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PubMed2005

The behavior of SATB1, a MAR-binding protein, in response to apoptosis stimulation.

As a MAR-binding protein, SATB1 regulates genes by folding chromatin into a loop domain. Apoptosis is known to be accompanied by a collapse of nuclear architecture and cleavage of condensing chromatin into oligonucleosomal fragments. To further understand the functional role of MAR-binding proteins during apoptosis we investigated the relationship of the behavior of SATB1 and the collapse of nuclear architecture in Jurkat cells with immunostaining and Western blot analysis. We demonstrated that SATB1 formed special three-dimensional network distributions during early apoptosis. The distribution change of SATB1 was associated with cleavage of the protein and accompanied by the nuclear architecture collapse. Cleavage of SATB1 was mediated by caspase-3 and was apoptosis specific. Our observations further support the notion that early proteolysis of MAR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Apoptosis↗
PubMed1998

The mapping of DNA topoisomerase sites in vivo: a tool to enlight the functions of topoisomerases.

The possibility to record a trace of the precise sites of topoisomerase action has been exploited for almost 12 years in many laboratories. The large majority of the studies were performed in vitro, giving a good picture of sequence specificities of topoisomerases, and of the preference of various drugs for some sequences. Only a relatively small number of reports concern in vivo studies. Their main conclusions are the following: i) topoisomerase II sites are often found near replication origins and termini, where they are supposed to play a role in the decatenation of daughter DNA molecules, and possibly in the initiation of replication; ii) topoisomerase II sites are found in the promoter region of many genes, but they seem related to the condensation state of chromatin in this region, rather than to transcription per se; iii) some topoisomerase II sites, resistant to high salt, are found in or near matrix associated regions (MARs), suggesting a role in loop anchorage or (and) in the control of topology of individual chromatin loops; iv) topoisomerase I sites appear less localized, acting all along the transcription units, where they seem directly involved in transcription; and v) topoisomerase I sites are possibly connected with replication fork progression and (or) with the termination of replication. Despite these advances, the precise role of topoisomerases in vivo is still poorly understood, especially in recombination and chromatin condensation and decondensation during the cell cycle. Future attempts should take into account the possible specialization of the multiple topoisomerases found in a given cell, and the use of highly synchronized systems.

Animals↗
PubMed1985

Differential formation of O6-ethylguanine in the DNA of rat brain chromatin fibers of different folding levels exposed to N-ethyl-N-nitrosourea in vitro.

Extended (histone H1-depleted), 11-nm-thick chromatin fibers and condensed 25- to 35-nm-thick chromatin fibers, representing the first and second level of DNA folding in chromatin, respectively, as well as nucleosome core particles, were isolated from fetal rat brain cells and briefly exposed to N-ethyl-N-nitrosourea (EtNU) in vitro. The O6-ethyl-2'-deoxyguanosine (O6-EtdGuo):2'-deoxyguanosine (dGuo) molar ration in DNA enzymatically hydrolyzed to 2'-deoxynucleosides was determined by competitive radioimmunoassay using an anti-O6-EtdGuo monoclonal antibody with an affinity constant for O6-EtdGuo of approximately 2 X 10(10) liters/mol. In comparison to naked DNA (O6-EtdGuo:dGuo relative value, 1.0) the O6 atom of guanine proved to be increasingly protected from ethylation by EtNU in the DNA of the histone H1-depleted chromatin fibers (relative value, approximately 0.6) and of the condensed chromatin fibers (relative value, approximately 0.4). From the O6-EtdGuo:dGuo relative values obtained for the DNA of nucleosome core particles (approximately 0.5) and of H1-depleted chromatin fibers (approximately 0.6), it follows that the accessibility of the O6 atom of guanine to the electrophilic ethyldiazonium ion generated from EtNU in internucleosomal DNA (protein free) is about twice that found in nucleosomal DNA. The overall O6-EtdGuo:dGuo molar ratio in the DNA of H5 rat hepatoma cells exposed to EtNU in vitro was similar to that of the DNA of the condensed 25- to 35-nm chromatin fibers. Since the bulk of genomic DNA is organized in the form of these chromatin fibers, the overall degree of intercellular O6-EtdGuo formation appears to be mainly determined by this folding level of DNA, and not, or only to a low degree, by the DNA folding levels of higher-order chromatin structures such as those found in large loops or domains of chromatin fibers within the cell nucleus.

