Functional Diversity of Human Basic Helix-Loop-Helix Transcription Factor TCF4 Isoforms Generated by Alternative 5′ Exon Usage and Splicing

BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2) is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG). While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence about the functional diversity of the alternative TCF4 protein isoforms

Stringent response of Escherichia coli: revisiting the bibliome using literature mining

Understanding the mechanisms responsible for cellular responses depends on the systematic collection and analysis of information on the main biological concepts involved. Indeed, the identification of biologically relevant concepts in free text, namely genes, tRNAs, mRNAs, gene products and small molecules, is crucial to capture the structure and functioning of different responses. Results In this work, we review literature reports on the study of the stringent response in Escherichia coli. Rather than undertaking the development of a highly specialised literature mining approach, we investigate the suitability of concept recognition and statistical analysis of concept occurrence as means to highlight the concepts that are most likely to be biologically engaged during this response. The co-occurrence analysis of core concepts in this stringent response, i.e. the (p)ppGpp nucleotides with gene products was also inspected and suggest that besides the enzymes RelA and SpoT that control the basal levels of (p)ppGpp nucleotides, many other proteins have a key role in this response. Functional enrichment analysis revealed that basic cellular processes such as metabolism, transcriptional and translational regulation are central, but other stress-associated responses might be elicited during the stringent response. In addition, the identification of less annotated concepts revealed that some (p)ppGpp-induced functional activities are still overlooked in most reviews. Conclusions In this paper we applied a literature mining approach that offers a more comprehensive analysis of the stringent response in E. coli. The compilation of relevant biological entities to this stress response and the assessment of their functional roles provided a more systematic understanding of this cellular response. Overlooked regulatory entities, such as transcriptional regulators, were found to play a role in this stress response. Moreover, the involvement of other stress-associated concepts demonstrates the complexity of this cellular response

DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. Methodology/Principal Findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38uC and 42uC. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. Conclusion/Significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart

The Stringent Response and Cell Cycle Arrest in Escherichia coli

The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions

Transcriptional Responses of Cultured Rat Sympathetic Neurons during BMP-7-Induced Dendritic Growth

Dendrites are the primary site of synapse formation in the vertebrate nervous system; however, relatively little is known about the molecular mechanisms that regulate the initial formation of primary dendrites. Embryonic rat sympathetic neurons cultured under defined conditions extend a single functional axon, but fail to form dendrites. Addition of bone morphogenetic proteins (BMPs) triggers these neurons to extend multiple dendrites without altering axonal growth or cell survival. We used this culture system to examine differential gene expression patterns in naïve vs. BMP-treated sympathetic neurons in order to identify candidate genes involved in regulation of primary dendritogenesis.To determine the critical transcriptional window during BMP-induced dendritic growth, morphometric analysis of microtubule-associated protein (MAP-2)-immunopositive processes was used to quantify dendritic growth in cultures exposed to the transcription inhibitor actinomycin-D added at varying times after addition of BMP-7. BMP-7-induced dendritic growth was blocked when transcription was inhibited within the first 24 hr after adding exogenous BMP-7. Thus, total RNA was isolated from sympathetic neurons exposed to three different experimental conditions: (1) no BMP-7 treatment; (2) treatment with BMP-7 for 6 hr; and (3) treatment with BMP-7 for 24 hr. Affymetrix oligonucleotide microarrays were used to identify differential gene expression under these three culture conditions. BMP-7 significantly regulated 56 unique genes at 6 hr and 185 unique genes at 24 hr. Bioinformatic analyses implicate both established and novel genes and signaling pathways in primary dendritogenesis.This study provides a unique dataset that will be useful in generating testable hypotheses regarding transcriptional control of the initial stages of dendritic growth. Since BMPs selectively promote dendritic growth in central neurons as well, these findings may be generally applicable to dendritic growth in other neuronal cell types

DNA replication defect in the Escherichia coli cgtA (ts) mutant arising from reduced DnaA levels

