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

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

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

© 2018 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (November 2018) in accordance with the publisher’s archiving policyPurpose Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family. Methods and Materials Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair. Results Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin C–induced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by γ-H2AX foci and the comet assay. Conclusions In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.This work was supported by the Norwegian Cancer Society and the Research Council of Norway (to P.C.); an Australia Research Council Future Fellowship award (to L.H.W.); National Health and Medical Research Council Program Grant 1053792 (to R.B.P. and R.D.H.), senior research fellowships (to R.B.P. and R.D.H.), and a project grant (to L.H.W.); and a Cure Brain Cancer Foundation Australia project grant (to L.H.W. and H.P.J.V.)

A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes