNA into loop that are anchored to the nuclear matrix. These DNA loops appear as a halo surrounding the nuclear matrix, and nuclei extracted in this manner are, therefore, called nuclear halos. Here, we report that sperm nuclear halos injected into oocytes can form pronuclei, then transform into chromosomes with normal morphology. This suggests that sperm nuclear halos retain all the information necessary for normal chromosomal organization, and that micromanipulation of these extracted sperm nuclei can be accomplished without major DNA damage.

Animals↗
PubMed2006

ERK modulates DNA bending and enhancesome structure by phosphorylating HMG1-boxes 1 and 2 of the RNA polymerase I transcription factor UBF.

Transcription of the ribosomal RNA genes of mammals by RNA polymerase I is rapidly activated by epidermal growth factor via the MAP-kinase (ERK) signaling cascade. This activation is mediated by direct phosphorylation of the HMG box DNA binding domains of the architectural transcription factor UBF. Mutation of the ERK sites of UBF inhibits its normal function and blocks growth factor activation of ribosomal transcription. UBF has little or no DNA sequence selectivity and binds throughout the ribosomal genes, defining a specialized chromatin. Indeed, the HMG boxes of UBF induce looping of the ribosomal DNA to create the enhancesome, a structure somewhat reminiscent of the nucleosome. Here, we show that both ERK phosphorylation and mutations that simulate this phosphorylation decrease the affinity of the individual HMG boxes of UBF for linear ribosomal DNA but have little or no effect on the capacity of these HMG boxes to bind to pre-bent DNA and do not affect the overall binding constant of UBF for the DNA. Electron spectroscopic imaging showed that ERK site UBF mutants do not induce the characteristic DNA looping of the enhancesome and associate with no more than half of the enhancesomal DNA. The data demonstrate that ERK phosphorylation of UBF prevents DNA bending by its first two HMG boxes, leading to a cooperative unfolding of the enhancesome.

Amino Acid Sequence↗
PubMed2005

Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis.

Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes.

3T3 Cells↗
PubMed1987

Preferential DNA repair in active genes.

We have demonstrated that essential genes in rodent cells and in normal human cells are preferentially repaired after UV damage. In rodent cells, some genes are repaired much more efficiently than the bulk of the genome. This may explain the long standing paradox that their overall genome repair is low, but their UV survival is as high as for proficiently repairing human cells. Rodent cells appear to repair efficiently only genomic regions of vital importance. In normal human cells, we have found that genes are repaired faster than the bulk of the genome, but eventually (after 24 hrs) all genomic regions are proficiently repaired. The demonstration of preferential DNA repair mandates caution in interpreting correlations between overall DNA repair capacity and other biological parameters. Changes in preferential DNA repair could have profound effects on such parameters without noticeably altering overall genome repair levels since the vital regions only constitute less than 1% of the genome. We have correlated overall genome repair, repair in the DHFR gene, and UV resistance for three different cell lines: CHO, XPC and normal human. The results further suggest that determinations of DNA repair in specific genomic sequences may be more important than overall DNA repair measurements for correlations to other biological end points such as resistance to UV damage. Although DNA repair heterogeneity has been demonstrated in XPC, we have found that the preferentially repaired regions in these cells do not include the essential DHFR gene. DNA repair may normally be regulated over the genome in a similar manner to that for transcription, and we propose that this regulation is deficient in the human DNA repair deficient syndrome XPC. We have also analyzed the genomic fine structure of DNA repair in and around the DHFR gene in CHO cells. We find a region of preferential DNA repair of approximately 60-80 kb in length with maximal DNA repair efficiency at the 5' end of the gene and in its 5' flanking sequences. This size corresponds very well with proposed and measured lengths for loops or domains of higher order structure in chromatin, and suggests that DNA repair efficiency in genomic regions might reflect aspects of local chromatin structure, and thus provide us with a probe for the detection of chromatin structural changes. We have found considerable differences in the repair efficiency of different genes within the same cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Lampbrush chromosomes and gene utilization in meiotic prophase.

The main features of lampbrush chromosome organization are reviewed and the significance of RNA transcription on lampbrush loops is questioned. Special consideration is given to evidence for the transcription on lampbrush loops of satellite DNA, low copy number genes with defined functions, the histone genes, the 5S genes and the genes for ribosomal RNA. It is concluded that there is widespread but somewhat indiscriminate transcription on lampbrush loops of a range of repetitive DNA sequences, transcription of a wide range of mRNAs, continuous transcription of histone and 5S RNA, and low or aberrant transcription of ribosomal RNA. The 'read-through' hypothesis of lampbrush loop transcription is explained and evaluated, and some of the assumptions underlying it and the problems it raises are examined and discussed. The hypothesis requires that transcription starts at a normal promoter site for a functional gene situated at the thin end of a lampbrush loop, and that once started the transcribing polymerase cannot stop until it reaches another promoter that is already initiated or some condensed and untranscribable chromatin. The following questions are considered. Why should polymerase on a lampbrush loop disregard normal termination signals? Why should polymerase stop when it encounters another initiated promoter sequence? Why is the number of loops or transcription units related to genome size signifying, according to the read-through hypothesis, that oocytes from animals with large genomes have more active 'functional gene promoters' than those from animals with small genomes? Finally, some special situations where there is enhanced or reduced lampbrush activity are considered and their significance in relation to ideas about the function of lampbrush chromosomes is discussed. Specific examples include the frog, Ascaphus truei, whose oocytes have eight germinal vesicles each with a full complement of lampbrush chromosomes, another frog, Flectonotus pygmaeus, in which each oocyte starts with several thousand meiotic nuclei only some of which go into a lampbrush phase, and certain species of reptile in whose germinal vesicles the chromosomes never acquire a lampbrush form.

Animals↗
PubMed2002

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.

Amino Acid Sequence↗
PubMed1976

Visualization of prokaryotic DNA in a regularly condensed chromatin-like fiber.

Electron microscopy of disrupted Escherichia coli cells under certain conditions revealed loops of a fiber 120 A in diameter which were attached to the cell envelope and showed a 130 A repeating beaded substructure. These fibers were detected only when the cells were lysed in 0.15 M NaCl solutions directly on the electron microscope supporting films and if the dehydration steps began within 2 min of lysis. Under these conditions examination of cells lysogenic for phage lambda after superinfection with lambda wild type or deletion mutants disclosed short loops of a 120 A diameter fiber free of the cell envelope. Because the contour length of these loops was proportionate to the DNA content of the superinfecting lambda phage, it was concluded that the fibers contained DNA condensed 6.5-fold in blocks of about 250 base pairs.

Chromatin↗
PubMed1999

Compartmentalization of interphase chromosomes observed in simulation and experiment.

Human interphase chromosomes were simulated as a flexible fiber with excluded volume interaction, which represents the chromatin fiber of each chromosome. For the higher-order structures, we assumed a folding into 120 kb loops and an arrangement of these loops into rosette-like subcompartments. Chromosomes consist of subcompartments connected by small fragments of chromatin. Number and size of subcompartments correspond with chromosome bands in early prophase. We observed essentially separated chromosome arms in both our model calculations and confocal laser scanning microscopy, and measured the same overlap in simulation and experiment. Overlap, number and size of chromosome 15 subcompartments of our model chromosomes agree with subchromosomal foci composed of either early or late replicating chromatin, which were observed at all stages of the cell cycle and possibly provide a functionally relevant unit of chromosome territory compartmentalization. Computed distances of chromosome specific markers both on Mb and 10-100 Mb scale agree with fluorescent in situ hybridization measurements under different preparation conditions.

Cell Cycle↗
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↗