Unknown,Transcriptomics,Genomics,Proteomics

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4C-ker: A method to reproducibly identify genome-wide interactions captured by 4C-Seq experiments

ABSTRACT: 4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes. 4C-Seq experiments from Igh and Cd83 bait in activated B cells and Tcrb (Eb) bait in double negative T cells and immature B cells. RNA-Seq and ATAC-Seq experiments in DN and Immature B cells.

ORGANISM(S): Mus musculus

SUBMITTER: Jane Skok

PROVIDER: E-GEOD-77645 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Inflammation is a beneficial host response to infection, but it also contributes to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (i.e., they can cease to express their signature cytokine, IL-17A) and plasticity (i.e., they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However technical limitations prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as “transdifferentiation”. Furthermore, while Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two novel fate-mapping mouse models to track Th17 cells during immune responses to show that CD4+ T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a global change in their transcriptome and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF- β signaling and the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases. We isolated intestinal lymphocytes from two independent experiments, each using 5 mice injected with anti-CD3 mAb. Th17, exTh17, Tr1 exTh17, Tr1, Foxp3 Treg and Foxp3 IL-10+ Treg cell populations were FACS-sorted from these two independent experiments and the cells of each population were pooled before the analysis. Around 5,000 cells for each population were processed.

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4C-ker: A method to reproducibly identify genome-wide interactions captured by 4C-Seq experiments

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