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PubMed2001

Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex.

Chromatin domain boundaries, like scs or gypsy insulators in Drosophila, have been identified in transgene assays through their enhancer-blocking activity. Boundary elements in the bithorax complex (BX-C), such as Fab-7 and Fab-8, have been identified genetically and been shown to have insulator activity in transgene assays. However, it is not clear whether boundary elements identified in transgene assays will function appropriately in chromosomal contexts such as BX-C. Using gene conversion, we have substituted the scs or gypsy insulators for Fab-7. We find that both scs and gypsy are very potent insulators in the ectoderm, but surprisingly, the insulating activity of gypsy (but not scs) is lost in the CNS. Our results reveal that the Fab-7 boundary must have special properties that scs and gypsy lack, which allow it to function appropriately in BX-C regulation.

Abdomen↗
PubMed2000

Methylated DNA sequences in genomic imprinting.

Genomic imprinting is a special form of epigenetic system that determines the parent-of-origin-specific, or monoallelic, expression of a small number of genes, termed "imprinted" genes. Considerable sequence and methylation analysis of imprinted genes has revealed a common theme: Regions of allele-specific methylation inherited from the gametes, or primary differentially methylated regions (DMRs), are associated with CpG islands and repeat elements, and this overall structure suggests functional significance. For at least three imprinted genes the sequence of the primary DMR constitutes an element able to regulate gene activity in cis--a chromatin insulator and a promoter of an antisense transcript. In these cases the unique feature of imprinting appears to be in the ability to switch the regulatory capacity of these elements on or off by the absence or presence of inherited methylation. Increasing evidence therefore suggests that genomic imprinting for at least some genes constitutes the regulation of gene regulatory elements by methylation. An important challenge now is to determine how the differential methylation of primary DMR sequences is established in the germ line. If methylation is the primary imprint, then the processes establishing it are the primary imprinting mechanisms. Trans-acting factors that are expressed in one sex of germ line and not the other are likely to be involved, and their ability to methylate may be mediated through repeat elements associated with the sequence of primary DMRs.

Alleles↗
PubMed1996

Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency.

The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression.

Animals↗
PubMed2004

Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5' and 3' regions and including the enhancer blocker sns.

The tandem repeated sea urchin alpha-histone genes are developmentally regulated by gene-specific promoter elements. Coordinate transcription of the five genes begins after meiotic maturation of the oocyte, continues through cleavage, and reaches its maximum at morula stage, after which these genes are shut off and maintained in a silenced state for the life cycle of the animal. Although cis regulative sequences affecting the timing and the level of expression of these genes have been characterized, much less is known about the mechanism of their repression. Here we report the results of a functional analysis that allowed the identification of the sequence elements needed for the silencing of the alpha-H2A gene at gastrula stage. We found that important negative regulative sequences are located in the 462 bp sns 5 fragment located in the 3' region. Remarkably, sns 5 contains the sns enhancer blocking element and the most 3' H2A codons. In addition, we made the striking observation that inhibition of the anti-enhancer activity of sns, by titration of the binding proteins in microinjected embryos, also affected the capability of sns 5 to down-regulate transgene expression at gastrula stage. A further sequence element essential for repression of the H2A gene was identified upstream of the enhancer, in the 5' region, and contains four GAGA repeats. Altogether these findings suggest that down-regulation of the alpha-H2A gene occurs by the functional interaction of the 5' and 3' cis sequence elements. These results demonstrate the involvement of a genomic insulator in the silencing of gene expression.

Animals↗
PubMed2005

Islet-infiltrating B-cells in nonobese diabetic mice predominantly target nervous system elements.

