Nucleosome positioning: resources and tools online

Nucleosome positioning is an important process required for proper genome packing and its accessibility to execute the genetic program in a cell-specific, timely manner. In the recent years hundreds of papers have been devoted to the bioinformatics, physics and biology of nucleosome positioning. The purpose of this review is to cover a practical aspect of this field, namely, to provide a guide to the multitude of nucleosome positioning resources available online. These include almost 300 experimental datasets of genome-wide nucleosome occupancy profiles determined in different cell types and more than 40 computational tools for the analysis of experimental nucleosome positioning data and prediction of intrinsic nucleosome formation probabilities from the DNA sequence. A manually curated, up to date list of these resources will be maintained at http://generegulation.info

NucTools: analysis of chromatin feature occupancy profiles from high-throughput sequencing data

Background: Biomedical applications of high-throughput sequencing methods generate a vast amount of data in which numerous chromatin features are mapped along the genome. The results are frequently analysed by creating binary data sets that link the presence/absence of a given feature to specific genomic loci. However, the nucleosome occupancy or chromatin accessibility landscape is essentially continuous. It is currently a challenge in the field to cope with continuous distributions of deep sequencing chromatin readouts and to integrate the different types of discrete chromatin features to reveal linkages between them. Results: Here we introduce the NucTools suite of Perl scripts as well as MATLAB- and R-based visualization programs for a nucleosome-centred downstream analysis of deep sequencing data. NucTools accounts for the continuous distribution of nucleosome occupancy. It allows calculations of nucleosome occupancy profiles averaged over several replicates, comparisons of nucleosome occupancy landscapes between different experimental conditions, and the estimation of the changes of integral chromatin properties such as the nucleosome repeat length. Furthermore, NucTools facilitates the annotation of nucleosome occupancy with other chromatin features like binding of transcription factors or architectural proteins, and epigenetic marks like histone modifications or DNA methylation. The applications of NucTools are demonstrated for the comparison of several datasets for nucleosome occupancy in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). Conclusions: The typical workflows of data processing and integrative analysis with NucTools reveal information on the interplay of nucleosome positioning with other features such as for example binding of a transcription factor CTCF, regions with stable and unstable nucleosomes, and domains of large organized chromatin K9me2 modifications (LOCKs). As potential limitations and problems we discuss how inter-replicate variability of MNase-seq experiments can be addressed

Nucleosome Positioning and Its Role in Gene Regulation in Yeast

Nucleosome, composed of a 147-bp segment of DNA helix wrapped around a histone protein octamer, serves as the basic unit of chromatin. Nucleosome positioning refers to the relative position of DNA double helix with respect to the histone octamer. The positioning has an important role in transcription, DNA replication and other DNA transactions since packing DNA into nucleosomes occludes the binding site of proteins. Moreover, the nucleosomes bear histone modifications thus having a profound effect in regulation. Nucleosome positioning and its roles are extensively studied in model organism yeast. In this chapter, nucleosome organization and its roles in gene regulation are reviewed. Typically, nucleosomes are depleted around transcription start sites (TSSs), resulting in a nucleosome-free region (NFR) that is flanked by two well-positioned H2A.Z-containing nucleosomes. The nucleosomes downstream of the TSS are equally spaced in a nucleosome array. DNA sequences, especially 10–11 bp periodicities of some specific dinucleotides, partly determine the nucleosome positioning. Nucleosome occupancy can be determined with high throughput sequencing techniques. Importantly, nucleosome positions are dynamic in different cell types and different environments. Histones depletions, histones mutations, heat shock and changes in carbon source will profoundly change nucleosome organization. In the yeast cells, upon mutating the histones, the nucleosomes change drastically at promoters and the highly expressed genes, such as ribosome genes, undergo more change. The changes of nucleosomes tightly associate the transcription initiation, elongation and termination. H2A.Z is contained in the +1 and −1 nucleosomes and thus in transcription. Chaperon Chz1 and elongation factor Spt16 function in H2A.Z deposition on chromatin. The chapter covers the basic concept of nucleosomes, nucleosome determinant, the techniques of mapping nucleosomes, nucleosome alteration upon stress and mutation, and Htz1 dynamics on chromatin

Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5’UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes

The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression

Anopheles gambiae mosquitoes are primary human malaria vectors, but we know very little about their mechanisms of transcriptional regulation. We profiled chromatin accessibility by the assay for transposase-accessible chromatin by sequencing (ATAC-seq) in laboratory-reared A. gambiae mosquitoes experimentally infected with the human malaria parasite Plasmodium falciparum. By integrating ATAC-seq, RNA-seq and ChIP-seq data, we showed a positive correlation between accessibility at promoters and introns, gene expression and active histone marks. By comparing expression and chromatin structure patterns in different tissues, we were able to infer cis-regulatory elements controlling tissue-specific gene expression and to predict the in vivo binding sites of relevant transcription factors. The ATAC-seq assay also allowed the precise mapping of active regulatory regions, including novel transcription start sites and enhancers that were annotated to mosquito immune-related genes. Not only is this study important for advancing our understanding of mechanisms of transcriptional regulation in the mosquito vector of human malaria, but the information we produced also has great potential for developing new mosquito-control and anti-malaria strategies

Genomic data integration with hidden Markov models to understand transcription regulation

Transcription is a tightly controlled process that involves the recruitment and prost-translational modification of DNA-associated protein complexes, which can be mapped to the genome using high-throughput experimental assays. An accurate annotation of genomic elements such as transcription units or cis-regulatory elements such as promoters or enhancers is crucial for the use and interpretation of data generated by these assays. Thus, integrative genomic data analysis of high-throughput assays with hidden Markov models (HMMs) has become a popular tool for genome annotation. However, current algorithms are limited by unrealistic data distribution assumptions and variance models. Moreover, they are not able to assign forward or reverse direction to states or properly integrate strand-specific (e.g., RNA expression) with non-strand-specific (e.g., ChIP) data, which is essential to characterize directed processes such as transcription. In this thesis new HMM-based methods are proposed to overcome these limitations. These include (i) bidirectional HMMs (bdHMMs) which integrate strand-specific with non-strand-specific data to infer directed genomic states de novo and (ii) GenoSTAN (Genomic STate ANnotation), a HMM using discrete probability distributions to model count data, for genome annotation from Next-Generation-Sequencing data. Both approaches are made available in the R/Bioconductor package STAN (STate ANnotation) which provides an efficient implementation that can be run on large genomes such as human. STAN is used to derive new and improved annotations of transcription in yeast and human and to generate a map of promoters and enhancers in 127 human cell types and tissues.Integration of transcription factor binding and RNA expression data in yeast recovers the majority of transcribed loci, reveals gene-specific variations in the yeast transcription cycle, identifies 32 new transcribed loci, a regulated initiation-elongation transition, the absence of elongation factors Ctk1 and Paf1 from a class of genes, a distinct transcription mechanism for highly expressed genes and novel DNA sequence motifs associated with transcription termination.Moreover, promoters and enhancers are predicted in 127 human cell types and tissues are mapped by integrating sequencing data from the ENCODE and Roadmap Epigenomics projects, today’s largest compendium of chromatin assays. Promoters and enhancers are identified with consistently higher accuracy and show significantly higher enrichment of complex trait-associated genetic variants than current annotations. Investigation of binding of 101 transcription factors in human K562 cells reveals common and distinctive TF binding properties of enhancers and promoters.Application of STAN to transient transcriptome sequencing (TT-Seq) data in human K562 cells recovers stable mRNAs, long intergenic non-coding RNAs, and additionally maps over 10,000 transient RNAs, including enhancer RNAs, antisense RNAs, and promoter-associated RNAs. Further analyses reveal that transient RNAs such as enhancer RNAs are short and lack U1 motifs and secondary structure. Taken together, the annotations inferred in this thesis gave new insights into transcription and its regulation and will be an important resource for future research in genomics. STAN is a valuable tool to create such annotations also in other organisms and as more data becomes available improve the existing ones

Molecular mechanisms of CD8+ T cell mediated control of HIV-1 infection in peripheral blood and lymphoid tissues.