Alkylation↗
PubMed1994

The 30 nm chromatin fiber as a flexible polymer.

Our analysis of the data of van den Engh, Sachs, and Trask (Science 257, 1410 (1992)), for the dependence of the mean square distance between pairs of hybridization sites (< L2n >, micron 2) on the known genomic distance (n, bp) separating these sites on chromosome number 4 in G1 human fibroblast nuclei, shows that < L2n > is proportional to n2v with v = 3/5 for n < 1 Mbp. The v-value of 3/5 is characteristic of flexible polymer chains with excluded volume effects in dilute good solutions. Since the DNA concentration in nuclei is very high (ca. 1-10 mg/ml), and theory (Flory, J. Chem. Phys. 17, 303, 1949) predicts v = 1/2 for overlapping polymers, the finding of v = 3/5 means that the chromatin fibers do not overlap in interphase nuclei. The dependence of < L2n > on n for n < 4 Mbp is consistent with the model of large (approximately 6 Mbp, 3 microns diameter) loops of interphase chromatin attached to nuclear membrane sites. Using the constant (e.g., Widom, Ann. Rev. Biophys. Biophys. Chem. 18, 365 (1989)) and variable (Williams & Langmore, Biophys. J. 59, 606 (1991)) diameter fiber models, the Kuhn statistical segment of the 30 nm chromatin fiber was estimated to have a length of 196-272 nm with a corresponding DNA content of 21-37 kbp.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopolymers↗
PubMed1990

Chromatin higher-order structure: two-start double superhelix formed by zig-zag shaped nucleosome chain with folded linker DNA.

Hydrodynamic properties of chromatins differing in linker DNA length and in transcriptional activity have been studied by the method of sedimentation velocity. Oligonucleosomes of different chain length were isolated from chromatins of pigeon brain cortical neurones, rat thymus and sea urchin sperm characterized by nucleosome DNA repeat length of 165, 198 and 248 base pairs respectively. The hydrodynamic behaviour of oligonucleosomes in the dependence on the number of nucleosomes in the chain and on the ionic strength has been analysed on the basis of cylinder model. The data obtained allows one to calculate the main structural parameters of the oligonucleosomal chain: its mass per unit length, the hydrodynamic diameter of the chain, the length of the chain per nucleosome and DNA packing ratio. It is shown that hydrodynamic behaviour of nucleosome oligomers from all types of chromatins investigated at low ionic strength can be well described by the model of three-dimensional zig-zag chain with similar diameter and length of the chain per nucleosome, DNA packing ratio growing with the increase of linker DNA length. It can be achieved by unfolding the short linker DNA in neurone chromatin and by coiling the long linker DNA of sea urchin sperm chromatin into a loop. With the increase of ionic strength zig-zag shaped nucleosomal chain is condensed into a two-start double superhelix with closely arranged nucleosomes and linker DNA loops packed inside the superhelix. The suggested model is in good agreement with available experimental data and overcomes a number of difficulties which arise for the solenoid model and other models of the 30-nm chromatin fibril.

Animals↗
PubMed2002

The myogenic basic helix-loop-helix family of transcription factors shows similar requirements for SWI/SNF chromatin remodeling enzymes during muscle differentiation in culture.

The myogenic basic helix-loop-helix family of transcription factors, MyoD, Myf5, myogenin, and MRF4, can each activate the muscle differentiation program when ectopically expressed in non-muscle cells. SWI/SNF complexes are ATP-dependent chromatin remodeling enzymes. We demonstrated previously that SWI/SNF enzymes promote MyoD-mediated muscle differentiation. To ascertain the requirement for SWI/SNF enzymes in muscle differentiation mediated by different MyoD family members, we examined MyoD, Myf5, MRF4, and myogenin-mediated induction of muscle differentiation in cells expressing dominant negative versions of BRG1 or BRM-based SWI/SNF enzymes. We demonstrated that expression of dominant negative BRG1 or BRM inhibited the induction of muscle-specific gene expression by Myf5 and MRF4; however, myogenin failed to induce measurable quantities of muscle-specific mRNAs, even in cells not expressing dominant negative SWI/SNF. In contrast, all four myogenic regulators induced expression of the cell cycle regulators p21, Rb, and cyclin D3 and promoted cell cycle arrest independently of the SWI/SNF enzymes. We proposed that SWI/SNF enzymes are required for the induction of all muscle-specific gene expression by MyoD, Myf5, and MRF4, whereas induction of the cell cycle regulators, p21, Rb, and cyclin D3 occurred independently of SWI/SNF function.

