Dmitry K. Pokholok

Transcriptional regulatory code of a eukaryotic genome

DNA-binding transcriptional regulators interpret the genomes regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeasts transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeasts transcriptional regulators.

Genome-wide Map of Nucleosome Acetylation and Methylation in Yeast

Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.

Exchange of RNA Polymerase II Initiation and Elongation Factors during Gene Expression In Vivo

We have systematically explored the in vivo occupancy of promoters and open reading frames by components of the RNA polymerase II transcription initiation and elongation apparatuses in yeast. RNA polymerase II, Mediator, and the general transcription factors (GTFs) were recruited to all promoters tested upon gene activation. RNA polymerase II, TFIIS, Spt5, and, unexpectedly, the Paf1/Cdc73 complex, were found associated with open reading frames. The presence of the Paf1/Cdc73 complex on ORFs in vivo suggests a novel function for this complex in elongation. Elongator was not detected under any conditions tested, and further analysis revealed that the majority of elongator is cytoplasmic. These results suggest a revised model for transcription initiation and elongation apparatuses in living cells.

High-resolution computational models of genome binding events

Direct physical information that describes where transcription factors, nucleosomes, modified histones, RNA polymerase II and other key proteins interact with the genome provides an invaluable mechanistic foundation for understanding complex programs of gene regulation. We present a method, joint binding deconvolution (JBD), which uses additional easily obtainable experimental data about chromatin immunoprecipitation (ChIP) to improve the spatial resolution of the transcription factor binding locations inferred from ChIP followed by DNA microarray hybridization (ChIP-Chip) data. Based on this probabilistic model of binding data, we further pursue improved spatial resolution by using sequence information. We produce positional priors that link ChIP-Chip data to sequence data by guiding motif discovery to inferred protein-DNA binding sites. We present results on the yeast transcription factors Gcn4 and Mig2 to demonstrate JBDs spatial resolution capabilities and show that positional priors allow computational discovery of the Mig2 motif when a standard approach fails.

Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube

Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using “on-bead” barcoded tagmentation. The key to the method that we call “contiguity preserving transposition” sequencing on beads (CPTv2-seq) is transposon-mediated transfer of homogenous populations of barcodes from beads to individual long DNA molecules that get fragmented at the same time (tagmentation). These are then processed to sequencing libraries wherein all sequencing reads originating from each long DNA molecule share a common barcode. Single-tube, bulk processing of long DNA molecules with ∼150,000 different barcoded bead types provides a barcode-linked read structure that reveals long-range molecular contiguity. This technology provides a simple, rapid, plate-scalable and automatable route to accurate, haplotype-resolved sequencing, and phasing of structural variants of the genome.