Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data

Motivation: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays.

Transcriptional regulatory logic of the diurnal cycle in the mouse liver

Matteo Dal Peraro, Bart Deplancke, Frédéric Bruno Martin Gachon, Nouria Hernandez, Alexandra Styliani Kalantzi, Irina Krier, Felix Naef, Sunil Kumar Raghav, Guillaume Rey, Jonathan Aryeh Sobel, Benjamin Dieter Weger

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1(-/-) livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1(-/-) compared to WT livers, the amplitudes in Bmal1(-/-) were generally lower. The residual rhythms in Bmal1(-/-) reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK: BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.

Public Library of Science2017

Genomics and transcriptomics of the Mycobacterium tuberculosis complex

Swapna Uplekar

The goal of eliminating tuberculosis (TB) by 2050 depends on the development of improved TB diagnostics, drugs and vaccines. Advances in these areas require a deep understanding of the disease and its causative agent, Mycobacterium tuberculosis (M. tb). Mycobacterial species that cause TB in humans and other mammalian hosts are grouped within the M. tb complex. Development of powerful technologies such as next-generation sequencing and microarrays opened up new avenues for comparative and functional genomics of the M. tb complex. Due to the large and increasingly complex datasets generated from these technologies, the bottleneck in biological investigation has shifted from data generation to analysis. The objectives of this thesis were to establish and employ strategies for the analysis, integration, and interpretation of high-throughput sequencing and microarray datasets using a range of bioinformatics and statistical tools. In the area of comparative genomics, we assessed the genetic diversity in the M. tb complex using various methods, such as SNP (single nucleotide polymorphism) genotyping, automated Sanger sequencing and next-generation sequencing. In a study comparing the genomes of the virulent M. bovis and M. bovis BCG vaccine strains, we identified a set of SNPs that were common to all BCG strains, and could provide novel insights on the molecular basis of BCG attenuation. In another study, we surveyed the genetic variation in the highly immunodominant esx gene family among clinical isolates of M. tb and identified sequence polymorphisms in known T- cell epitopes on Esx proteins that could affect their immunogenicity. We exploited the power of next-generation sequencing to detect sequence variation among M. tb strains that could result in phenotypic differences. By comparing the genomes of drug-resistant mutants with the sensitive wild-type strain we were able to identify the target of the anti-TB drug, pyridomycin. Using a similar approach we identified a mutation that makes M. tb strains incapable of producing PDIMs (phthiocerol dimycocerosates), which are cell wall associated lipids involved in M. tb virulence. In the area of functional genomics, we mapped genome-wide binding sites for transcription factors using chromatin immunoprecipitation followed by hybridization to microarrays (ChIP-on-chip) or sequencing (ChIP-seq), and performed transcription profiling by means of high-throughput cDNA sequencing (RNA-seq). We carried out a comprehensive study to characterize the whole transcriptome of M. tb in exponential and stationary phases of growth, and understand the genome-wide dynamics of two key components of the transcription machinery, namely, RNA polymerase and NusA. By systematic integration of the ChIP-seq and RNA-seq data, we identified a set of transcription units (TU) in the M. tb genome, and mapped their putative promoters. Analysis of RNAP and NusA binding across the promoter and body of TUs and their correlation with transcription uncovered new functional aspects of the transcriptional complex in M. tb. We also exploited the ChIP-on-chip and ChIP-seq technologies to define the regulon of the M. tb sigma factor F, and gain a better understanding of the regulatory role of the nucleoid associated protein, EspR. Altogether, this thesis has improved our knowledge of the evolution, physiology and virulence of the M. tb complex. In addition, we have established next generation sequencing as a powerful tool for comparative and functional studies, with potential applications in the clinical setting.

EPFL2012

RNA-Protein interactions at AU-rich elements

Ramona Pauchard-Batschulat

The expression of many proto-oncogenes, nuclear transcription factors and cytokines is regulated post-transcriptionally by specific interactions between distinct cis-elements within the mRNA and trans-acting factors that bind to these elements and thereby either promote or inhibit the rapid decay of the mRNA. This kind of regulation enables the cell to rapidly respond to changes in the cellular environment. These elements are usually located within the 3’UTR of the mRNA, rich in adenylate and uridylate and called AU-rich elements or AREs. To date, many of these trans-acting factors have been determined and among the best studied are the stabilizing HuR, the rather destabilizing AUF1 and the destabilizing KSRP and TTP. Previous studies have suggested that these ARE-BPs as well as yet unidentified factors might have overlapping target specificities and bind either in a concurrent, concomitant or maybe an even cooperative fashion to their targets to regulate mRNA half-life. However, the exact manner of how these factors might interact and how these interactions might be influenced, is largely unknown. Thus, my first project aimed to study the interactions between AUF1, KSRP and HuR in the context of the 3’UTRs of RANKL and Bcl-6 that were recently identified as AUF1 targets with a short half-life. By combining ARE-BP immunoprecipitations with qRT-PCR analysis of co-precipitated reporter RNAs, we were able to gain interesting insight into the RNA-protein interactions at AU-rich elements. However, this approach is very laborious and can study RNA-protein interactions only within a limited context. Yet, the pools of mRNA targets as well as binding factors are very multifaceted and likely depend on the cell type and condition as well as the cellular environment. Thus, a much broader approach is necessary to reveal the entire network of RNA-protein and protein-protein interactions at AU-rich elements. A promising technique is the combination of ARE-BP immunoprecipitation and high-throughput sequencing to analyze co-precipitated RNA. This approach allows for the extraction of a lot of important data from a single experiment as for example the virtually complete pool of target mRNAs under the studied conditions, the binding sites within these mRNAs as well as the frequency with which these sequences were detected. Based on these data, it would be possible to make suggestions about the binding specificities as well as potential, yet un-identified targets. If accumulating data sets for different cellular or environmental conditions, we might gain insight into the regulation of these RNA-protein interactions. In a similar way, the comparison of data sets for different ARE-binding proteins would give us further insight into the protein-protein interactions at and around AU-rich elements. Therefore, my second project focused on the development of a novel protocol that allows for the rapid, reliable and reproducible generation of high-throughput sequencing samples based on co-immunoprecipitated RNA fragments.

EPFL2013