In Escherichia coli and other bacteria, the ribosome-associated CgtA GTP-binding protein plays a critical role in many basic cellular processes, including the control of DNA replication and/or segregation. However, the mechanism of this control is largely unknown. Here we report that ectopic expression of the dnaA gene partially restored both early growth in liquid medium and DNA synthesis defects of the cgtA (ts) mutant. Amounts of DnaA protein in the cgtA (ts) mutant incubated at elevated (42°C) temperature were significantly lower relative to wild-type bacteria. Both level of dnaA mRNA and transcriptional activity of the dnaA promoter- lacZ fusion were decreased in the CgtA-deficient cells. The effects of ectopic expression of dnaA were specific as analogous expression of another gene coding for a replication regulator, seqA , had no significant changes in growth and DNA synthesis in the cgtA mutant. Thus, it appears that the DNA replication defect in this mutant is a consequence of reduced DnaA levels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45857/1/203_2006_Article_99.pd

Susceptibility of linear and nonlinear otoacoustic emission components to low-dose styrene exposure

OBJECTIVE: To investigate potential susceptibility of active cochlear mechanisms to low-level styrene exposure by comparing TEOAEs in workers and controls. DESIGN: Two advanced analysis techniques were applied to detect sub-clinical changes in linear and nonlinear cochlear mechanisms of OAE generation: the wavelet transform to decompose TEOAEs into time-frequency components and extract signal-to-noise ratio and latency of each component, and the bispectrum to detect and extract nonlinear TEOAE contributions as quadratic frequency couplings (QFCs). STUDY SAMPLE: Two cohorts of workers were examined: subjects exposed exclusively to styrene (N = 9), and subjects exposed to styrene and noise (N = 6). The control group was perfectly matched by age and sex to the exposed group. RESULTS: Exposed subjects showed significantly lowered SNR in TEOAE components at mid-to-high frequencies (above 1.6 kHz) and a shift of QFC distribution towards lower frequencies than controls. No systematic differences were observed in latency. CONCLUSION: Low-level styrene exposure may have induced a modification of cochlear functionality as concerns linear and nonlinear OAE generation mechanisms. The lack of change in latency seems to suggest that the OAE components, where generation region and latency are tightly coupled, may not have been affected by styrene and noise exposure levels considered here

Effects of low-dose styrene exposure on linear and nonlinear otoacoustic emission components

This study investigates the potential effects of low-level styrene exposure on active cochlear mechanisms. To this purpose, transient-evoked otoacoustic emissions (TEOAEs) were recorded and compared in a group of exposed workers and controls. Otoacoustic emissions are low intensity sounds connected to the active mechanisms characterizing the healthy cochlea and can be used to monitor changes in cochlear functionality due to exposure to external agents such as noise, electromagnetic fields, and ototoxic substances. TEOAEs from healthy ears are composed of the contribution of two generation mechanisms, the so-called nonlinear distortion and linear reflection. To detect possible sub-clinical changes in the two generation mechanisms, we applied two advanced and ad hoc analysis techniques: the wavelet transform WT) and the bispectrum. The WT was used to study linear mechanisms by decomposing TEOAEs into time-frequency components; the bispectrum was applied to detect and extract nonlinear TEOAE contributions as quadratic frequency couplings QFCs). The exposed group consisted of two cohorts of subjects exposed exclusively to styrene and subjects exposed to styrene and noise. Subjects in the control group were perfectly matched by age and sex to their peers in the two cohorts of the exposed group. In exposed subjects, linear TEOAE components showed significantly lowered SNR at mid to high frequencies above 1600 Hz) compared to the control group; non-linear contributions, as measured by the QFC, showed in the exposed group a shift towards lower frequencies compared to the control group. No differences were observed in the latency of linear components. We found that TEOAEs characteristics of styrene-exposed workers didn't change before and after the working shift, and that styrene exposure affected the physiological effect of age on cochlear mechanisms. Overall, these results reveal that low level styrene exposure may have induced a sub-clinical modification of cochlear functionality