Inherent promoter bidirectionality facilitates maintenance of sequence integrity and transcription of parasitic DNA in mammalian genomes

<p>Abstract</p> <p>Background</p> <p>Many mammalian genes are arranged in a bidirectional manner, sharing a common promoter and regulatory elements. This is especially true for promoters containing a CpG island, usually unmethylated and associated with an 'open' or accessible chromatin structure. In evolutionary terms, a primary function of genomic methylation is postulated to entail protection of the host genome from the disruption associated with activity of parasitic or transposable elements. These are usually epigenetically silenced following insertion into mammalian genomes, becoming sequence degenerate over time. Despite this, it is clear that many transposable element-derived DNAs have evaded host-mediated epigenetic silencing to remain expressed (domesticated) in mammalian genomes, several of which have demonstrated essential roles during mammalian development.</p> <p>Results</p> <p>The current study provides evidence that many CpG island-associated promoters associated with single genes exhibit inherent bidirectionality, facilitating "hijack" by transposable elements to create novel antisense 'head-to-head' bidirectional gene pairs in the genome that facilitates escape from host-mediated epigenetic silencing. This is often associated with an increase in CpG island length and transcriptional activity in the antisense direction. From a list of over 60 predicted protein-coding genes derived from transposable elements in the human genome and 40 in the mouse, we have found that a significant proportion are orientated in a bidirectional manner with CpG associated regulatory regions.</p> <p>Conclusion</p> <p>These data strongly suggest that the selective force that shields endogenous CpG-containing promoter from epigenetic silencing can extend to exogenous foreign DNA elements inserted in close proximity in the antisense orientation, with resulting transcription and maintenance of sequence integrity of such elements in the host genome. Over time, this may result in "domestication" of such elements to provide novel cellular and developmental functions.</p

An improved Diagnostic PCR Assay for identification of Cryptic Heterozygosity for CGG Triplet Repeat Alleles in the Fragile X Gene (FMR1)

<p>Abstract</p> <p>Background</p> <p>Fragile X syndrome (OMIM #300624) is the most common, recognised, heritable cause of mental retardation. Widespread testing is warranted by the relatively high frequency of the disorder, the benefits of early detection and the identification of related carriers whose offspring are at a 1 in 2 risk of inheriting the expanded pathogenic mutation. However, cost-effective screening of mentally retarded individuals has been impeded by the lack of a single, simple laboratory test. Currently, Fragile X syndrome can be excluded in males and a majority of females using a simple high-throughput PCR test. Due to the limited sensitivity of the PCR test, we find in our diagnostic service that approximately 40% of females appear homozygous and a labour intensive and expensive Southern blot test is required to distinguish these from females carrying one normal allele and an expanded allele.</p> <p>Results</p> <p>We describe an improved PCR test which displays a high level of precision allowing alleles differing by a single triplet to be resolved. Using the new assay, we detected 46/83 (53%) cryptic heterozygotes previously labelled as homozygotes. The assay also extended the range of repeats amplifiable, up to 170 CGG repeats in males and 130 CGG repeats in females. Combined with the high precision, the assay also improves discrimination of normal (CGG repeats < 45) from grey zone (45 < CGG repeats < 54) alleles and grey zone alleles from small premutations (55 < CGG repeats < 100).</p> <p>Conclusion</p> <p>Use of this PCR test provides significantly improved precision and amplification of longer alleles. The number of follow-up Southern blot tests required is reduced (up to 50%) with consequent improvement in turnaround time and cost.</p

Loss of TOP3B leads to increased R-loop formation and genome instability

Topoisomerase III beta (TOP3B) is one of the least understood members of the topoisomerase family of proteins and remains enigmatic. Our recent data shed light on the function and relevance of TOP3B to disease. A homozygous deletion for the TOP3B gene was identified in a patient with bilateral renal cancer. Analyses in both patient and modelled human cells show the disruption of TOP3B causes genome instability with a rise in DNA damage and chromosome bridging (mis-segregation). The primary molecular defect underlying this pathology is a significant increase in R-loop formation. Our data show that TOP3B is necessary to prevent the accumulation of excessive R-loops and identify TOP3B as a putative cancer gene, and support recent data showing that R-loops are involved in cancer aetiology

Streptavidin-Binding Peptide (SBP)-tagged SMC2 allows single-step affinity fluorescence, blotting or purification of the condensin complex

<p>Abstract</p> <p>Background</p> <p>Cell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated.</p> <p>Aim</p> <p>To develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification.</p> <p>Results</p> <p>We have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions.</p> <p>Conclusions</p> <p>The small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.</p

Sex recognition by odour and variation in the uropygial gland secretion in starlings

1. Although a growing body of evidence supports that olfaction based on chemical compounds emitted by birds may play a role in individual recognition, the possible role of chemical cues in sexual selection of birds has been only preliminarily studied.2. We investigated for the first time whether a passerine bird, the spotless starling Sturnus unicolor, was able to discriminate the sex of conspecifics by using olfactory cues and whether the size and secretion composition of the uropygial gland convey information on sex, age and reproductive status in this species.3. We performed a blind choice experiment during mating, and we found that starlings were able to discriminate the sex of conspecifics by using chemical cues alone. Both male and female starlings preferred male scents. Furthermore, the analysis of the chemical composition of the uropygial gland secretion by using gas chromatography–mass spectrometry (GC–MS) revealed differences between sexes, ages and reproductive status.4. In conclusion, our study reveals for first time that a passerine species can discriminate the sex of conspecifics by relying on chemical cues and suggests that the uropygial gland secretion may potentially function as a chemical signal used in mate choice and/or intrasexual competition in this species.This research was funded by the Spanish Ministry of Education and Science ⁄ FEDER (CGL2008-00718) and PIE 200930I029 to J. M. Avilés and D. Parejo.The study was conducted under licence of the Junta de Andalucía GC–MS analyses were performed by Dr. Rafael Núñez at the Scientific Instrumentation Service (EEZ, CSIC) (Granada, Spain).Peer reviewe

Centromere Protein B Null Mice are Mitotically and Meiotically Normal but Have Lower Body and Testis Weights

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented

Targeting the Mitotic Checkpoint to Kill Tumor Cells

One of the most common hallmarks of cancer cells is aneuploidy or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis, a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype, meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAi-mediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. This suggests that inhibition of the mitotic checkpoint might represent a useful anti-cancer strategy

A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition.

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor

Spindle Assembly Checkpoint Regulates Mitotic Cell Cycle Progression during Preimplantation Embryo Development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos