Megan J. Wilson

Matching SOX: Partner proteins and co-factors of the SOX family of transcriptional regulators

SOX transcription factors perform a remarkable variety of important roles in vertebrate development, either activating or repressing specific target genes through interaction with different partner proteins. Surprisingly, these interactions are often mediated by the conserved, DNA-binding HMG domain, raising questions as to how each factors specificity is generated. We propose a model whereby non-HMG domains may influence partner protein selection and/or binding stability.

Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas

A variety of bacterial species secrete and take up chelating compounds that enable acquisition of iron (siderophores). It has become clear that a common feature in regulation of different iron acquisition systems is the involvement of alternative sigma factor proteins of the extracytoplasmic function (ECF) family. Two of these proteins, PvdS from Pseudomonas aeruginosa and FecI from Escherichia coli K‐12, have been studied extensively. PvdS directs transcription of genes required for the biosynthesis of a siderophore, pyoverdine, and FecI causes ex‐pression of genes for uptake of ferric citrate. FecI forms part of a signalling system that responds to the presence of ferric citrate. Here, we review recent advances in understanding of PvdS and of the Fec signalling system. PvdS and FecI are part of a distinct subfamily of ECF sigma factors involved in iron acquisition and hence named the iron‐starvation sigmas. Analysis of microbial genome sequences shows that Fec‐like signalling systems are present in a wide range of species and many such systems may be present in a single species. The availability of tools for large‐scale genome analysis is likely to lead to rapid advances in our understanding of this expanding family of proteins.

Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa

ABSTRACT The expression of many virulence factors in Pseudomonas aeruginosa is dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence of P. aeruginosa PAO1 is influenced by the iron- and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a ΔpvdS::Gm mutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the ΔpvdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from ΔpvdS::Gm was investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is homologous to an endoprotease produced by Lysobacter enzymogenes. In this study, the gene encoding an endoprotease was cloned from PAO1 and designated prpL (PvdS-regulated endoprotease, lysyl class). All (n = 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, examined carry prpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site ofprpL and confirmed that its transcription is iron dependent. In the ΔpvdS::Gmmutant, the level of prpL transcription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription of prpL was independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1s ability to persist in a rat chronic pulmonary infection model.

Analysis of Promoters Recognized by PvdS, an Extracytoplasmic-Function Sigma Factor Protein from Pseudomonas aeruginosa

The alternative sigma factor PvdS is required by Pseudomonas aeruginosa for initiation of transcription from pyoverdine (pvd) promoters. Two divergent PvdS-dependent promoters (pvdE and pvdF) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other pvd promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from pvd promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from pvd promoters and are likely to represent a -35 sequence element for this sigma factor.

Comprehensive survey of developmental genes in the pea aphid, Acyrthosiphon pisum: frequent lineage‐specific duplications and losses of developmental genes

Aphids exhibit unique attributes, such as polyphenisms and specialized cells to house endosymbionts, that make them an interesting system for studies at the interface of ecology, evolution and development. Here we present a comprehensive characterization of the developmental genes in the pea aphid, Acyrthosiphon pisum, and compare our results to other sequenced insects. We investigated genes involved in fundamental developmental processes such as establishment of the body plan and organogenesis, focusing on transcription factors and components of signalling pathways. We found that most developmental genes were well conserved in the pea aphid, although many lineage‐specific gene duplications and gene losses have occurred in several gene families. In particular, genetic components of transforming growth factor beta (TGFβ) Wnt, JAK/STAT (Janus kinase/signal transducer and activator of transcription) and EGF (Epidermal Growth Factor) pathways appear to have been significantly modified in the pea aphid.

Giant, Krüppel, and caudal act as gap genes with extensive roles in patterning the honeybee embryo

In Drosophila, gap genes translate positional information from gradients of maternal coordinate activity and act to position the periodic patterns of pair-rule gene stripes across broad domains of the embryo. In holometabolous insects, maternal coordinate genes are fast-evolving, the domains that gap genes specify often differ from their orthologues in Drosophila while the expression of pair-rule genes is more conserved. This implies that gap genes may buffer the fast-evolving maternal coordinate genes to give a more conserved pair-rule output. To test this idea, we have examined the function and expression of three honeybee orthologues of gap genes, Krüppel, caudal, and giant. In honeybees, where many Drosophila maternal coordinate genes are missing, these three gap genes have more extensive domains of expression and activity than in other insects. Unusually, honeybee caudal mRNA is initially localized to the anterior of the oocyte and embryo, yet it has no discernible function in that domain. We have also examined the influence of these three genes on the expression of honeybee even-skipped and a honeybee orthologue of engrailed and show that the way that these genes influence segmental patterning differs from Drosophila. We conclude that while the fundamental function of these gap genes is conserved in the honeybee, shifts in their expression and function have occurred, perhaps due to the apparently different maternal patterning systems in this insect.

Tailless patterning functions are conserved in the honeybee even in the absence of Torso signaling.

In Drosophila, the maternal Torso terminal signaling pathway activates expression of the gene tailless (tll), which is required for the patterning of anterior and posterior termini. We cloned the honeybee orthologue of tll (Am-tll) and found that embryonic expression of Am-tll resembles that of Drosophila, with expression in triangular anterior dorsal-lateral domains and a posterior cap. Functional studies revealed that Am-tll has an essential role in patterning the posterior terminal segments and the brain, similar to the activity of tll in other insects. As the honeybee genome lacks many of the components of the Torso pathway required for terminal patterning, we investigated the regulation of honeybee tailless (Am-tll). Am-tll is expressed maternally and, in the honeybee ovary, Am-tll mRNA becomes localized to the dorsal side of the oocyte, a process requiring the actin cytoskeleton. This RNA becomes redistributed in early embryos to a posterior domain. We also show that the activation of the anterior domain of Am-tll is dependent on honeybee orthodenticle-1. Together these findings indicate major differences in post-transcriptional regulation of tailless in the honeybee compared to other insects but that this regulation leads to a conserved expression pattern. These results provide an example of an early event in development evolving and yet still producing a conserved output for the rest of development to build upon.

Evolution of the insect Sox genes

BackgroundThe Sox gene family of transcriptional regulators have essential roles during development and have been extensively studied in vertebrates. The mouse, human and fugu genomes contain at least 20 Sox genes, which are subdivided into groups based on sequence similarity of the highly conserved HMG domain. In the well-studied insect Drosophila melanogaster, eight Sox genes have been identified and are involved in processes such as neurogenesis, dorsal-ventral patterning and segmentation.ResultsWe examined the available genome sequences of Apis mellifera, Nasonia vitripennis, Tribolium castaneum, Anopheles gambiae and identified Sox family members which were classified by phylogenetics using the HMG domains. Using in situ hybridisation we determined the expression patterns of eight honeybee Sox genes in honeybee embryo, adult brain and queen ovary. AmSoxB group genes were expressed in the nervous system, brain and Malphigian tubules. The restricted localization of AmSox21b and AmSoxB1 mRNAs within the oocyte, suggested a role in, or that they are regulated by, dorsal-ventral patterning. AmSoxC, D and F were expressed ubiquitously in late embryos and in the follicle cells of the queen ovary. Expression of AmSoxF and two AmSoxE genes was detected in the drone testis.ConclusionInsect genomes contain between eight and nine Sox genes, with at least four members belonging to Sox group B and other Sox subgroups each being represented by a single Sox gene. Hymenopteran insects have an additional SoxE gene, which may have arisen by gene duplication. Expression analyses of honeybee SoxB genes implies that this group of genes may be able to rapidly evolve new functions and expression domains, while the combined expression pattern of all the SoxB genes is maintained.

p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which is important for the prevention of B-cell lymphomas. We created a mouse model (mΔpro) that lacked residues 58–88 of the proline-rich domain of p53. mΔpro is defective for apoptosis, but is able to arrest cell-cycle progression in hematopoietic tissues. mΔpro develops late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-null mice. Interestingly, mΔpro lymphomas comprised incorrectly differentiated B cells. B-cell irregularities were also detected in mΔpro before tumor onset, in which aged mice showed an increased population of inappropriately differentiated B cells in the bone marrow and spleen. We predict that by keeping B-cell populations in check, p53-dependent apoptosis prevents irregular B cells from eventuating in lymphomas.

Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (Apis mellifera)

Axis formation is a key step in development, but studies indicate that genes involved in insect axis formation are relatively fast evolving. Orthodenticle genes have conserved roles, often with hunchback, in maternal anterior patterning in several insect species. We show that two orthodenticle genes, otd1 and otd2, and hunchback act as maternal anterior patterning genes in the honeybee (Apis mellifera) but, unlike other insects, act to pattern the majority of the anteroposterior axis. These genes regulate the expression domains of anterior, central and posterior gap genes and may directly regulate the anterior gap gene giant. We show otd1 and hunchback also influence dorsoventral patterning by regulating zerknült (zen) as they do in Tribolium, but that zen does not regulate the expression of honeybee gap genes. This suggests that interactions between anteroposterior and dorsal-ventral patterning are ancestral in holometabolous insects. Honeybee axis formation, and the function of the conserved anterior patterning gene orthodenticle, displays unique characters that indicate that, even when conserved genes pattern the axis, their regulatory interactions differ within orders of insects, consistent with relatively fast evolution in axis formation pathways.