A common regulatory motif, where a heterodimeric transcriptional regulator positively autoregulates only one of its components, is found to have particular properties that enable precise and robust control of cellular responses to environmental stimuli, providing an explanation for the prevalence of this motif in evolved regulatory networks.

Many important biological systems rely on regulation by dimers of proteins which upregulate the transcription of numerous targets, including one, and only one, of the dimer pair. This is termed asymmetric self-upregulation.ASymmetric Self-UpREgulated (ASSURE) networks confer rapid induction of their targets and their network behaviors are robust to parameter variation—both features appear to have contributed to the prevalence of the network across widely different biological systems.Likely evolutionary precursors to ASSURE networks are symmetrically self-upregulated network mediated by homodimers. In silico and experimental studies demonstrate that the ASSURE network confers a competitive advantage over its symmetrical counterpart.

Positive feedback is a common mechanism enabling biological systems to respond to stimuli in a switch-like manner. Such systems are often characterized by the requisite formation of a heterodimer where only one of the pair is subject to feedback. This ASymmetric Self-UpREgulation (ASSURE) motif is central to many biological systems, including cholesterol homeostasis (LXRα/RXRα), adipocyte differentiation (PPARγ/RXRα), development and differentiation (RAR/RXR), myogenesis (MyoD/E12) and cellular antiviral defense (IRF3/IRF7). To understand why this motif is so prevalent, we examined its properties in an evolutionarily conserved transcriptional regulatory network in yeast (Oaf1p/Pip2p). We demonstrate that the asymmetry in positive feedback confers a competitive advantage and allows the system to robustly increase its responsiveness while precisely tuning the response to a consistent level in the presence of varying stimuli. This study reveals evolutionary advantages for the ASSURE motif, and mechanisms for control, that are relevant to pharmacologic intervention and synthetic biology applications.

Stable incorporation of labeled amino acids in cell culture is a simple approach to label proteins in vivo for mass spectrometric quantification. Full incorporation of isotopically heavy amino acids facilitates accurate quantification of proteins from different cultures, yet analysis methods for determination of incorporation are cumbersome and time-consuming. We present QTIPS, Quantification by Total Identified Peptides for SILAC, a straightforward, accurate method to determine the level of heavy amino acid incorporation throughout a population of peptides detected by mass spectrometry. Using QTIPS, we show that the incorporation of heavy amino acids in baker’s yeast is unaffected by the use of prototrophic strains, indicating that auxotrophy is not a requirement for SILAC experiments in this organism. This method has general utility for multiple applications where isotopic labeling is used for quantification in mass spectrometry.

Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for β-oxidation of fatty acids. Peroxisomes biogenesis can be induced by the presence of peroxisome proliferators, including fatty acids, which activate complex cellular programs that underlie the induction process. Here, we used multi-parameter quantitative phenotype analyses of an arrayed mutant collection of yeast cells induced to proliferate peroxisomes, to establish a comprehensive inventory of genes required for peroxisome induction and function. The assays employed include growth in the presence of fatty acids, and confocal imaging and flow cytometry through the induction process. In addition to the classical phenotypes associated with loss of peroxisomal functions, these studies identified 169 genes required for robust signaling, transcription, normal peroxisomal development and morphologies, and transmission of peroxisomes to daughter cells. These gene products are localized throughout the cell, and many have indirect connections to peroxisome function. By integration with extant data sets, we present a total of 211 genes linked to peroxisome biogenesis and highlight the complex networks through which information flows during peroxisome biogenesis and function.

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

Chz1p is a histone chaperone that interacts physically and functionally with the histone variant Htz1p, which has been implicated in establishing and maintaining boundaries between transcriptionally inactive heterochromatin and active euchromatin. To investigate the role of Chz1p in chromatin organization, we performed genome-wide expression arrays and chromatin immunoprecipitations of SIR complex components and modified histones in a CHZ1 deletion strain. Deletion of CHZ1 led to reduced ubiquitination of subtelomere-associated H2B, reduced subtelomeric H3K79 di-methylation, and increased binding of Sir3p, and Sir4p at telomere-distal euchromatin regions, correlating with decreased gene expression in subtelomeric regions. This anti-silencing defect appears to be mediated by enhanced association of de-ubiquitinase Ubp10p with subtelomeric DNA, as detected by chromatin immunoprecipitation analysis. In support of this, we show that deletion of UBP10 can antagonize the subtelomeric silencing phenotype of Δchz1. Taken together, the results demonstrate a novel role for Chz1p in epigenetic regulation, through H2B de-ubiquitination by Ubp10p.

The histone variant H2A.Z (Htz1p) has been implicated in transcriptional regulation in numerous organisms, including Saccharomyces cerevisiae. Genome-wide transcriptome profiling and chromatin immunoprecipitation studies identified a role for Htz1p in the rapid and robust activation of many oleate-responsive genes encoding peroxisomal proteins, in particular POT1, POX1, FOX2, and CTA1. The Swr1p-, Gcn5p-, and Chz1p-dependent association of Htz1p with these promoters in their repressed states appears to establish an epigenetic marker for the rapid and strong expression of these highly inducible promoters. Isw2p also plays a role in establishing the nucleosome state of these promoters and associates stably in the absence of Htz1p. An analysis of the nucleosome dynamics and Htz1p association with these promoters suggests a complex mechanism in which Htz1p-containing nucleosomes at fatty acid-responsive promoters are disassembled upon initial exposure to oleic acid leading to the loss of Htz1p from the promoter. These nucleosomes reassemble at later stages of gene expression. While these new nucleosomes do not incorporate Htz1p, the initial presence of Htz1p appears to mark the promoter for sustained gene expression and the recruitment of TATA-binding protein.

Reversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.

In transcriptional regulatory networks, the coincident binding of a combination of factors to regulate a gene implies the existence of complex mechanisms to control both the gene expression profile and specificity of the response. Unraveling this complexity is a major challenge to biologists. Here, a novel network topology-based clustering approach was applied to condition-specific genome-wide chromatin localization and expression data to characterize a dynamic transcriptional regulatory network responsive to the fatty acid oleate. A network of four (predicted) regulators of the response (Oaf1p, Pip2p, Adr1p and Oaf3p) was investigated. By analyzing trends in the network structure, we found that two groups of multi-input motifs form in response to oleate, each controlling distinct functional classes of genes. This functionality is contributed in part by Oaf1p, which is a component of both types of multi-input motifs and has two different regulatory activities depending on its binding context. The dynamic cooperation between Oaf1p and Pip2p appears to temporally synchronize the two different responses. Together, these data suggest a network mechanism involving dynamic combinatorial control for coordinating transcriptional responses.

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

An open-source inkjet arrayer is described here capable of rapidly producing sets of unique 9,800-feature arrays.

DNA arrays are valuable tools in molecular biology laboratories. Their rapid acceptance was aided by the release of plans for a pin-spotting microarrayer by researchers at Stanford. Inkjet microarraying is a flexible, complementary technique that allows the synthesis of arrays of any oligonucleotide sequences de novo. We describe here an open-source inkjet arrayer capable of rapidly producing sets of unique 9,800-feature arrays.