Distinct Wolbachia localization patterns in oocytes of diverse host species reveal multiple strategies of maternal transmission.

A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity, and routes underlying this transmission strategy. Here, we address these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed 3 Wolbachia distribution patterns: (1) a tight clustering at the posterior pole plasm (the site of germline formation); (2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; and (3) a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicates Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that 1 Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Taken together, these studies suggest that for some Drosophila species, Wolbachia invade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on 1 of 2 distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here, we provide compelling evidence that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.

The yeast transcription elongation factor Spt4/5 is a sequence-specific RNA binding protein.

The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5.

Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5.

The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.

A broadly implementable research course in phage discovery and genomics for first-year undergraduate students.

UNLABELLED: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

The Spt4-Spt5 complex: a multi-faceted regulator of transcription elongation.

In all domains of life, elongating RNA polymerases require the assistance of accessory factors to maintain their processivity and regulate their rate. Among these elongation factors, the Spt5/NusG factors stand out. Members of this protein family appear to be the only transcription accessory proteins that are universally conserved across all domains of life. In archaea and eukaryotes, Spt5 associates with a second protein, Spt4. In addition to regulating elongation, the eukaryotic Spt4-Spt5 complex appears to couple chromatin modification states and RNA processing to transcription elongation. This review discusses the experimental bases for our current understanding of Spt4-Spt5 function and recent studies that are beginning to elucidate the structure of Spt4-Spt5/RNA polymerase complexes and mechanism of Spt4-Spt5 action. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

A key role for Chd1 in histone H3 dynamics at the 3' ends of long genes in yeast.

Chd proteins are ATP-dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5' and 3' ends of genes, accelerating H3 replacement at the 5' ends of genes while protecting the 3' ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3' ends of genes.

Structural determination of NSC 670224, synthesis of analogues and biological evaluation.

Follow my lead! NSC 670224, previously shown to be toxic to Saccharomyces cerevisiae at low micromolar concentrations, potentially acts via a mechanism of action related to that of tamoxifen (NSC 180973), breast cancer drug. The structure of NSC 670224, previously thought to be a 2,4-dichloro arene, was established as the 3,4-dichloro arene, and a focused library of analogues were synthesized and biologically evaluated.

Competing for the clamp: promoting RNA polymerase processivity and managing the transition from initiation to elongation.

Transcription elongation factor NusG/Spt5 spans the central cleft of RNA polymerase and functionally competes with transcription initiation factors. This work highlights the RNA polymerase clamp as a target for regulation and points to dynamic interactions between initiation and elongation machineries.

Histone H3K4 and K36 methylation, Chd1 and Rpd3S oppose the functions of Saccharomyces cerevisiae Spt4-Spt5 in transcription.

Spt4-Spt5, a general transcription elongation factor for RNA polymerase II, also has roles in chromatin regulation. However, the relationships between these functions are not clear. Previously, we isolated suppressors of a Saccharomyces cerevisiae spt5 mutation in genes encoding members of the Paf1 complex, which regulates several cotranscriptional histone modifications, and Chd1, a chromatin remodeling enzyme. Here, we show that this suppression of spt5 can result from loss of histone H3 lysines 4 or 36 methylation, or reduced recruitment of Chd1 or the Rpd3S complex. These spt5 suppressors also rescue the synthetic growth defects observed in spt5 mutants that also lack elongation factor TFIIS. Using a FLO8 reporter gene, we found that a chd1 mutation caused cryptic initiation of transcription. We further observed enhancement of cryptic initiation in chd1 isw1 mutants and increased histone acetylation in a chd1 mutant. We suggest that, as previously proposed for H3 lysine 36 methylation and the Rpd3S complex, H3 lysine 4 methylation and Chd1 function to maintain normal chromatin structures over transcribed genes, and that one function of Spt4-Spt5 is to help RNA polymerase II overcome the repressive effects of these histone modifications and chromatin regulators on transcription.

Core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation.

The Spt4-Spt5 complex is an essential RNA polymerase II elongation factor found in all eukaryotes and important for gene regulation. We report here the crystal structure of Saccharomyces cerevisiae Spt4 bound to the NGN domain of Spt5. This structure reveals that Spt4-Spt5 binding is governed by an acid-dipole interaction between Spt5 and Spt4. Mutations that disrupt this interaction disrupt the complex. Residues forming this pivotal interaction are conserved in the archaeal homologs of Spt4 and Spt5, which we show also form a complex. Even though bacteria lack a Spt4 homolog, the NGN domains of Spt5 and its bacterial homologs are structurally similar. Spt4 is located at a position that may help to maintain the functional conformation of the following KOW domains in Spt5. This structural and evolutionary perspective of the Spt4-Spt5 complex and its homologs suggest that it is an ancient, core component of the transcription elongation machinery.

Just the FACTs: histone H2B ubiquitylation and nucleosome dynamics.

In this issue of Molecular Cell, Fleming et al. (2008) show that histone H2B ubiquitylation and FACT function interdependently to facilitate nucleosome reassembly during transcription elongation, thereby demonstrating that histone posttranslational modifications can provide important but transient transcriptional signaling cues.

Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

Exploring functional relationships between components of the gene expression machinery.

Eukaryotic gene expression requires the coordinated activity of many macromolecular machines including transcription factors and RNA polymerase, the spliceosome, mRNA export factors, the nuclear pore, the ribosome and decay machineries. Yeast carrying mutations in genes encoding components of these machineries were examined using microarrays to measure changes in both pre-mRNA and mRNA levels. We used these measurements as a quantitative phenotype to ask how steps in the gene expression pathway are functionally connected. A multiclass support vector machine was trained to recognize the gene expression phenotypes caused by these mutations. In several cases, unexpected phenotype assignments by the computer revealed functional roles for specific factors at multiple steps in the gene expression pathway. The ability to resolve gene expression pathway phenotypes provides insight into how the major machineries of gene expression communicate with each other.

Transcription elongation by RNA polymerase II.

The elongation of transcripts by RNA polymerase II (RNAPII) is subject to regulation and requires the services of a host of accessory proteins. Although the biochemical mechanisms underlying elongation and its regulation remain obscure, recent progress sets the stage for rapid advancement in our understanding of this phase of transcription. High-resolution crystal structures will allow focused analyses of RNAPII in all its functional states. Several recent studies suggest specific roles for the C-terminal heptad repeats of the largest subunit of RNAPII in elongation. Proteomic approaches are being used to identify new transcription-elongation factors and to define interactions between elongation factors and RNAPII. Finally, a combination of genetic analysis and the localization of factors on transcribed chromatin is being used to confirm a role for factors in elongation.