Synthetic Transcription Factors Switch from Local to Long-Range Control during Cell Differentiation.

Genes, including promoters and enhancers, are regulated by short- and long-range interactions in higher eukaryotes. It is unclear how mammalian gene expression subject to such a combinatorial regulation can be controlled by synthetic transcription factors (TF). Here, we studied how synthetic TALE transcriptional activators and repressors affect the expression of genes in a gene array during cellular differentiation. The protocadherin gene array is silent in mouse embryonic stem (ES) and neuronal progenitor cells. The TALE transcriptional activator recruited to a promoter activates specifically the target gene in ES cells. Upon differentiation into neuronal progenitors, the transcriptional regulatory logic changes: the same activator behaves like an enhancer, activating distant genes in a correlated, stochastic fashion. The long-range effect is reflected by the alterations in CpG methylation. Our findings reveal the limits of precision and the opportunities in the control of gene expression for TF-based therapies in cells of various differentiation stages.

Stochastic Gene Choice during Cellular Differentiation.

Genes in higher eukaryotes are regulated by long-range interactions, which can determine what combination of genes is expressed in a chromosomal segment. The choice of the genes can display exclusivity, independence, or co-occurrence. We introduced a simple measure to quantify this interdependence in gene expression and differentiated mouse embryonic stem cells to neurons to measure the single-cell expression of the gene isoforms in the protocadherin (Pcdh) cluster, a key component of neuronal diversity. As the neuronal progenitors mature into neurons, expression of the gene isoforms in the Pcdh array is initially concurrent. Even though the number of the expressed genes is increasing during differentiation, the expression shifts toward exclusivity. The expression frequency correlates highly with CTCF binding to the promoters and follows dynamically the changes in the binding during the differentiation. These findings aid in understanding the interplay between cellular differentiation and stochastic gene choice.

Impact of Methods on the Measurement of mRNA Turnover.

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

Multiplexed gene control reveals rapid mRNA turnover.

The rates of mRNA synthesis and decay determine the mRNA expression level. The two processes are under coordinated control, which makes the measurements of these rates challenging, as evidenced by the low correlation among the methods of measurement of RNA half-lives. We developed a minimally invasive method, multiplexed gene control, to shut off expression of genes with controllable synthetic promoters. The method was validated by measuring the ratios of the nascent to mature mRNA molecules and by measuring the half-life with endogenous promoters that can be controlled naturally or through inserting short sequences that impart repressibility. The measured mRNA half-lives correlated highly with those obtained with the metabolic pulse-labeling method in yeast. However, mRNA degradation was considerably faster in comparison to previous estimates, with a median half-life of around 2 min. The half-life permits the estimation of promoter-dependent and promoter-independent transcription rates. The dynamical range of the promoter-independent transcription rates was larger than that of the mRNA half-lives. The rapid mRNA turnover and the broad adjustability of promoter-independent transcription rates are expected to have a major impact on stochastic gene expression and gene network behavior.

Functional and expressional analyses of the anti-FlhD4C2 factor gene ydiV in Escherichia coli.

Although Escherichia coli and Salmonella enterica serovar Typhimurium have a similar flagellar regulatory system, the response of flagellar synthesis to nutrient conditions is quite different between the two: that is, in low-nutrient conditions, flagellar synthesis is inhibited in Salmonella and enhanced in E. coli. In Salmonella, this inhibition is mediated by an anti-FlhD(4)C(2) factor, YdiV, which is expressed in low-nutrient conditions and binds to FlhD(4)C(2) to inhibit the expression of the class 2 flagellar genes. The fliZ gene encodes a repressor of the ydiV gene, and thus is required for efficient flagellar gene expression in low-nutrient conditions in Salmonella. In this study, we showed that the E. coli ydiV gene encodes a protein which inhibits motility and flagellar production when expressed from a multicopy plasmid. We showed further that E. coli YdiV binds to FlhD(4)C(2) and inhibits its binding to the class 2 flagellar promoter. These results indicate that E. coli YdiV can also act as an anti-FlhD(4)C(2) factor. However, although the ydiV gene was transcribed efficiently in E. coli cells, the intracellular level of the YdiV protein was extremely low due to its inefficient translation. Consistent with this, E. coli cells did not require FliZ for efficient motility development. This indicates that, unlike in Salmonella, the FliZ-YdiV regulatory system does not work in the nutritional control of flagellar gene expression in E. coli.

Discovery of a potent and orally available acyl-CoA: cholesterol acyltransferase inhibitor as an anti-atherosclerotic agent: (4-phenylcoumarin)acetanilide derivatives.

Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterification. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A series of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of the structure-activity relationships of a substituent on this scaffold, with an emphasis on improving the pharmacokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC50=12 nM) and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os (p.o.).

The transcript from the σ(28)-dependent promoter is translationally inert in the expression of the σ(28)-encoding gene fliA in the fliAZ operon of Salmonella enterica serovar Typhimurium.

