Michael Kertesz

Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

The role of site accessibility in microRNA target recognition

MicroRNAs are key regulators of gene expression, but the precise mechanisms underlying their interaction with their mRNA targets are still poorly understood. Here, we systematically investigate the role of target-site accessibility, as determined by base-pairing interactions within the mRNA, in microRNA target recognition. We experimentally show that mutations diminishing target accessibility substantially reduce microRNA-mediated translational repression, with effects comparable to those of mutations that disrupt sequence complementarity. We devise a parameter-free model for microRNA-target interaction that computes the difference between the free energy gained from the formation of the microRNA-target duplex and the energetic cost of unpairing the target to make it accessible to the microRNA. This model explains the variability in our experiments, predicts validated targets more accurately than existing algorithms, and shows that genomes accommodate site accessibility by preferentially positioning targets in highly accessible regions. Our study thus demonstrates that target accessibility is a critical factor in microRNA function.

Genome-wide measurement of RNA secondary structure in yeast

The structures of RNA molecules are often important for their function and regulation, yet there are no experimental techniques for genome-scale measurement of RNA structure. Here we describe a novel strategy termed parallel analysis of RNA structure (PARS), which is based on deep sequencing fragments of RNAs that were treated with structure-specific enzymes, thus providing simultaneous in vitro profiling of the secondary structure of thousands of RNA species at single nucleotide resolution. We apply PARS to profile the secondary structure of the messenger RNAs (mRNAs) of the budding yeast Saccharomyces cerevisiae and obtain structural profiles for over 3,000 distinct transcripts. Analysis of these profiles reveals several RNA structural properties of yeast transcripts, including the existence of more secondary structure over coding regions compared with untranslated regions, a three-nucleotide periodicity of secondary structure across coding regions and an anti-correlation between the efficiency with which an mRNA is translated and the structure over its translation start site. PARS is readily applicable to other organisms and to profiling RNA structure in diverse conditions, thus enabling studies of the dynamics of secondary structure at a genomic scale.

Whole-genome haplotyping using long reads and statistical methods

The rapid growth of sequencing technologies has greatly contributed to our understanding of human genetics. Yet, despite this growth, mainstream technologies have not been fully able to resolve the diploid nature of the human genome. Here we describe statistically aided, long-read haplotyping (SLRH), a rapid, accurate method that uses a statistical algorithm to take advantage of the partially phased information contained in long genomic fragments analyzed by short-read sequencing. For a human sample, as little as 30 Gbp of additional sequencing data are needed to phase genotypes identified by 50× coverage whole-genome sequencing. Using SLRH, we phase 99% of single-nucleotide variants in three human genomes into long haplotype blocks 0.2–1 Mbp in length. We apply our method to determine allele-specific methylation patterns in a human genome and identify hundreds of differentially methylated regions that were previously unknown. SLRH should facilitate population-scale haplotyping of human genomes.

Computational prediction of RNA structural motifs involved in posttranscriptional regulatory processes

Messenger RNA molecules are tightly regulated, mostly through interactions with proteins and other RNAs, but the mechanisms that confer the specificity of such interactions are poorly understood. It is clear, however, that this specificity is determined by both the nucleotide sequence and secondary structure of the mRNA. Here, we develop RNApromo, an efficient computational tool for identifying structural elements within mRNAs that are involved in specifying posttranscriptional regulations. By analyzing experimental data on mRNA decay rates, we identify common structural elements in fast-decaying and slow-decaying mRNAs and link them with binding preferences of several RNA binding proteins. We also predict structural elements in sets of mRNAs with common subcellular localization in mouse neurons and fly embryos. Finally, by analyzing pre-microRNA stem–loops, we identify structural differences between pre-microRNAs of animals and plants, which provide insights into the mechanism of microRNA biogenesis. Together, our results reveal unexplored layers of posttranscriptional regulations in groups of RNAs and are therefore an important step toward a better understanding of the regulatory information conveyed within RNA molecules. Our new RNA motif discovery tool is available online.

Diagnosis of Capnocytophaga canimorsus Sepsis by Whole-Genome Next-Generation Sequencing

We report the case of a 60-year-old man with septic shock due to Capnocytophaga canimorsus that was diagnosed in 24 hours by a novel whole-genome next-generation sequencing assay. This technology shows great promise in identifying fastidious pathogens, and, if validated, it has profound implications for infectious disease diagnosis.

Computational prediction of RNA structural motifs involved in post-transcriptional regulatory processes.

mRNA molecules are tightly regulated, mostly through interactions with proteins and other RNAs, but the mechanisms that confer the specificity of such interactions are poorly understood. It is clear, however, that this specificity is determined by both the nucleotide sequence and secondary structure of the mRNA. We developed RNApromo, an efficient computational tool for identifying structural elements within mRNAs that are involved in specifying post-transcriptional regulations. Using RNApromo, we predicted putative motifs in sets of mRNAs with substantial experimental evidence for common post-transcriptional regulation, including mRNAs with similar decay rates, mRNAs that are bound by the same RNA binding protein, and mRNAs with a common cellular localization. Our new RNA motif discovery tool reveals unexplored layers of post-transcriptional regulations in groups of RNAs, and is therefore an important step toward a better understanding of the regulatory information conveyed within RNA molecules.