Stanley Fields

Global mapping of protein-DNA interactions in vivo by digital genomic footprinting

The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.

High Resolution Mapping of Protein Sequence–Function Relationships

We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

Deep mutational scanning: a new style of protein science

Mutagenesis provides insight into proteins, but only recently have assays that couple genotype to phenotype been used to assess the activities of as many as 1 million mutant versions of a protein in a single experiment. This approach—deep mutational scanning—yields large-scale data sets that can reveal intrinsic protein properties, protein behavior within cells and the consequences of human genetic variation. Deep mutational scanning is transforming the study of proteins, but many challenges must be tackled for it to fulfill its promise.

Massively Parallel Functional Analysis of BRCA1 RING Domain Variants

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.

Whole-genome sequencing of a laboratory-evolved yeast strain

BackgroundExperimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined.ResultsWe sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement.ConclusionsThis study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

Engineering an allosteric transcription factor to respond to new ligands.

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

Capture and sequence analysis of RNAs with terminal 2′,3′-cyclic phosphates

The combination of ligation-based RNA capture methods and high-throughput sequencing has facilitated the characterization of transcriptomes and the identification of novel noncoding RNAs. However, current ligation-based RNA capture methods require RNA substrates with terminal 3-hydroxyl groups, limiting their utility for identifying RNAs with modified termini like 2,3-cyclic phosphates. Cyclic phosphate-terminated RNAs are generated by endonucleolytic cleavages and self-cleaving ribozymes and are found as stable modifications on cellular RNAs such as the U6 spliceosomal RNA. We developed a method that uses the Arabidopsis thaliana tRNA ligase to add an adaptor oligonucleotide to RNAs that terminate in 2,3-cyclic phosphates. The adaptor allows specific priming by reverse transcriptase, which is followed by additional steps for PCR amplification and high-throughput DNA sequencing. Applying the method to total human RNA, we found 2836 sequencing reads corresponding to the 3 terminus of U6 snRNA, validating the method. In addition to a large background of reads that map throughout abundantly transcribed RNAs, we also found 42,324 reads of specific fragments from several tRNA isoacceptor families, suggesting that this method may identify processing events previously undetected by other RNA cloning techniques.

A general strategy to construct small molecule biosensors in eukaryotes.

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.10606.001

Adjacent Codons Act in Concert to Modulate Translation Efficiency in Yeast

Translation elongation efficiency is largely thought of as the sum of decoding efficiencies for individual codons. Here, we find that adjacent codon pairs modulate translation efficiency. Deploying an approach in Saccharomyces cerevisiae that scored the expression of over 35,000 GFP variants in which three adjacent codons were randomized, we have identified 17 pairs of adjacent codons associated with reduced expression. For many pairs, codon order is obligatory for inhibition, implying a more complex interaction than a simple additive effect. Inhibition mediated by adjacent codons occurs during translation itself as GFP expression is restored by increased tRNA levels or by non-native tRNAs with exact-matching anticodons. Inhibition operates in endogenous genes, based on analysis of ribosome profiling data. Our findings suggest translation efficiency is modulated by an interplay between tRNAs at adjacent sites in the ribosome and that this concerted effect needs to be considered in predicting the functional consequences of codon choice.

Interactive learning: lessons from two hybrids over two decades.

The initial yeast two‐hybrid experiment – published in 1989 – described an approach to detecting protein–protein interactions that has flourished over the last two decades, leading to the assembly of large‐scale data sets of these interactions. Yet the yeast assay originated because of the laboratorys interests in technology development, not because of its need to identify partners of any protein then under study. In addition to such motivating forces, other features of the process of originating a technology can be revealed by considering the lessons of the two‐hybrid approach. These include the value of timeliness in a methods development, the willingness of an investigator to try experimental approaches that prove fruitless, the ability of biological macromolecules to display surprising attributes, the benefits of a community expending efforts to expand the uses of a technology platform, and the role of scientific training of those who work in technology.