There are three classes of promoters for flagellar operons in Salmonella. Class 2 promoters are transcribed by σ(70) RNA polymerase in the presence of an essential activator, FlhD(4)C(2), and activated by an auxiliary regulator, FliZ. Class 3 promoters are transcribed by σ(28) RNA polymerase and repressed by an anti-σ(28) factor, FlgM. σ(28) (FliA) and FliZ are encoded by the fliA and fliZ genes, respectively, which together constitute an operon transcribed in this order. This operon is transcribed from both class 2 and class 3 promoters, suggesting that it should be activated by its own product, σ(28), even in the absence of FlhD(4)C(2). However, σ(28)-dependent transcription occurs in vivo only in the presence of FlhD(4)C(2), indicating that transcription from the class 2 promoter is a prerequisite to that from the class 3 promoter. In this study, we examined the effects of variously modified versions of the fliA regulatory region on transcription and translation of the fliA gene. We showed that FliA is not significantly translated from the class 3 transcript. In contrast, the 5'-terminal AU-rich sequence found in the class 2 transcript confers efficient fliA translation. Replacement of the Shine-Dalgarno sequence of the fliA gene with a better one improved fliA translation from the class 3 transcript. These results suggest that the 5'-terminal AU-rich sequence of the class 2 transcript may assist ribosome binding. FliZ was shown to be expressed from both the class 2 and class 3 transcripts.

trans-translation-mediated tight regulation of the expression of the alternative ribosome-rescue factor ArfA in Escherichia coli.

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

FliZ acts as a repressor of the ydiV gene, which encodes an anti-FlhD4C2 factor of the flagellar regulon in Salmonella enterica serovar typhimurium.

YdiV acts as an anti-FlhD4C2 factor, which negatively regulates the class 2 flagellar operons in poor medium in Salmonella enterica serovar Typhimurium. On the other hand, one of the class 2 flagellar genes, fliZ, encodes a positive regulator of the class 2 operons. In this study, we found that the FliZ-dependent activation of class 2 operon expression was more profound in poor medium than in rich medium and not observed in the ydiV mutant background. Transcription of the ydiV gene was shown to increase in the fliZ mutant. Purified FliZ protein was shown in vitro to bind to the promoter region of the nlpC gene, which is located just upstream of the ydiV gene, and to repress its transcription. These results indicate that FliZ is a repressor of the nlpC-ydiV operon and activates the class 2 operons by repressing ydiV expression. Therefore, the fliZ and ydiV genes form a regulatory loop.

EAL domain protein YdiV acts as an anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica Serovar Typhimurium.

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(4)C(2) heterohexamer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. In this study, we showed that flagellar expression was much reduced in the cells grown in poor medium compared to those grown in rich medium. This nutritional control was shown to be executed at a step after class 1 transcription. We isolated five Tn5 insertion mutants in which the class 2 expression was derepressed in poor medium. These insertions were located in the ydiV (cdgR) gene or a gene just upstream of ydiV. The ydiV gene is known to encode an EAL domain protein and to act as a negative regulator of flagellar expression. Gene disruption and complementation analyses revealed that the ydiV gene is responsible for nutritional control. Expression analysis of the ydiV gene showed that its translation, but not transcription, was enhanced by growth in poor medium. The ydiV mutation did not have a significant effect on either the steady-state level of flhDC mRNA or that of FlhC protein. Purified YdiV protein was shown in vitro to bind to FlhD(4)C(2) through interaction with FlhD subunit and to inhibit its binding to the class 2 promoter, resulting in inhibition of FlhD(4)C(2)-dependent transcription. Taking these data together, we conclude that YdiV is a novel anti-FlhD(4)C(2) factor responsible for nutritional control of the flagellar regulon.

Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in human myocytes.

TAK-475 is a squalene synthase inhibitor, rapidly metabolized to T-91485 in vivo. We investigated the myotoxicities of T-91485 and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in a human rhabdomyosarcoma cell line, RD, and in human skeletal myocytes. In differentiated RD cells, T-91485, atorvastatin (ATV) and simvastatin acid (SIM) inhibited cholesterol biosynthesis, with IC(50) values of 36, 2.8 and 3.8 nM, respectively. ATV and SIM decreased the intracellular ATP content, with IC(25) values (concentrations giving a 25% decrease in intracellular ATP content) of 0.61 and 0.44 microM, respectively. Although T-91485 potently inhibited cholesterol synthesis in RD cells, the IC(25) value exceeded 100 microM. In human skeletal myocytes, T-91485, ATV and SIM concentration-dependently inhibited cholesterol biosynthesis, with IC(50) values of 45, 8.6 and 8.4 nM, respectively. ATV and SIM decreased intracellular ATP content, with IC(25) values of 2.1 and 0.72 microM, respectively. Although T-91485 potently inhibited cholesterol synthesis, the IC(25) value exceeded 100 microM. Myotoxicity induced by ATV was prevented by mevalonate or geranylgeranyl-PP, but not by squalene in skeletal cells. Furthermore, T-91485 attenuated the myotoxicity of ATV. These findings suggest that TAK-475 and T-91485 may not only be far from myotoxic, they may also decrease statin-induced myotoxicity in lipid-lowering therapy.

APPLICATION OF PRINCIPAL COMPONENT ANALYSIS TO FOOD TEXTURE MEASUREMENTS.

In order to clarify the relationship between different texture measurements, a principal component analysis was applied to the analysis of instrumental and sensory texture descriptions obtained on 72 samples of boiled plant protein representing hard gels, soft gels, and pastes. Twelve objective parameters were quantified using the Texturometer, the OKADA Gelometer, and the Curd Meter. Over 80% of the total variance could be explained by the first three components. The first component corresponded to parameters measuring breaking stress or 'hardness', the second to parameters measuring 'springiness', and the third to parameters measuring 'adhesion'. Hard gel samples were characterized by high values for the first component. Soft gel and paste samples could be distinguished from each other on the basis of the third component, with pastes being generally more adhesive.