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

Novel pathogenic mutations and further evidence for clinical relevance of genes and variants causing hearing impairment in Tunisian population

Introduction: Hearing impairment (HI) is characterized by complex genetic heterogeneity. The evolution of next generation sequencing, including targeted enrichment panels, has revolutionized HI diagnosis. Objectives: In this study, we investigated genetic causes in 22 individuals with non-GJB2 HI. Methods: We customized a HaloplexHS kit to include 30 genes known to be associated with autosomal recessive nonsyndromic HI (ARNSHI) and Usher syndrome in North Africa. Results: In accordance with the ACMG/AMP guidelines, we report 11 pathogenic variants; as follows; five novel variants including three missense (ESRRB-Tyr295Cys, MYO15A-Phe2089Leu and MYO7A-Tyr560Cys) and two nonsense (USH1C-Gln122Ter and CIB2-Arg104Ter) mutations; two previously reported mutations (OTOF-Glu57Ter and PNPT1-Glu475Gly), but first time identified among Tunisian families; and four other identified mutations namely WHRN-Gly808AspfsX11, SLC22A4-Cys113Tyr and two MYO7A compound heterozygous splice site variants that were previously described in Tunisia. Pathogenic variants in WHRN and CIB2 genes, in patients with convincing phenotype ruling out retinitis pigmentosa, provide strong evidence supporting their association with ARNSHI. Moreover, we shed lights on the pathogenic implication of mutations in PNPT1 gene in auditory function providing new evidence for its association with ARNSHI. Lack of segregation of a previously identified causal mutation OTOA-Val603Phe further supports its classification as variant of unknown significance. Our study reports absence of otoacoustic emission in subjects using bilateral hearing aids for several years indicating the importance of screening genetic alteration in OTOF gene for proper management of those patients. Conclusion: In conclusion, our findings do not only expand the spectrum of HI mutations in Tunisian patients, but also improve our knowledge about clinical relevance of HI causing genes and variants

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

Induction of the TF proteins of interest in HEK293 cells after treatment with forskolin, TPA and cadmium.

<p>Proteins extracted from treated and control cells were analyzed using Western blots and TF-specific antibodies. The levels of phosphorylated TFs and inactive TFs were analyzed for (A) CREB and ATF, (B) IκB, (C) c-jun and (D) SP1. Tubulin was used as a loading control. Quantification of the levels of protein on the Western blots showed a 1.6 and 1.3 fold increase in P-CREB and P-ATF after treatment with forskolin and a 1.5 and 1.6 fold increase in P-IκB, and P-c-jun after treatment with TPA. Treatment of HEK293 cells with cadmium chloride, dexamethasone, forskolin and TPA resulted in a 1.1, 1.1. 1.0 and 1.0 fold increase in the levels of SP1 protein. (E) Increased <i>hMTIIA</i> gene expression in HEK293 cells after treatment with cadmium. Expression of the cadmium-responsive <i>hMTIIa</i> gene was normalized to the expression of the chromosomal reference gene <i>B2M.</i> Abbreviations: -, carrier only control; C, cadmium; D, dexamethasone; F, forskolin; T, TPA.</p

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

qPCR analysis of induction of TFBS-directed UR expression in treated cells transfected with TFBS-UR plasmids.

<p>The statistical model calculated a posterior probability distribution over the mean of the log normalized fold induction. The p-value indicated the posterior probability that there was no difference in expression levels between the control and treatment samples. 95% credible intervals were also calculated for the mean log normalized fold induction and indicate the region where there is a 95% probability that the mean effect lies within it. Bars not crossing the 0 line show significant evidence for an effect following treatment with the inducer of interest.</p

A schematic representation of the method.

<p>In each reporter plasmid, the transcription factor binding site (TFBS) and the thymidine kinase promoter (P_TK) were present upstream of the transcriptional start site (TSS) and the unique DNA reporter (UR) sequence. The cassette was flanked by two poly(A) signals to prevent transcriptional interference due to the circular plasmid. Each TFBS was assigned a specific UR sequence to act as a signature for its corresponding TF activity. These plasmids were tranfected into cells and the cells treated with compounds of interest, mRNA was isolated, reverse transcribed and analyzed on two detection platforms. For microarray analysis, cDNA was amplified by PCR using a Cy3 or Cy5-labelled universal sense forward primer (Cy3/Cy5-AG_URF) in conjunction with a universal antisense reverse primer (prMJ264) to generate a mixture of 120 bp fluorescently labelled PCR amplicons that could be analyzed on DNA microarrays. For the qPCR reaction, a forward primer, specific for each UR, was used in combination with a universal FAM-labelled hydrolysis probe (prMJ245) and a universal reverse primer (prMJ264).</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