Doctoral Degree. University of KwaZulu-Natal, Durban.Naturally induced CD8+ T cells do not clear human immunodeficiency virus (HIV) infection, partly because the virus rapidly escapes CD8+ T cell responses and the effector cells are excluded from HIV reservoirs sites. However, optimizing CD8+ T cell responses could potentially be leveraged in HIV vaccine or cure efforts if epitope escape and barriers to effector CD8+ T cells infiltrating the sites of HIV reservoirs are overcome. In our first study, we described a potential mechanism of HIV-1 control by CD8+ T cells targeting different variants in individuals infected with HIV-1. Our second study focused on describing the molecular regulation of CXCR5 expression in human CD8+ T cells. Study 1 HLA-B*81 is associated with control of HIV-1 subtype C infection, while the closely related allele B*42 is not. Interestingly, both alleles present the immunodominant Gag TL9 epitope, and the magnitude of this response correlates negatively with viral load. To examine the role of T cell receptor (TCR) in this process, we characterized the sequence and function of TL9-specific CD8+ TCR in B*81 and B*42 individuals. TL9-specific CD8+ T cells were identified and isolated using B*81 and/or B*42 TL9 tetramers. TCR beta genes were amplified from single sorted cells and sequenced. Paired alpha genes were identified for selected clones. TCR function was tested using a reporter cell assay where TCR+ Jurkat cells were co-cultured with peptide-pulsed or HIV-1 infected B*81 or B*42 target cells, and signalling quantified by luminescence. TCR recognition was assessed against all single amino acid TL9 variants and results were compared to HIV-1 subtype C sequences. A population of dual-reactive T cells was detected by both B*81- and B*42-TL9 tetramers in 7/9 (78%) B*81 and 4/11 (36%) B*42 individuals; and this population was associated with lower viremia. Mono- and dual-reactive TCR beta sequences were collected from six individuals. In B*81 individuals, all TCRs were highly restricted to TRBV12-3. In B*42 individuals, mono-reactive TCRs encoded a variety of V beta genes, while dual-reactive TCRs were restricted to TRBV12-3 and enriched for public clones. Functional analyses indicated that B*81 TCRs (1 mono, 2 dual) and a dualreactive public B*42 TCR displayed similar TL9 cross-reactivity profiles and enhanced capacity to recognize HIV-1 escape mutations compared to mono-reactive B*42 TCRs. This work highlights the impact of TCR promiscuity on T cell-mediated control of HIV-1. Study 2 HIV-1 infection is difficult to cure even with effective antiretroviral therapy (ART) because of persistent viral replication in immune privileged sites such as the B cell follicles of secondary lymphoid tissues. CD8+ T cells are generally excluded from B cell follicles, partially due to a lack of expression of the follicular homing receptor CXCR5. Recent murine studies have identified CXCR5+ CD8+ T cells, referred to as follicular CD8+ T cells (fCD8s), that localize in B cell follicles. However, the mechanisms governing expression of CXCR5 on human CD8+ T cells are not known. We investigated the epigenetic and transcriptional mechanisms involved in the regulation of CXCR5 expression in human CD8+ T cells. We FACS-sorted CXCR5+CD8+ (fCD8s), CXCR5-CD8+ (non-fCD8s), naïve CD8+ T cells and germinal center T follicular helper cells (GCTfh) from the lymph node of HIV- 1 infected individuals and performed RNA-sequencing (RNA-Seq), DNA methylation assays and the assay for transposase-accessible chromatin using sequencing (ATACSeq). RNA-Seq was used to quantify the expressed genes in FACS-sorted subsets and to determine transcriptional modules governing CXCR5 expression in CD8+ T cells. ATAC-Seq was used to quantify accessible genes, identify the transcriptional factors footprinting and determine epigenetic modules governing CXCR5 expression. DNA methylation, a major epigenetic gene silencing mechanism, was used to profile methylation pattern of the CXCR5 gene region in the sorted subsets. We observed hypermethylation of DNA around the transcriptional start site (TSS) of the CXCR5 gene in non-fCD8s but not in fCD8s. ATAC-Seq analysis revealed a closed chromatin conformation at the TSS in non-fCD8s, but not in fCD8s. Our gene expression data revealed significant differences in the CXCR5 associated factors between GCTfh and fCD8s. Computational analysis further revealed the presence ofa nucleosome at the TSS of fCD8s, which could be a plausible explanation for lower expression of CXCR5 in fCD8s as compared to GCTfh. Together, we identified epigenetic regulations involved in CXCR5 expression in human CD8+ T cells and propose that DNA methylation, chromatin structure and nucleosome positioning cooperatively regulate the expression of CXCR5 in CD8+ T cells. Our data open up the possibility of using epigenetic manipulation as a novel strategy for redirecting CD8+ T cells to B cell follicles where they are needed to eradicate HIV-1 infected cells