3T3 Cells↗
PubMed2002

Chromatin (dis)organization and cancer: BUR-binding proteins as biomarkers for cancer.

Malignant transformation of cells is associated with changes in gene expression. Gross alterations in chromatin organization may be involved in such gene dysregulation, as well as the involvement of specific transcription factors. Specialized genomic DNA segments that exhibit high affinity to the nuclear matrix in vitro have been designated as matrix/scaffold attachment regions (MARs/SARs). MARs are postulated to anchor chromatin onto the nuclear matrix, thereby organizing genomic DNA into topologically distinct loop domains that are important in replication and transcription. In support of this notion, MARs often colocalize or exist in close proximity to regulatory sequences including enhancers. Base unpairing regions (BURs) are typically 100-150 bp regions within MARs, possess an intrinsic propensity to unwind under negative superhelical strain, and are considered to be hallmark of MARs. To investigate a potential mechanism that could lead to significant alterations in gene expression in cancer cells, this review focuses on a group of chromatin-associated proteins that specifically recognize double stranded BURs. Several important proteins have been identified from cancer cells as BUR-binding proteins, including poly (ADP-ribose) polymerase (PARP-1), Ku autoantigen, SAF-A, HMG-I(Y), nucleolin and p53. Many of these proteins are dramatically upregulated in malignancy of the breast. Increase in the amount of these BUR-binding proteins, some of which are known to interact with each other, may not only provide an architectural core but also recruit functional multi-molecular complexes at the base of chromatin loops to affect multiple distant genes. Experimental strategies by which these proteins can be exploited as carcinoma-specific diagnostic markers and as targets for antineoplastic therapy are discussed.

Animals↗
PubMed1994

The telochore: a telomeric differentiation of the chromosome axis.

We have analysed by means of silver staining the structure of the chromosome axis at the telomeres of meiotic chromosomes in three different grasshopper species. At metaphase I the chromatid axes run the length of the chromatids although they do not reach the chromosome ends. The axes of sister chromatids are associated and show a round differentiation at their distal ends that we have named the 'telochore'. Telochores never contact the chromosome ends: there is always some chromatin beyond them. In late metaphase I bivalents with a distal chiasma, anaphase I and metaphase II half-bivalents and anaphase II chromatids, the axes clearly possess one telochore in each chromosome end. These results seem to indicate that telochores are differentiations of the distal ends of chromatids. We discuss the possible structural significance of telochores according to the current scaffold/radial loop model of chromatin organization of eukaryotic metaphase chromosomes. Additionally, we suggest the possible functional role of the telochore as a nucleoprotein domain forming a protective cap for telomeric DNA.

Animals↗
PubMed1994

Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos.

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

Cell Cycle↗
PubMed1992

Different patterns of rDNA organization at interphase in nuclei of wheat and rye.

The physical location of the rDNA repeating units (25 S, 18 S and 5.8 S rRNA genes and the intergenic spacer sequences) was investigated in rye (Secale cereale L.) and wheat (Triticum aestivum L.) root tip meristematic cells by in situ hybridization using light and electron microscopy. The rDNA sequences are organized differently in the two related and intercrossable species. In rye (2n = 14, one pair of chromosomes with nucleolar organizing regions, NORs), two condensed blocks of rDNA-containing chromatin occurred in each interphase nucleus. The blocks were associated with the periphery of nucleoli and a single-labelled, decondensed rDNA fibre extended into the nucleolus from the block. We term this expression pattern terminal decondensation. In wheat (2n = 6x = 42, five pairs of chromosomes with NORs), inactive condensed labelled chromatin was found unassociated with nucleoli. Active NORs had some condensed rDNA associated with the nucleolar periphery, but, in contrast to rye, condensed rDNA was also found within the nucleolus. The condensed labelled rDNA in wheat nucleoli was visible as fluorescent foci in the light microscope and labelled condensed chromatin in the electron microscope. Its absence in rye shows that condensed rDNA need not be present in active plant nucleoli. Diffuse labelled sites of rDNA, likely to represent actively transcribed rDNA, were found in both rye and wheat. Active rDNA loci in wheat have many expressed segments separated by unexpressed, condensed, rDNA-fragmented decondensation-while each locus in rye has a single, unexpressed perinucleolar condensed block of rRNA genes. Thus the positions of actively transcribed genes within the tandem arrays of rDNA at each locus are fundamentally different in the two cereals. The NOR chromosome appeared to extend through the nucleolus, and active rDNA sequences did not loop out from chromatin into the nucleolus as is frequently described in nucleolar models.

Cell Nucleolus↗
PubMed1984

Packing of a specific gene into higher order structures following repression of RNA synthesis.

Transcription of the Balbiani ring (BR) genes of the dipteran Chironomus tentans was inhibited by teh nucleoside analogue DRB (5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole). The BR genes were emptied of RNA polymerases and the subsequent packing of the genes was monitored by transmission electron microscopy. The thin chromatin axis of the transcriptionally active genes condensed into a thick (20-25 nm) chromatin fiber, which was recorded as a linear structure, an open loop or a supercoiled loop. The compacted genes were finally packed into dense clumps of chromatin. It was proposed that upon repression of RNA synthesis the BR gene template attains the following consecutive stages with increasing compaction: transcription loop----linear thick fiber----open thick fiber loop----supercoiled thick fiber loop----dense chromatin. Within the chromatin blocks structures that resembled the supercoiled loops were discerned, suggesting that the final packing of the template might be accomplished by a close alignment of supercoiled loops.

Animals↗
PubMed2006

Estimating the prevalence and regulatory potential of the telomere looping effect in yeast transcription regulation.

Telomeres have long been implicated in the regulation of gene expression. Some studies have reported that telomere looping effect (TLE) can juxtapose genes and regulatory sequences that are far apart and facilitate long-distance control of gene expression. In this work, we report a detailed investigation on the prevalence and regulatory potential of TLE on a genomic scale by assembling data on protein-DNA interactions from several large-scale ChIp-chip experiments in Saccharomyces cerevisiae. Analysis of the assembled data revealed that a statistically significant number of DNA segments that were inferred to be bound by ten or more transcription factors in these experiments physically mapped to the ends of several chromosomes (19 of 32 chromosome ends). For the 83 transcription factors that were inferred to interact with these DNA segments, we found a statistically significant skew in the distribution of their internal binding sites over the length of the entire chromosome, such that more than expected binding events occurred proximal to chromosomal ends than elsewhere. Taken together these observations suggest that the telomere looping effect is their most likely explanation and imply that a notable fraction of the internally bound yeast transcription factors potentially interact with looped back telomeres. Further, we also identified several components of the basal transcriptional machinery that are also frequently linked to these chromosome end segments, strengthening the proposal for a direct interaction between the chromosome ends and internally located transcriptional complexes. We observed that certain chromatin factors might participate in the TLE and potentially modulate gene expression by chromatin modifications such as histone deacetylation. Our findings provide the first computational evidence for a significant role of long-range regulatory interactions due to telomere looping. Based on these observations, we also propose that genome-wide chromatin immunoprecipitation data might be useful to systematically uncover long-range chromatin looping effects in gene expression.

Binding Sites↗
PubMed1998

Identification of scaffold/matrix attachment region in recurrent site of woodchuck hepatitis virus integration.

Scaffold or matrix attachment regions (S/MARs) are noncoding genomic DNA sequences displaying in vitro selective binding affinity for nuclear scaffold. They have been reported to be involved in the physical attachment of genomic DNA to the nuclear scaffold, and thus in the organization of the chromatin in functional loops or domains, and in the regulation of gene expression. In this work, we report the identification of an S/MAR in a woodchuck chromosomal locus, named b3n, previously described as a recurrent site of woodchuck hepatitis virus (WHV) DNA integration in woodchuck hepatocellular carcinoma (HCC). The 4.3-kb sequence of this locus contains several Alu-like repeats and a gag-like coding region with frameshift mutations. Computer analysis revealed the presence of a region with unusually high AT content, typical of most S/MARs, and of specific motifs (A boxes, T boxes, topoisomerase II sites, and unwinding elements) overlapping or in proximity to the region with high AT content, predicting that b3n might contain an S/MAR. Fragments of the b3n locus were isolated by conventional and inverse PCR techniques. In in vitro binding experiments with both heterologous and autologous scaffold preparations, a 592-bp fragment spanning the region rich in S/MAR features showed marked scaffold affinity, which was specific when autologous scaffolds were used. The presence of an S/MAR at the b3n locus and its nature as a recurrent WHV integration site in HCC suggest the involvement of S/MAR elements in some of the mechanisms leading to liver oncogenesis.

