Practical Guidelines for the Comprehensive Analysis of ChIP-seq Data

Bailey, Timonthy; Krajewski, Paweł; Ladunga, István; Lefèbvre, Céline; Li, Qunhua; Liu, Tao; Madrigal, Pedro; Taslim, Cenny; Zhang, Jie

doi:10.1371/journal.pcbi.1003326

Cited by 239 publications

(224 citation statements)

References 98 publications

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“…For all ChIPseq data, PCR duplicates were removed, meaning that all identical reads were collapsed to one single read. This is a common and established processing step for ChIP-seq data (52)(53)(54). BAM data were converted to BED format using the BEDtools bamToBed option (55).…”

Section: Discussionmentioning

confidence: 99%

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

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The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA crosslinking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.The RNAi machinery has many functions in the eukaryotic cell, and aspects of the RNAi molecular mechanism are highly conserved between yeast and humans (1). Essentially, a small RNA is bound by a member of the Argonaute family of proteins and contributes sequence specificity to a larger protein complex. In the cytoplasm, the RNAi machinery uses Watson-Crick base pairing to target the RNA-induced silencing complex to a specific mRNA and facilitate its degradation. A related process is well established in the nucleus of Schizosaccharomyces pombe, where instead of targeting cytoplasmic mRNAs for destruction, a small RNA targets the RNA-induced transcriptional silencing complex to the pericentromeric regions of each chromosome and facilitates the generation of heterochromatin (2, 3).Work in a chicken-human hybrid cell line supports the possibility that the RNAi machinery is responsible for centromeric chromatin structure in vertebrates as well (4). Indeed, when Dicer is conditionally inactivated, transcription of ␣-satellite DNA from human chromosome 21 increases. Furthermore, the loss of Dicer results in a loss of siRNAs originating from these repeat regions, a delocalization of HP1, and disruption of mitosis (4). The RNAi machinery is also implicated in the creation and/or maintenance of heterochromatin at various sites throughout the genome, in addition to the centromeric regions. Transfection of a siRNA homologous to the EF1a promoter in human cells silences the endogenous gene (5). In a related study, human Argonaute 1 (A...

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Section: Discussionmentioning

confidence: 99%

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

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“…Overall we obtained between 2 and 10 million reads per sample, and 70-80% of these reads uniquely aligned to the genome. This number of reads is adequate to draw meaningful conclusions in C. albicans, as it is estimated that high quality gene expression profiling can be achieved with 2 million reads per sample for yeast (Bailey et al 2013). Reads aligned primarily to exonic regions ( Figure S2A).…”

Section: Quality Control For Rna-seqmentioning

confidence: 99%

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans

et al. 2017

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Almost all humans are colonized with Candida albicans. However, in immunocompromised individuals, this benign commensal organism becomes a serious, life-threatening pathogen. Here, we describe and analyze the regulatory networks that modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous sequence analysis of clinical C. albicans isolates from immunocompromised patients indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen species (ROS), suggesting they help neutralize the host-derived ROS produced by phagocytes, as well as establish a sustained infection in vivo. Transcriptomic analysis of mutants under resting conditions where cells were not experiencing oxidative stress revealed a large network that control macro and micronutrient homeostasis, which likely contributes to overall pathogen fitness in host niches. Under oxidative stress, both transcription factors regulate a separate set of genes involved in detoxification of ROS and down-regulating ribosome biogenesis. ChIP-seq analysis, which reveals vastly different binding partners for each transcription factor (TF) before and after oxidative stress, further confirms these results. Furthermore, the absence of a dominant binding motif likely facilitates their mobility, and supports the notion that they represent a recent expansion of the ZCF family in the pathogenic Candida species. Our analyses provide a framework for understanding new aspects of the interface between C. albicans and host defense response, and extends our understanding of how complex cell behaviors are linked to the evolution of TFs.

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“…We filtered duplicated reads using the "auto" mode of the keep-dup function. We followed the guidelines of the ENCODE project in order to identify confident peak calls (Landt et al, 2012;Bailey et al, 2013) (Supplemental Figure 11). First, we ran MACS2 on every individual sample and subjected the peak calls of the two biological replicates of each treatment to an IDR (irreproducibility discovery rate) analysis .…”

Section: Chip-seq and Peak Callingmentioning

confidence: 99%

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

et al. 2015

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To secure their access to water, light, and nutrients, many plant species have developed allelopathic strategies to suppress competitors. To this end, they release into the rhizosphere phytotoxic substances that inhibit the germination and growth of neighbors. Despite the importance of allelopathy in shaping natural plant communities and for agricultural production, the underlying molecular mechanisms are largely unknown. Here, we report that allelochemicals derived from the common class of cyclic hydroxamic acid root exudates directly affect the chromatin-modifying machinery in Arabidopsis thaliana. These allelochemicals inhibit histone deacetylases both in vitro and in vivo and exert their activity through locus-specific alterations of histone acetylation and associated gene expression. Our multilevel analysis collectively shows how plant-plant interactions interfere with a fundamental cellular process, histone acetylation, by targeting an evolutionarily highly conserved class of enzymes.

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Practical Guidelines for the Comprehensive Analysis of ChIP-seq Data

Cited by 239 publications

References 98 publications

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

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