Genome-wide analysis of DNA methylation topology to understand cell fate

DNA methylation is an epignetic modification associated with gene regulation. It has extensively been studied in the context of small regulatory regions. Yet, not so much is known about large domains characterized by fuzzy methylation patterns, termed Partially Methylated Domains (PMDs). The present thesis comprises PMD analyses in various contexts and provides several new aspects to study DNA methylation. First, a comprehensive analysis of PMDs across a large cohort of WGBS samples was performed, to identify structural and functional features associated with PMDs. A newly developed approach, ChromH3M, was proposed for the analysis and integration of a large spectrum of WGBS data sets. Second, PMDs were found to be indicators of the cellular proliferation history and segmented loss of DNA methylation in PMDs supports the sequential linear differentiation model of memory T-cells. Third, assessment of genome-wide methylation changes in PMDs of Multiple Sclerosis-discordant monozygotic co-twins did not show significant differences, but local changes (DMRs) were identified. Taken together, the outcomes of the presented studies shed light on a so far neglected aspect of DNA methylation, that is PMDs, in different contexts; lineage specialization, differentiation, replication, disease, chromatin organization and gene expression.Die DNA-Methylierung ist eine epigenetische Modi1kation, die funktionell mit der Genregulation verbunden ist. Sie wurde bereits ausführlich im Kontext kleiner regulatorischer Regionen untersucht. Es ist jedoch noch nicht sehr viel bekannt über große Domänen, welche erstmals in WGBS-Daten beschrieben wurden. Sie werden als partiell methylierte Regionen (PMDs) bezeichnet und sind durch das Vorhandensein variabler Methylierungsmuster charakterisiert. Die vorliegende Arbeit umfasst PMD-Analysen in unterschiedlichen Kontexten und liefert verschiedene neue Aspekte zur Untersuchung der DNA-Methylierung. Zuerst wurde eine umfassende Analyse von PMDs in einer großen Kohorte von WGBS-Proben durchgeführt, um strukturelle und funktionelle Merkmale zu identi 1zieren, die mit PMDs assoziert sind. Ein neu entwickelter Ansatz, ChromH3M, wurde für die Analyse und Integration einer großen Kohorte vonWGBS Datensätzen angewandt. Zweitens wurde festgestellt, dass PMDs Indikatoren für die Zellproliferationshistorie sind, und der zu beobachtende graduelle Verlust der globalen DNAMethylierung bei der Differenzierung von T-Gedächtniszellen unterstützt die Hypothese der sequenziellen linearen Differenzierung. Drittens zeigte die Bewertung der genomweiten Methylierungsänderungen in PMDs von Multiple Sklerose-diskordanten monozygoten Zwillingen keine signi1kanten Unterschiede, jedoch wurden lokale Änderungen (DMRs) identi1ziert. Insgesamt geben die Ergebnisse der vorgestellten Studien Aufschluss über einen bislang eher vernachlässigten Aspekt der DNA-Methylierung, d.h. PMDs, in verschiedenen Zusammenhängen: der Festlegung der Zell-entwicklungsbahnen, der Zelldifferenzierung, der Replikation, die Krankheit, der Organisation des Chromatins, sowie der Regulation der Genexpression

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution