Chromatin insulator elements: establishing barriers to set heterochromatin boundaries

Epigenomic profiling has revealed that substantial portions of genomes in higher eukaryotes are organized into extensive domains of transcriptionally repressive chromatin. The boundaries of repressive chromatin domains can be fixed by DNA elements known as barrier insulators, to both shield neighboring gene expression and to maintain the integrity of chromosomal silencing. Here, we examine the current progress in identifying vertebrate barrier elements and their binding factors. We overview the design of the reporter assays used to define enhancer-blocking and barrier insulators. We look at the mechanisms vertebrate barrier proteins, such as USF1 and VEZF1, employ to counteract Polycomb- and heterochromatin-associated repression. We also undertake a critical analysis of whether CTCF could also act as a barrier protein. There is good evidence that barrier elements in vertebrates can form repressive chromatin domain boundaries. Future studies will determine whether barriers are frequently used to define repressive domain boundaries in vertebrates

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. <p/>Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. <p/>Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. <p/>Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters

Protecting a transgene expression from the HAC-based vector by different chromatin insulators

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-013-1362-9) contains supplementary material, which is available to authorized users

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al

Analysis of induction in cadmium chloride-treated cells transfected with TFBS-UR plasmids.

<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with cadmium. (A) Microarray-based detection of TF derived activation of UR expression. (B) qPCR-based detection of TF-derived activation of UR expression. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The grey bar represents treatment-independent changes in the system. TFBS marked with * represent treatment-dependent effects on the TF library. Numerical data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s004" target="_blank">Table S3</a>. A statistical analysis of the qPCR assay data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone-0050521-g003" target="_blank">Figure 3</a>.</p

Non-audit Services and Auditor Independence: New Zealand Evidence

This paper examines evidence in New Zealand about whether auditors providing more non-audit services are less independent. Three sets of tests are used to address the issue. The first examines whether there is a relation between non-audit fees and audit fees, the second examines whether there is a relation between non-audit fees and audit report qualification or modification, and the third examines whether there is a relation between non-audit fees and stability of audit tenure. The results suggest a potential for the impairment of auditor independence in appearance when auditors provide non-audit services but no evidence of any impact on independence of mind. Copyright 2006 The Authors Journal compilation (c) 2006 Blackwell Publishing Ltd.

Induction of selected TFBS-directed UR expression in HEK293 cells after treatment with cadmium, dexamethasone, TPA and forskolin.

<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with drugs of interest. (A) MRE-directed UR expression after treatment with cadmium. (B) GRE-directed UR expression after treatment with dexamethasone. (C) NF-κB-directed UR expression after treatment with TPA. (D) CREB-directed UR expression after treatment with forskolin. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The error bars are calculated as 1 standard error of the mean each way.</p

Activation of transcription factors by specific treatments on the qPCR platform.

<p>HEK293 cells transfected with pool of plasmids (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40) and were subsequently treated with chemicals of interest. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The errors are calculated as 1 standard error of the mean each way. P-values indicate the posterior probability that there was no difference in expression levels between the control and treatment samples so a lower p-value would indicate a greater likelihood that there was a difference between the control and treatment samples. Abbreviations: IBMX: 3-isobutyl-1-methylxanthine, EHNA: erythro-9-(2-hydroxy-3-nonyl)adenine.</p