The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. RESULTS: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. CONCLUSIONS: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes

Role of transcript and interplay between transcription and replication in triplet-repeat instability in mammalian cells

Triplet-repeat expansions cause several inherited human diseases. Expanded triplet-repeats are unstable in somatic cells, and tissue-specific somatic instability contributes to disease pathogenesis. In mammalian cells instability of triplet-repeats is dependent on the location of the origin of replication relative to the repeat tract, supporting the ‘fork-shift’ model of repeat instability. Disease-causing triplet-repeats are transcribed, but how this influences instability remains unclear. We examined instability of the expanded (GAA•TTC)n sequence in mammalian cells by analyzing individual replication events directed by the SV40 origin from five different locations, in the presence and absence of doxycycline-induced transcription. Depending on the location of the SV40 origin, either no instability was observed, instability was caused by replication with no further increase due to transcription, or instability required transcription. Whereas contractions accounted for most of the observed instability, one construct showed expansions upon induction of transcription. These expansions disappeared when transcript stability was reduced via removal or mutation of a spliceable intron. These results reveal a complex interrelationship of transcription and replication in the etiology of repeat instability. While both processes may not be sufficient for the initiation of instability, transcription and/or transcript stability seem to further modulate the fork-shift model of triplet-repeat instability

Age, gender, mileage and the DBQ: The validity of the Driver Behavior Questionnaire in different driver groups

The Driver Behavior Questionnaire (DBQ) is one of the most widely used instruments for measuring self-reported driving behaviors. Despite the popularity of the DBQ, the applicability of the DBQ in different driver groups has remained mostly unexamined. The present study measured aberrant driving behavior using the original DBQ (Reason, J.T., Manstead, A., Stradling, S.G., Baxter, J., Campbell, K., 1990. Errors and violations on the road - a real distinction. Ergonomics, 33 (10/11), 1315-1332) to test the factorial validity and reliability of the instrument across different subgroups of Danish drivers. The survey was conducted among 11,004 Danish driving license holders of whom 2250 male and 2190 female drivers completed the questionnaire containing background variables and the DBQ. Exploratory and confirmatory factor analysis showed that the original three-factor solution, a four-factor solution and a two-factor solution had acceptable fit when using the whole sample. However, fit indices of these solutions varied across subgroups. The presents study illustrates that both the original DBQ and a Danish four-factor DBQ structure is relatively stable across subgroups, indicating factorial validity and reliability of the DBQ. However, as the Danish DBQ structure has an overall better fit, the present study highlights the importance of performing an explorative analysis when applying the DBQ in order to assess the problem areas within a driving population

Using a structural and logics systems approach to infer bHLH–DNA binding specificity determinants

Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein–DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH–DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.National Institute of General Medical Sciences (U.S.) (DK068429)National Institute of General Medical Sciences (U.S.) (HG003985)European Union (PROSPECTS HEALTH-F4-2008-201648

Conserved elements associated with ribosomal genes and their trans-splice acceptor sites in Caenorhabditis elegans

The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression

Modelling of Short-Term Interactions Between Concrete Support and the Excavated Damage Zone Around Galleries Drilled in Callovo–Oxfordian Claystone

peer reviewedProduction of energy from nuclear power plants generates high-level radioactive nuclear waste, harmful during dozens of thousand years. Deep geological disposal of nuclear waste represents the most reliable solutions for its safe isolation. Confinement of radioactive wastes relies on the multi-barrier concept in which isolation is provided by a series of engineered (canister, backfill) and natural (host rock) barriers. Few underground research laboratories have been built all over the world to test and validate storage solutions. The underground drilling process of disposal drifts may generate cracks, fractures/strain localisation in shear bands within the rock surrounding the gallery especially in argillaceous rocks. These degradations affect the hydro-mechanical properties of the material, such as permeability, e.g. creating a preferential flow path for radionuclide migration. Hydraulic conductivity increase within this zone must remain limited to preserve the natural barrier. In addition galleries are currently reinforced by different types of concrete supports such as shotcrete and/or prefab elements. Their purpose is twofold: avoiding partial collapse of the tunnel during drilling operations and limiting convergence of the surrounding rock. Properties of both concrete and rock mass are time dependent, due to shotcrete hydration and hydromechanical couplings within the host rock. By the use of a hydro-mechanical coupled Finite Element Code with a Second Gradient regularization, this paper aims at investigating and predicting support and rock interactions (convergence, stress field). The effect of shotcrete hydration evolution, spraying time and use of compressible wedges is studied in order to determine their relative influence

Cataloguing functionally relevant polymorphisms in gene DNA ligase I: a computational approach

A computational approach for identifying functionally relevant SNPs in gene LIG1 has been proposed. LIG1 is a crucial gene which is involved in excision repair pathways and mutations in this gene may lead to increase sensitivity towards DNA damaging agents. A total of 792 SNPs were reported to be associated with gene LIG1 in dbSNP. Different web server namely SIFT, PolyPhen, CUPSAT, FASTSNP, MAPPER and dbSMR were used to identify potentially functional SNPs in gene LIG1. SIFT, PolyPhen and CUPSAT servers predicted eleven nsSNPs to be intolerant, thirteen nsSNP to be damaging and two nsSNPs have the potential to destabilize protein structure. The nsSNP rs11666150 was predicted to be damaging by all three servers and its mutant structure showed significant increase in overall energy. FASTSNP predicted twenty SNPs to be present in splicing modifier binding sites while rSNP module from MAPPER server predicted nine SNPs to influence the binding of transcription factors. The results from the study may provide vital clues in establishing affect of polymorphism on phenotype and in elucidating drug response