Identification of novel cerebellar developmental transcriptional regulators with motif activity analysis.

BACKGROUND: The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development. RESULTS: We used the HeliScopeCAGE library sequencing on cerebellar samples over 8 embryonic and 4 early postnatal times. This study showcases temporal expression pattern changes during cerebellar development. Through a bioinformatics analysis that focused on transcription factors, their promoters and binding sites, we identified genes that appear as strong candidates for involvement in cerebellar development. We selected several candidate transcriptional regulators for validation experiments including qRT-PCR and shRNA transcript knockdown. We observed marked and reproducible developmental defects in Atf4, Rfx3, and Scrt2 knockdown embryos, which support the role of these genes in cerebellar development. CONCLUSIONS: The successful identification of these novel gene regulators in cerebellar development demonstrates that the FANTOM5 cerebellum time series is a high-quality transcriptome database for functional investigation of gene regulatory networks in cerebellar development.

Activation of multiple transcriptional regulators by growth restriction in Pseudomonas aeruginosa.

Growth restriction by antibiotics is a common feature that pathogenic bacteria must overcome for survival. The struggle of bacteria to escape from growth restriction eventually results in development of antibiotic-resistance through the expression of a set of genes. Here we found that some physiologically important transcriptional regulators of Pseudomonas aeruginosa including QscR, a quorum sensing (QS) receptor, SoxR, a superoxide sensor-regulator, and AntR, a regulator of anthranilate-related secondary metabolism, are activated by various growth-restricted conditions. We generated the growth-restricted conditions by various methods, such as overexpression of PA2537 and treatment with antibiotics or disinfectants. The overexpression of PA2537, encoding an acyltransferase homologue, tightly restricted the growth of P. aeruginosa and significantly activated QscR during the growth restriction. Similarly, treatments with gentamycin, tetracycline, and ethanol also activated QscR near their minimal inhibitory concentrations (MICs). Some non-QS regulators, such as AntR and SoxR, were also activated near the MICs in the same conditions. However, LasR and PqsR, other QS receptors of P. aeruginosa, were not activated, suggesting that only a specific set of transcriptional regulators is activated by growth restriction. Since paraquat, a super-oxide generator, significantly activated QscR and AntR, we suggest that the oxidative stress generated by growth restriction may be partly involved in this phenomenon.

Interspecies signaling through QscR, a quorum receptor of Pseudomonas aeruginosa.

The QS machinery of Pseudomonas aeruginosa, an opportunistic human pathogen, consists of three acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-I, RhlR-I, and QscR. QscR, known as an orphan receptor and a repressor of other QS systems, operates its own regulon using N-3-oxododecanoyl HSL (3OC12), which is synthesized by LasI, as its signal. In this study, we addressed the role of QscR in interspecies communication. We found that QscR auto-activates its own transcription in the presence of 3OC12. In a single population of P. aeruginosa, where 3OC12 is the sole signal available for QscR, the QscR regulon is activated by 3OC12 produced by the LasI-R system. However, the broad signal specificity of QscR allowed it to respond to a non-P. aeruginosa signal, such as N-decanoyl HSL (C10) and N-3-hydroxydecanoyl HSL (3OHC10), which preferentially activated QscR to LasR. The signal extracts from Pseudomonas fluorescens and Burkholeria vietnamiensis also preferentially activated QscR. These non-P. aeruginosa signals activated QscR more strongly than 3OC12, the authentic P. aeruginosa signal. Since a variety of acyl-HSLs are produced in the multi-species habitat of nature, our study provides a clue for the particular situation that allows QscR to secede from the conventional QS cascade in mixed microbial community.

AntR-mediated bidirectional activation of antA and antR, anthranilate degradative genes in Pseudomonas aeruginosa.

Bidirectional activation of transcription is a peculiar regulation mode of gene expression. In this study, we show that genes involved in the metabolism of anthranilate, a precursor of biosynthesis of tryptophan and Pseudomonas quinolone signal (PQS) are regulated by this bidirectional activation of transcription. Anthranilate is degraded by anthranilate dioxygenase complex encoded by antABC operon, and AntR, a LysR-type regulator encoded by antR activates the transcription of antABC operon in the presence of anthranilate. In P. aeruginosa, antABC and antR are divergently located and AntR binds to the intergenic region between antA and antR to activate the antABC transcription. In this study, we determined the transcriptional start site of the antA promoter (antA(p)) and AntR-responsive elements (AREs) in P. aeruginosa. The upstream deletion analysis of antA(p) and in vitro gel shift assay with purified AntR showed that there are two AREs at -194 to -148 and -88 to -47 regions. We also found that AntR activates antR promoter (antR(p)) in the opposite direction and both AREs are important in the bidirectional activation of antA(p) and antR(p). Two AREs have different binding affinities to AntR and the strength of transcriptional activation was dramatically asymmetric depending on the direction. We suggest that the different affinities of two AREs may explain the asymmetry of the bidirectional activation by AntR.

Growth phase-differential quorum sensing regulation of anthranilate metabolism in Pseudomonas aeruginosa.

Pseudomonas quinolone signal (PQS) plays a role in the regulation of virulence genes and it is intertwined in the las/rhl quorum sensing (QS) circuits of Pseudomonas aeruginosa. PQS is synthesized from anthranilate by pqsA-D and pqsH whose expression is influenced by the las/rhl systems. Since anthranilate can be degraded by functions of antABC and catBCA, PQS synthesis might be regulated by the balance between the expression of the pqsA-D/phnAB, pqsH, antABC, and catBCA gene loci. antA and catA are repressed by LasR during log phase and activated by RhlR in late stationary phase, whereas pqsA-E/phnAB is activated by LasR in log phase and repressed by RhlR. QscR represses both but each repression occurs in a different growth phase. This growth phase-differential regulation appears to be accomplished by the antagonistic interplay of LasR, RhlR, and QscR, mediated by two intermediate regulators, AntR and PqsR, and their cofactors, anthranilate and PQS, where the expressions of antR and pqsR and the production of anthranilate and PQS are growth phase-differentially regulated by QS systems. Especially, the anthranilate level increases in an RhlR-dependent manner at late stationary phase. From these results, we suggest that RhlR and LasR regulate the anthranilate metabolism in a mutually antagonistic and growth phase-differential manner by affecting both the expressions and activities of AntR and PqsR, and that QscR also phase-differentially represses both LasR and RhlR functions in this regulation.