Epistasis analysis with global transcriptional phenotypes.

Classical epistasis analysis can determine the order of function of genes in pathways using morphological, biochemical and other phenotypes. It requires knowledge of the pathway's phenotypic output and a variety of experimental expertise and so is unsuitable for genome-scale analysis. Here we used microarray profiles of mutants as phenotypes for epistasis analysis. Considering genes that regulate activity of protein kinase A in Dictyostelium, we identified known and unknown epistatic relationships and reconstructed a genetic network with microarray phenotypes alone. This work shows that microarray data can provide a uniform, quantitative tool for large-scale genetic network analysis.

Publication practices and standards: recommendations from GSK Vaccines' author survey.

BACKGROUND: Evolving standards of good publication practice (GPP) and a survey conducted in 2009 of authors, who were investigators and researchers not employed by the company prompted changes to GSK Vaccines' publication practices. We conducted a follow-up survey in 2012 to assess the company's revised practices and to evaluate understanding of GPP among investigators and researchers who had previously authored at least one publication in collaboration with GSK Vaccines. METHODS: The 50-question web-based survey addressed authoring practices and transparency of decision-making. Investigators and researchers (n = 1,273) who had authored at least one publication reporting on GSK Vaccines-sponsored human research since 2007, were invited to participate. Responses to 37 closed questions are presented. The remaining 13 questions were open-ended or did not concern publication practices. RESULTS: A total of 415 external authors (32.6%) responded. International Committee of Medical Journal Editors (ICMJE) authorship criteria were clear to most respondents (78.1%); 7.7% found they were unclear. The majority of participants (86.8%) found GSK Vaccines' authorship questionnaire a suitable tool to assess eligibility for authorship as per the ICMJE criteria. However, only 68.5% felt that the outcome of the questionnaire is communicated appropriately and 58.3% felt well informed on changes in authorship. Nearly two-thirds (62.9%) of respondents felt that having a pharmaceutical company employee as lead author makes manuscript acceptance less likely. Access to relevant data was regarded as sufficient by 78.5% of respondents. Briefing meetings before publication start, publication steering committees and core writing teams were recognized as valuable publication practices. Professional medical writing support was seen as adding value to publication development by 87.7% of participants. Most respondents agreed that manuscript discussions should start early, with 81.7% stating that they were in favor of introducing a formalized 'author agreement' at the publication start. CONCLUSIONS: GSK Vaccines made changes to its publication practices to ensure improved transparency and better involvement of external authors. The results of this survey suggest that these changes have been effective to a large extent. They confirm the need for effective and timely communication, as well as transparent processes for authorship and decision-making during publication development. The identified gaps in GPP will help to guide further improvements to the company's policies on publication practices.

Global transcriptional responses to cisplatin in Dictyostelium discoideum identify potential drug targets.

Dictyostelium discoideum is a useful model for studying mechanisms of cisplatin drug sensitivity. Our previous findings, that mutations in sphingolipid metabolism genes confer cisplatin resistance in D. discoideum and in human cells, raised interest in the resistance mechanisms and their implications for cisplatin chemotherapy. Here we used expression microarrays to monitor physiological changes and to identify pathways that are affected by cisplatin treatment of D. discoideum. We found >400 genes whose regulation was altered by cisplatin treatment of wild-type cells, including groups of genes that participate in cell proliferation and in nucleotide and protein metabolism, showing that the cisplatin response is orderly and multifaceted. Transcriptional profiling of two isogenic cisplatin-resistant mutants, impaired in different sphingolipid metabolism steps, showed that the effect of cisplatin treatment was greater than the effect of the mutations, indicating that cisplatin resistance in the mutants is due to specific abilities to overcome the drug effects rather than to general drug insensitivity. Nevertheless, the mutants exhibited significantly different responses to cisplatin compared with the parent, and >200 genes accounted for that difference. Mutations in five cisplatin response genes (sgkB, csbA, acbA, smlA, and atg8) resulted in altered drug sensitivity, implicating novel pathways in cisplatin response. Our data illustrate how modeling complex cellular responses to drugs in genetically stable and tractable systems can uncover new targets with the potential for improving chemotherapy.

Microarray phenotyping in Dictyostelium reveals a regulon of chemotaxis genes.

MOTIVATION: Coordinate regulation of gene expression can provide information on gene function. To begin a large-scale analysis of Dictyostelium gene function, we clustered genes based on their expression in wild-type and mutant strains and analyzed their functions. RESULTS: We found 17 modes of wild-type gene expression and refined them into 57 submodes considering mutant data. Annotation analyses revealed correlations between co-expression and function and an unexpected correlation between expression and function of genes involved in various aspects of chemotaxis. Co-regulation of chemotaxis genes was also found in published data from neutrophils. To test the predictive power of the analysis, we examined the phenotypes of mutations in seven co-regulated genes that had no published role in chemotaxis. Six mutants exhibited chemotaxis defects, supporting the idea that function can be inferred from co-expression. The clustering and annotation analyses provide a public resource for Dictyostelium functional genomics.

A novel developmental mechanism in Dictyostelium revealed in a screen for communication mutants.

We performed a screen for signaling genes by selecting mutant strains of Dictyostelium that fail to develop spores in a pure population but sporulate well in chimerae with wild type cells. We found 9 strains whose sporulation was induced up to 10 million-fold in chimerae. Most strains were also able to sporulate in chimerae with each other, but 2 pairs failed to do so, suggesting that the genes in each pair participate in the production of 1 signal. One of the pairs, comD and comB, is described in detail. Sequence analysis revealed that both genes encode putative membrane proteins. ComD is predicted to have 15 transmembrane domains, and ComB has a region of high similarity to the Rab family of small GTPases and 1 transmembrane domain. Similarities between the developmental regulation and cell-type specificity of the genes' expression, the terminal developmental morphology, and the expression pattern of cell-type specific markers in the mutants suggest that comD and comB participate in 1 signal production pathway. This idea is also supported by a high similarity between the global transcriptional profiles of the mutant strains. Differences between the mutant phenotypes late in development suggest that comD and comB participate in separate processes as well. comD has a cell-autonomous role in the specialization of a novel prespore cell type, whereas comB has a cell-autonomous role in prestalk A cell differentiation.

TagA, a putative serine protease/ABC transporter of Dictyostelium that is required for cell fate determination at the onset of development.

The tag genes of Dictyostelium are predicted to encode multi-domain proteins consisting of serine protease and ATP-binding cassette transporter domains. We have identified a novel tag gene, tagA, which is involved in cell type differentiation. The tagA mRNA accumulates during the first four hours of development, whereas TagA protein accumulates between two and ten hours of development and decreases thereafter. Wild-type cells express tagA in prespore cells and mature spores, defining tagA expression as prespore specific. However, tagA mutant cells that activate the tagA promoter do not sporulate, but instead form part of the outer basal disc and lower cup of the fruiting body. tagA mutant aggregates elaborate multiple prestalk cell regions during development and produce spores asynchronously and with low viability. tagA mutants produce about twice as many prestalk cells as the wild type as judged by a prestalk cell reporter construct. When mixed with wild-type cells, tagA(-) cells become overrepresented in the prestalk cell population, suggesting that this phenotype is cell-autonomous. These results suggest that TagA is required for the specification of an initial population of prespore cells in which tagA is expressed. Expression profiling uncovered a delay in the transcriptional program between 2 and 6 hours, coincident with TagA expression, revealing an early function for TagA. TagA also appears to play a general role in cell fate determination since tagA mutants express a spore coat protein gene (cotB) within vacuolated cells that form part of the stalk and they express a prestalk/stalk-specific gene (ecmB) within cells that become spores. The expression of TagA at two hours of development, the observed coincident delay in the transcriptional program and the subsequent mis-expression of cell-type specific genes provide evidence for cell fate determination beginning in some cells much earlier than previously believed.

An orderly retreat: Dedifferentiation is a regulated process.

Differentiation is a highly regulated process whereby cells become specialized to perform specific functions and lose the ability to perform others. In contrast, the question of whether dedifferentiation is a genetically determined process, or merely an unregulated loss of the differentiated state, has not been resolved. We show here that dedifferentiation in the social amoeba Dictyostelium discoideum relies on a sequence of events that is independent of the original developmental state and involves the coordinated expression of a specific set of genes. A defect in one of these genes, the histidine kinase dhkA, alters the kinetics of dedifferentiation and uncouples the progression of dedifferentiation events. These observations establish dedifferentiation as a genetically determined process and suggest the existence of a developmental checkpoint that ensures a return path to the undifferentiated state.

A transcriptional profile of multicellular development in Dictyostelium discoideum.

A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events. Analysis of the two differentiated cell types, spores and stalk cells, and their precursors revealed a large number of differentially expressed genes as well as unexpected patterns of gene expression, which shed new light on the timing and possible mechanisms of cell-type divergence. Our findings provide new perspectives on the complexity of the developmental program and the fraction of the genome that is regulated during development.