Chromatin remodeling and genome stability

A report on the 12th Tenovus Scotland Symposium 'Stability and Regulation of Genes and Genomes', Glasgow UK, 6-7 April 2006

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

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

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

Levels of hMTIIa in HeLa cells transfected with gold nanoparticles.

<p>Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. Samples were treated with 12.5 µM CdCl₂. The level of hMTIIa gene expression (normalized to <i>B2M</i>) in untransfected and induced HeLa cells was defined as 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. Induced samples were treated with 12.5 µM CdCl₂ (+Cd). HeLa cells were transfected with unfunctionalized (−), control (C) or hMT-IIa (MT) ssDNA-functionalized nanoparticles. Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099458#pone.0099458.s008" target="_blank">Table S2C</a>.</p

Fluoresence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in live HeLa cells.

<p>HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence (NanoParticles column) or the presence of the transfection reagents Matra, Lipofectamine 2000 or GeneJuice. Control cells were treated with neither nanoparticles nor transfection reagents. The same signal intensity range was used for all FITC images. The scale bar represents 50 microns.</p

Levels of hMT-IIa in HeLa cells transfected with increasing concentrations of hMT-IIa or control ssDNA.

<p>Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. All samples were induced samples with 12.5 µM CdCl₂. Total hMT-IIa activity relative to <i>B2M</i> in cells transfected with varying levels of ssDNA was normalized to hMT-IIa-<i>B2M</i> expression in cells transfected with 0 nM ssDNA giving us a value for hMT-IIa activity in the presence of x nM ssDNA. The levels of hMT-IIa transcript in cells transfected with control ssDNA (C) was set to 1.0. The level of hMT-IIa gene expression (MT) in cells transfected with hMT-IIa ssDNA was compared to the levels of hMT-IIa in cells transfected with control ssDNA at each concentration providing a measure of the reduction in hMT-IIa activity in hMT-IIa ssDNA transfected compared to those transfected with control ssDNA. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. All samples were induced with 12.5 µM CdCl₂. HeLa cells were transfected with hMT-IIa-specific ssDNA (ssDNA). Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099458#pone.0099458.s008" target="_blank">Table S2B</a>.</p

Quantification of the levels of hMT-IIa transcript in HeLa cells after treatment with ssDNA-functionalized nanoparticles.

<p>Induced samples were treated with 12.5 µM CdCl₂ (+Cd). HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. The level of hMTIIa gene expression (normalized to <i>B2M</i>) in HeLa cells transfected with unfunctionalized (−)gold nanoparticles was normalized to 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way and represented as a percentage of the activity.</p