Dataset Information

Pitfalls in global normalization of ChIP-seq data in CD4(+) T cells treated with butyrate: A possible solution strategy.

ABSTRACT: Regulatory T cells (Treg) play a central role in the suppression of inflammatory and allergic responses. Colonization of certain gut commensal microbes such as Clostridia class IV and XIVa in the gut can induce development of colonic Treg cells contributing to the maintenance of gut immune homeostasis. Clostridia-derived butyrate promotes the differentiation of naïve T cells into Treg cells through upregulation of Foxp3, the master transcription factor of Treg cells. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that treatment of naïve T cells with butyrate induces Treg-polarizing conditions by enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus. In general, global normalization was utilized for ChIP-seq analysis to compare the data obtained from two or more samples. However, global normalization is not appropriate for the evaluation of ChIP-seq data when treatment can affect the total amount of target protein. Here, we introduce a unique normalization method for ChIP-seq analysis in cells treated with butyrate, a pan-HDAC inhibitor that is likely to affect total acetylation levels of histone H3.

SUBMITTER: Furusawa Y

PROVIDER: S-EPMC4536162 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Pitfalls in global normalization of ChIP-seq data in CD4(+) T cells treated with butyrate: A possible solution strategy.

Furusawa Yukihiro Y Endo Takaho A TA Obata Yuuki Y Ohara Osamu O Ohno Hiroshi H Hase Koji K

Genomics data 20140627

Regulatory T cells (Treg) play a central role in the suppression of inflammatory and allergic responses. Colonization of certain gut commensal microbes such as Clostridia class IV and XIVa in the gut can induce development of colonic Treg cells contributing to the maintenance of gut immune homeostasis. Clostridia-derived butyrate promotes the differentiation of naïve T cells into Treg cells through upregulation of Foxp3, the master transcription factor of Treg cells. Chromatin immunoprecipitatio ...[more]

PMID: 26484090

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Project description:BackgroundChip-seq experiments are becoming a standard approach for genome-wide profiling protein-DNA interactions, such as detecting transcription factor binding sites, histone modification marks and RNA Polymerase II occupancy. However, when comparing a ChIP sample versus a control sample, such as Input DNA, normalization procedures have to be applied in order to remove experimental source of biases. Despite the substantial impact that the choice of the normalization method can have on the results of a ChIP-seq data analysis, their assessment is not fully explored in the literature. In particular, there are no diagnostic tools that show whether the applied normalization is indeed appropriate for the data being analyzed.ResultsIn this work we propose a novel diagnostic tool to examine the appropriateness of the estimated normalization procedure. By plotting the empirical densities of log relative risks in bins of equal read count, along with the estimated normalization constant, after logarithmic transformation, the researcher is able to assess the appropriateness of the estimated normalization constant. We use the diagnostic plot to evaluate the appropriateness of the estimates obtained by CisGenome, NCIS and CCAT on several real data examples. Moreover, we show the impact that the choice of the normalization constant can have on standard tools for peak calling such as MACS or SICER. Finally, we propose a novel procedure for controlling the FDR using sample swapping. This procedure makes use of the estimated normalization constant in order to gain power over the naive choice of constant (used in MACS and SICER), which is the ratio of the total number of reads in the ChIP and Input samples.ConclusionsLinear normalization approaches aim to estimate a scale factor, r, to adjust for different sequencing depths when comparing ChIP versus Input samples. The estimated scaling factor can easily be incorporated in many peak caller algorithms to improve the accuracy of the peak identification. The diagnostic plot proposed in this paper can be used to assess how adequate ChIP/Input normalization constants are, and thus it allows the user to choose the most adequate estimate for the analysis.

Project description:Various nanomedicinal products (NMPs) have been reported to induce an adverse immune response, which may be related to their tendency to accumulate in or target cells of the immune system. Therefore, before their market authorization, NMPs should be thoroughly evaluated for their immunotoxic potential. Nonclinical regulatory immunotoxicity testing of nonbiological medicinal products, including NMPs, is currently performed by following the guideline S8 "Immunotoxicity Studies for Human Pharmaceuticals" of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). However, this guideline does not cover all the immunotoxicity endpoints reported for NMPs in the literature, such as complement activation related pseudo allergy, hypersensitivity and immunosuppression. In addition, ICH-S8 does not provide any nanospecific testing considerations, which is important given their tendency to interfere with many commonly used toxicity assays. We therefore propose a nonclinical regulatory immunotoxicity assessment strategy, which considers the immunotoxicity endpoints currently missing in the ICH-S8. We also list the known pitfalls related to the testing of NMPs and how to tackle them. Next to defining the relevant physicochemical and pharmacokinetic properties of the NMP and its intended use, the proposed strategy includes an in vitro assay battery addressing various relevant immunotoxicity endpoints. A weight of evidence evaluation of this information can be used to shape the type and design of further in vivo investigations. The final outcome of the immunotoxicity assessment can be included in the overall risk assessment of the NMP and provide alerts for relevant endpoints to address during clinical investigation. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Project description:Previous studies, which examined whether symbolic and non-symbolic quantity representations are processed by two independent systems or by one common system, reached contradicting findings, possibly due to methodological differences. Indeed, some researchers advocate the two systems approach, based on the presence of notation-specific switch cost in conditions where adults have to compare pairs of symbolic and non-symbolic quantities, in combination with the absence of such a cost in conditions containing quantities of the same notation. However, other researchers used matching instructions, and reported a facilitation in the mixed notation conditions, suggesting that the two systems are automatically integrated. In the current study, we conducted three experiments, in which we examined the existence of two separate quantity systems, but we used various experimental manipulations (e.g., task instructions, presentation order) to unravel the previous inconsistent findings. In Experiment 1, we investigated the role of task instructions by presenting participants with pure and mixed notation trials with both comparison and matching tasks. In Experiment 2, we tested the role of blocked and randomized presentation order for the pure and mixed trials. Our data showed that cost for switching between the symbolic and non-symbolic quantities is present, but is prone to a certain methodological drawback: when the differences between the processing times for two sequentially presented stimuli of different notations are not taken into account, this masks the cost for switching between the two systems. To overcome this problem, in Experiment 3 we used an audio-visual paradigm. Overall, our results provide further evidence for the existence of distinct quantity representations, independently of task instructions or presentation order. Additionally, considering this methodological pitfall we argue that the audio-visual paradigm is better suited when investigating the integration between symbolic and non- symbolic quantities.

Dataset Information

Pitfalls in global normalization of ChIP-seq data in CD4(+) T cells treated with butyrate: A possible solution strategy.

Publications

Pitfalls in global normalization of ChIP-seq data in CD4(+) T cells treated with butyrate: A possible solution strategy.

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