Marc T. Facciotti

The Inferelator: an algorithm for learning parsimonious regulatory networks from systems-biology data sets de novo

We present a method (the Inferelator) for deriving genome-wide transcriptional regulatory interactions, and apply the method to predict a large portion of the regulatory network of the archaeon Halobacterium NRC-1. The Inferelator uses regression and variable selection to identify transcriptional influences on genes based on the integration of genome annotation and expression data. The learned network successfully predicted Halobacteriums global expression under novel perturbations with predictive power similar to that seen over training data. Several specific regulatory predictions were experimentally tested and verified.

An Integrated Pipeline for de Novo Assembly of Microbial Genomes

Remarkable advances in DNA sequencing technology have created a need for de novo genome assembly methods tailored to work with the new sequencing data types. Many such methods have been published in recent years, but assembling raw sequence data to obtain a draft genome has remained a complex, multi-step process, involving several stages of sequence data cleaning, error correction, assembly, and quality control. Successful application of these steps usually requires intimate knowledge of a diverse set of algorithms and software. We present an assembly pipeline called A5 (Andrew And Aarons Awesome Assembly pipeline) that simplifies the entire genome assembly process by automating these stages, by integrating several previously published algorithms with new algorithms for quality control and automated assembly parameter selection. We demonstrate that A5 can produce assemblies of quality comparable to a leading assembly algorithm, SOAPdenovo, without any prior knowledge of the particular genome being assembled and without the extensive parameter tuning required by the other assembly algorithm. In particular, the assemblies produced by A5 exhibit 50% or more reduction in broken protein coding sequences relative to SOAPdenovo assemblies. The A5 pipeline can also assemble Illumina sequence data from libraries constructed by the Nextera (transposon-catalyzed) protocol, which have markedly different characteristics to mechanically sheared libraries. Finally, A5 has modest compute requirements, and can assemble a typical bacterial genome on current desktop or laptop computer hardware in under two hours, depending on depth of coverage.

A Predictive Model for Transcriptional Control of Physiology in a Free Living Cell

The environment significantly influences the dynamic expression and assembly of all components encoded in the genome of an organism into functional biological networks. We have constructed a model for this process in Halobacterium salinarum NRC-1 through the data-driven discovery of regulatory and functional interrelationships among approximately 80% of its genes and key abiotic factors in its hypersaline environment. Using relative changes in 72 transcription factors and 9 environmental factors (EFs) this model accurately predicts dynamic transcriptional responses of all these genes in 147 newly collected experiments representing completely novel genetic backgrounds and environments-suggesting a remarkable degree of network completeness. Using this model we have constructed and tested hypotheses critical to this organisms interaction with its changing hypersaline environment. This study supports the claim that the high degree of connectivity within biological and EF networks will enable the construction of similar models for any organism from relatively modest numbers of experiments.

Evaluation of algorithm performance in ChIP-seq peak detection.

Next-generation DNA sequencing coupled with chromatin immunoprecipitation (ChIP-seq) is revolutionizing our ability to interrogate whole genome protein-DNA interactions. Identification of protein binding sites from ChIP-seq data has required novel computational tools, distinct from those used for the analysis of ChIP-Chip experiments. The growing popularity of ChIP-seq spurred the development of many different analytical programs (at last count, we noted 31 open source methods), each with some purported advantage. Given that the literature is dense and empirical benchmarking challenging, selecting an appropriate method for ChIP-seq analysis has become a daunting task. Herein we compare the performance of eleven different peak calling programs on common empirical, transcription factor datasets and measure their sensitivity, accuracy and usability. Our analysis provides an unbiased critical assessment of available technologies, and should assist researchers in choosing a suitable tool for handling ChIP-seq data.

Improved stearate phenotype in transgenic canola expressing a modified acyl-acyl carrier protein thioesterase

The engineering of crops for selected fatty acid production is one of the major goals of plant biotechnology. The Garm FatA1, an acyl-acyl carrier protein (ACP) thioesterase isolated from Garcinia mangostana, generates an elevated stearate (18:0) phenotype in transgenic Brassica plants. By site-directed mutagenesis, we generated seven mutants that showed up to a 13-fold increase in specific enzyme activity toward 18:0-ACP in vitro. The seed-specific expression of mutant S111A/V193A in Brassica plants results in transgenic plants that accumulate 55–68% more stearate than plants expressing the wild-type enzyme. Our results demonstrate that a thioesterase can be engineered to increase specific activity and that its improved function demonstrated in vitro is retained in vivo.

Structure of an Early Intermediate in the M-State Phase of the Bacteriorhodopsin Photocycle

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.

Prevalence of transcription promoters within archaeal operons and coding sequences.

Despite the knowledge of complex prokaryotic‐transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well‐defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome‐wide characterization of transcript structures of ∼64% of all genes, including putative non‐coding RNAs in Halobacterium salinarum NRC‐1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment‐dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non‐functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

General transcription factor specified global gene regulation in archaea

Cells responding to dramatic environmental changes or undergoing a developmental switch typically change the expression of numerous genes. In bacteria, σ factors regulate much of this process, whereas in eukaryotes, four RNA polymerases and a multiplicity of generalized transcription factors (GTFs) are required. Here, by using a systems approach, we provide experimental evidence (including protein-coimmunoprecipitation, ChIP-Chip, GTF perturbation and knockout, and measurement of transcriptional changes in these genetically perturbed strains) for how archaea likely accomplish similar large-scale transcriptional segregation and modulation of physiological functions. We are able to associate GTFs to nearly half of all putative promoters and show evidence for at least 7 of the possible 42 functional GTF pairs. This report represents a significant contribution toward closing the gap in our understanding of gene regulation by GTFs for all three domains of life and provides an example for how to use various experimental techniques to rapidly learn significant portions of a global gene regulatory network of organisms for which little has been previously known.

Mauve Assembly Metrics

Summary: High-throughput DNA sequencing technologies have spurred the development of numerous novel methods for genome assembly. With few exceptions, these algorithms are heuristic and require one or more parameters to be manually set by the user. One approach to parameter tuning involves assembling data from an organism with an available high-quality reference genome, and measuring assembly accuracy using some metrics. We developed a system to measure assembly quality under several scoring metrics, and to compare assembly quality across a variety of assemblers, sequence data types, and parameter choices. When used in conjunction with training data such as a high-quality reference genome and sequence reads from the same organism, our program can be used to manually identify an optimal sequencing and assembly strategy for de novo sequencing of related organisms. Availability: GPL source code and a usage tutorial is at http://ngopt.googlecode.com Contact: aarondarling@ucdavis.edu Supplementary information: Supplementary data is available at Bioinformatics online.

IRF-1 and miRNA126 Modulate VCAM-1 Expression in Response to a High-Fat Meal

Rationale: A high-fat diet accompanied by hypertriglyceridemia increases an individual’s risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. Objective: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal. Methods and Results: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-&agr; alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. &OHgr;3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1. Conclusions: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.