Amino Acid Sequence↗
PubMed2002

Dermo-1, a multifunctional basic helix-loop-helix protein, represses MyoD transactivation via the HLH domain, MEF2 interaction, and chromatin deacetylation.

Dermo-1 is a multifunctional basic helix-loop-helix (bHLH) transcription factor that has been shown to be a potent negative regulator for gene transcription and apoptosis. To understand the molecular mechanisms that mediate the function of Dermo-1, we generated a series of Dermo-1 mutants and used a MyoD-mediated transcriptional activation model to characterize the roles of its N-terminal, bHLH, and C-terminal structural domains in transcriptional repression. Both the C-terminal and HLH domains of Dermo-1 were essential for its repression of MyoD-mediated transactivation. Dermo-1 repressed, in a dose-dependent fashion, the transactivation activity of myocyte enhancer factor 2 (MEF2), a protein known to cooperate with MyoD in activating E-box-dependent gene expression. Both the N- and C-terminal domains of Dermo-1, but not the bHLH domain, were required for the inhibition of MEF2, suggesting that Dermo-1 inhibits both MyoD- and MEF2-dependent transactivation but through different mechanisms. Dermo-1 interacted directly with MEF2 and selectively repressed the MEF2 transactivation domain. An overall increase of histone acetylation induced by trichostatin A treatment reduced Dermo-1 transcriptional repression activity, suggesting that histone deacetylation is involved in Dermo-1-mediated transcriptional repression. Together, these results suggest that MEF2 is an important target in Dermo-1-mediated transcriptional repression and provide initial evidence of the involvement of histone acetylation in Dermo-1 transcriptional repression.

Acetylation↗
PubMed1999

Lethal osteosclerotic skeletal dysplasia with intracellular inclusion bodies.

We report an apparently previously undescribed form of lethal osteosclerotic skeletal dysplasia in a 30-week male fetus with micromelic shortness of the limbs. Radiographic findings at necropsy included increased density in all bones, most marked in the skull, mandible, and pubis. The ribs were very short, abnormally modeled, and wide anteriorly. The vertebrae were posteriorly hypoplastic and wedged, particularly in the cervical and lumbar regions. The femora and tibiae were short with wide distal metaphyses, undermodeled diaphyses, and coxa vara. The humeri, radii, and ulnae were also short and undermodeled with proximal and distal flare. Chondro-osseous morphology showed short chondrocyte columns, extension of hypertrophic cells into the metaphysis, and overgrowth of perichondral bone. In the resting cartilage there were large chondrocytes containing a homogeneous material staining pink with von Kossa trichrome, gray with toluidine blue, and black with silver methenamine. The cortical bone was lacking and the trabecular bone was hypercellular, thick, and coarse. Ultrastructurally, the resting zone chondrocytes were large and round with condensed chromatin and dilated loops of rough endoplasmic reticulum. The radiographic and histopathologic findings in this case are unique and differ from those seen in other reported lethal osteosclerotic skeletal dysplasias.

Abnormalities, Multiple↗
PubMed2005

Solving mysteries of DNA replication and frog cloning.

Compared to sperm nuclei, nuclei from adult somatic cells replicate inefficiently in frog egg extract. In this issue of Cell, Lemaitre et al. (2005) show that pre-exposure of erythrocyte nuclei to a mitotic extract removes this difference, reorganizes the chromatin into shorter loops, and allows replication at much shorter intervals along the DNA. Remarkably, these observations also explain an old mystery of why serial nuclear transplantation was so successful for cloning frogs.

Animals↗
PubMed2004

Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.

Chromatin↗
PubMed1999

Origin and roles of nuclear matrix proteins. Specific functions of the MAR-binding protein MeCP2/ARBP.

HnRNP proteins are the major protein components of the nuclear matrix, and sites of nascent transcripts and RNA maturation are its main sources. The evidence for and the roles of functional and structural loops in interphase chromatin and metaphase chromosomes is discussed. Recent data suggest a specific role for the matrix attachment region (MAR)- and methyl-CpG-binding protein MeCP2/ARBP. This repressor protein binds to MARs and, through interaction with mSin3A, recruits a corepressor complex containing histone deacetylases. This in turn is thought to generate a localized silenced chromatin structure. Transfection experiments are presented in support of this model.

Animals↗