B-cells accumulate in pancreatic islets during the autoimmune response that precedes the onset of type 1 diabetes. However, the role and antigenic specificity of these cells remain a mystery. To elucidate the antigenic repertoire of islet-infiltrating B-cells in type 1 diabetes, we generated hybridoma cell lines of islet-infiltrating B-cells from nonobese diabetic (NOD) mice and NOD mice expressing a diabetogenic T-cell receptor (8.3-NOD). Surprisingly, characterization of the tissue specificity of the antibodies secreted by these cells revealed that a predominant fraction of these hybridomas produce antibodies specific for the pancreatic nervous system. Similar results were obtained with B-cell hybridomas derived from mild insulinic lesions of diabetes-resistant (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice. Immunoglobulin class analyses further indicated that most islet-derived hybridomas had arisen from B-cells that had undergone immunoglobulin class switch recombination, suggesting that islet-associated B-cells are involved in active, T-helper-driven immune responses against local antigenic targets. This is the first evidence showing the existence of a predominant active B-cell response in situ against pancreatic nervous system elements in diabetogenesis. Our data are consistent with the idea that this B-cell response precedes the progression of insulitis to overt diabetes, thus strongly supporting the idea that pancreatic nervous system elements are early targets in type 1 diabetes.

Animals↗
PubMed2000

The Su(Hw) chromatin insulator protein alters double-strand break repair frequencies in the Drosophila germ line.

P element-induced double-strand breaks (DSBs) on the X chromosome of Drosophila melanogaster were repaired up to four times more frequently when functional Su(Hw) chromatin insulator protein was removed from all genomic binding sites. Simultaneous comparisons of interallelic gap repair frequencies at two target loci on the X chromosome confirmed that a Su(Hw) binding site nested within a template had no effect on DSB repair efficiency. The results suggest that the genome-wide homology search of broken ends for homologous template sequences is affected because it is the only step in the recombinational repair process with an apparent genome-wide interaction. We propose that the searching 3'-hydroxy ends gain a higher degree of freedom for the search in a su(Hw) mutant background.

Animals↗
PubMed2002

Hybrid density-functional theory and the insulating gap of UO2.

We report the first calculations carried out with a periodic boundary condition code capable of examining hybrid density-functional theory (DFT) for f-element solids. We apply it to the electronic structure of the traditional Mott insulator UO2, and find that it correctly yields an antiferromagnetic insulator as opposed to the ferromagnetic metal predicted by the local spin density and generalized gradient approximations. The gap, density of states, and optimum lattice constant are all in good agreement with experiment. We stress that this results from the functional and the variational principle alone. We compare our results with the more traditional approximations.

Journal Article↗
PubMed2005

Mechanisms of synovial joint and articular cartilage formation: recent advances, but many lingering mysteries.

Synovial joints are elegant, critically important, and deceptively simple biomechanical structures. They are comprised of articular cartilage that covers each end of the opposing skeletal elements, synovial fluid that lubricates and nourishes the tissues, ligaments that hold the skeletal elements in check, and a fibrous capsule that insulates the joints from surrounding tissues. Joints also exhibit an exquisite arrays of shapes and sizes, best exemplified by the nearly spherical convex femoral head articulating into a nearly spherical concave hip acetabulum, or a phalangeal joint with two condyles on the distal side articulating in reciprocally-shaped sockets on the opposing proximal side. Though few in number, joint tissues are highly specialized in structure and function. This is illustrated by articular cartilage with its unique extracellular matrix, unique biomechanical resilience, its largely avascular nature, and its ability to persist through life with minimal turnover of its cells and components. The fact that interest in synovial joints has remained unabated for decades is a reflection of their fundamental importance for organism function and quality of life, and for their susceptibility to a variety of acquired and congenital conditions, most importantly arthritis. This has led to many advances in this field that encompass molecular genetics to biomechanics to medicine. Regrettably, what continues to be poorly understood are the mechanisms by which synovial joints actually form in the developing embryo. If available, this information would be not only of indisputable biological interest, but would also have significant biomedical ramifications, particularly in terms of designing novel tissue regeneration or reconstruction therapies. This review focuses on recent advances in understanding the mechanisms of synovial joint formation in the limbs, and places and discusses the information within the context of classic studies and the many mysteries and questions that remain unanswered.

Animals↗
PubMed2000

The insulator protein CTCF represses transcription on binding to the (gt)(22)(ga)(15) microsatellite in intron 2 of the HLA-DRB1(*)0401 gene.

The insulator and transcription factor CTCF is a highly conserved 11 zinc finger protein possessing multiple specifities in DNA sequence recognition. CTCF regulates transcription of several genes, like the human oncogene c-myc or the chicken lysozyme gene by binding extremely divergent DNA sequences with different sets of its 11 zinc fingers. Recently, an insulator function was demonstrated for several CTCF binding elements. Here we show that CTCF binds to the (gt)(22)(ga)(15) microsatellite repeat A9 in intron 2 of the HLA-DRB1(*)0401 gene. Reporter gene activity is repressed by the A9 element. This repression is dependent on coexpressed CTCF and is even stronger compared with the CTCF binding site F1 of the chicken lysozyme gene, for which a silencer activity has been shown. This is the first report suggesting a function for microsatellite sequences in regulating specific gene expression.

Animals↗
PubMed2005

Comparative electronic band structure study of the intrachain ferromagnetic versus antiferromagnetic coupling in the magnetic oxides Ca3Co2O6 and Ca3FeRhO6.

In the (MM'O6)infinity chains of the transition-metal magnetic oxides Ca3MM'O6 the MO6 trigonal prisms alternate with the M'O6 octahedra by sharing their triangular faces. In the (Co(2O6)infinity chains of Ca3Co2O6 (M = M' = Co) the spins are coupled ferromagnetically, but in the (FeRhO6)infinity chains of Ca3FeRhO6 (M = Fe, M' = Rh) they are coupled antiferromagnetically. The origin of this difference was probed by carrying out spin-polarized density functional theory electronic band structure calculations for ordered spin states of Ca3Co2O6 and Ca3FeRhO6. The spin state of a (MM'O6)infinity chain determines the occurrence of direct metal-metal bonding between the adjacent trigonal prism and octahedral site transition-metal atoms. The extent of direct metal-metal bonding in the (Co2O6)infinity chains of Ca3Co2O6 is stronger in the intrachain ferromagnetic state than in the intrachain antiferromagnetic state, so that the intrachain ferromagnetic state becomes more stable than the intrachain antiferromagnetic state. Such a metal-metal-bonding-induced ferromagnetism is expected to occur in magnetic insulators and magnetic metals of transition-metal elements in which direct metal-metal bonding can be enhanced by ferromagnetic ordering. In the (FeRhO6)infinity chains of Ca3FeRhO6 the ferromagnetic coupling does not lead to a strong metal-metal bonding and the adjacent spins interact by the Fe-O...O-Fe super-superexchange, hence leading to an antiferromagnetic coupling.

Journal Article↗
PubMed2006

Carbon nanotube linear bearing nanoswitches.

We exploit the remarkable low-friction bearing capabilities of multiwalled carbon nanotubes (MWNTs) to realize nanoelectromechanical switches. Our switches consist of two open-ended MWNT segments separated by a nanometer-scale gap. Switching occurs through electrostatically actuated sliding of the inner nanotube shells to close the gap, producing a conducting ON state. For double-walled nanotubes in particular, a gate voltage can restore the insulating OFF state. Acting as a nonvolatile memory element capable of several switching cycles, our devices are straightforward to implement, self-aligned, and do not require complex fabrication or geometries, allowing for convenient scalability.

Electrochemistry↗
PubMed2005

Novel electronically driven surface phase predicted in C/Si(111).

We predict a novel electronically driven phase for the recently created C/Si(111) surface at 1/3 monolayer coverage. Whereas the isoelectronic surface Sn/Ge(111) is a 3 x 3 distorted metal and Si/SiC(0001) is an undistorted magnetic Mott insulator, the new phase combines both features. Two of three adatoms in C/Si(111) should form a distorted (3 x 3) honeycomb sublattice, the third an undistorted insulating and magnetic triangular sublattice. The generally conflicting elements, namely, band energy, favoring distortion, and strong electron correlations favoring a Mott state, actually conspire in this case. This kind of state represents the surface analog of the Fazekas-Tosatti state in the charge density wave compound 1T-TaS2.

Journal Article↗
PubMed2006

[The establishment of conditional MT transgenic mice model].

OBJECTIVE: To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo. METHODS: Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two elements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene cassette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts (target) in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR. RESULTS: The conditional transgenic vector was constructed successfully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjection. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding. CONCLUSION: Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.

Animals↗
PubMed2001

CpG methylation regulates the Igf2/H19 insulator.

The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.

Alleles↗
PubMed2005

Band-selective measurements of electron dynamics in VO2 using femtosecond near-edge x-ray absorption.

We report on the first demonstration of femtosecond x-ray absorption spectroscopy, made uniquely possible by the use of broadly tunable bending-magnet radiation from "laser-sliced" electron bunches within a synchrotron storage ring. We measure the femtosecond electronic rearrangements that occur during the photoinduced insulator-metal phase transition in VO2. Symmetry- and element-specific x-ray absorption from V2p and O1s core levels (near 500 eV) separately measures the filling dynamics of differently hybridized V3d-O2p electronic bands near the Fermi level.

Journal Article↗
PubMed1993

Lineage control of an integration site-dependent transgene combined with the beta-globin locus control region.

In mice, the expression of integration site-dependent transgenes is controlled by host DNA elements. These may involve locus control regions (LCR), enhancers and silencers. We analysed the interaction between the LCR of the beta-globin locus (beta-LCR) and an integration site-dependent LacZ transgene (HPRTnlsLacZ). We obtained LacZ expression in erythroid cells of all transgenic mice, independent of temporal control, correlated to transgene copy-number. These results demonstrate that an integration site-dependent transgene can be region-controlled by the beta-LCR and establish that the beta-LacZ transgenic lines constitute a valuable source of genetically marked cells for the three erythroid series. This also extends to in vivo regulation in all three erythroid series the notion that the beta-LCR contains the information for region-control of non-erythroid genes. In non-erythroid lineages of all mice, we observed integration site-dependent LacZ expression. It suggests that a single LCR is not sufficient to insulate the LacZ gene from endogenous cis-acting control elements.

Animals↗
PubMed1998

hobo Induced rearrangements in the yellow locus influence the insulation effect of the gypsy su(Hw)-binding region in Drosophila melanogaster.

The su(Hw) protein is responsible for the insulation mediated by the su(Hw)-binding region present in the gypsy retrotransposon. In the y2 mutant, su(Hw) protein partially inhibits yellow transcription by repressing the function of transcriptional enhancers located distally from the yellow promoter with respect to gypsy. y2 mutation derivatives have been induced by the insertion of two hobo copies on the both sides of gypsy: into the yellow intron and into the 5' regulatory region upstream of the wing and body enhancers. The hobo elements have the same structure and orientation, opposite to the direction of yellow transcription. In the sequence context, where two copies of hobo are separated by the su(Hw)-binding region, hobo-dependent rearrangements are frequently associated with duplications of the region between the hobo elements. Duplication of the su(Hw)-binding region strongly inhibits the insulation of the yellow promoter separated from the body and wing enhancers by gypsy. These results provide a better insight into mechanisms by which the su(Hw)-binding region affects the enhancer function.

Animals↗
PubMed2001

Regulatory mechanisms at the mouse Igf2/H19 locus.

The closely linked H19 and Igf2 genes show highly similar patterns of gene expression but are reciprocally imprinted. H19 is expressed almost exclusively from the maternally inherited chromosome, while Igf2 expression is mostly from the paternal chromosome. In humans, loss of imprinting at this locus is associated with tumors and with developmental disorders. Monoallelic expression at the imprinted Igf2/H19 locus occurs by at least two distinct mechanisms: a developmentally regulated silencing of the paternal H19 promoter, and transcriptional insulation of the maternal Igf2 promoters. Both mechanisms of allele-specific silencing are ultimately dependent on a common cis-acting element located just upstream of the H19 promoter. The coordinated expression patterns and some experimental data support the idea that positive regulatory elements are also shared by the two genes. To clarify the organization and function of positive and negative regulatory elements at the H19/Igf2 locus, we analyzed two mouse mutations. First, we generated a deletion allele to localize enhancers used in vivo for expression of both H19 and Igf2 in mesodermal tissues to sequences downstream of the H19 gene. Coincidentally, we demonstrated that some expression of Igf2 is independent of the shared enhancer element. Second, we used this new information to further characterize an ectopic H19 differentially regulated region and the associated insulator. We demonstrated that its activity is parent-of-origin dependent. In contrast to recent results from Drosophila model systems; we showed that this duplication of a mammalian insulator does not interfere with its normal function. Implications of these findings for current models for monoallelic gene expression at this locus are discussed.

Alleles↗