Project description:Capture Hi-C (CHi-C) is a new technique for assessing genome organization based on chromosome conformation capture coupled to oligonucleotide capture of regions of interest, such as gene promoters. Chromatin loop detection is challenging because existing Hi-C/4C-like tools, which make different assumptions about the technical biases presented, are often unsuitable. We describe a new approach, ChiCMaxima, which uses local maxima combined with limited filtering to detect DNA looping interactions, integrating information from biological replicates. ChiCMaxima shows more stringency and robustness compared to previously developed tools. The tool includes a GUI browser for flexible visualization of CHi-C profiles alongside epigenomic tracks. The results of the analysis on existing mouse ES Capture Hi-C data (ArrayExpress E-MTAB-2414) were validated by two 4C-seq experiments, submitted here.
Project description:We have developed a mass spectrometry (MS) and bioinformatics-based pipeline to generate a proteome-wide resource of protein subcellular localization across multiple human cancer cell lines (www.subcellbarcode.org). Here, we present a detailed wet-lab protocol spanning from subcellular fractionation to MS-sample prep, as well as a dry-lab protocol covering quantitative MS-data analysis, machine-learning-based classification, differential localization analysis and visualization of the output. For broad applicability, we evaluated the pipeline using MS-data generated by three different peptide prefractionation approaches, HiRIEF-LC-MS, High-pH reverse phase fractionation and direct analysis without pre-fractionation using long gradient LC-MS.
Project description:The transcription factor ZNF143 contains seven tandem zinc fingers and is involved in 3D genome construction; however, the mechanism by which ZNF143 functions in chromatin looping remains unclear. Here, we show that ZNF143 directionally recognizes diverse genomic sites and is required for chromatin looping between these sites. In addition, ZNF143 is located between CTCF and cohesin at numerous CTCF sites and ZNF143 removal narrows the space between CTCF and cohesin. Moreover, genetic deletion of ZNF143, in conjunction with acute CTCF depletion, revealed that ZNF143 and CTCF collaborate to regulate higher-order genome organization. Thus, ZNF143 is recruited by CTCF to the CTCF sites to regulate TAD formation and genome compartmentalization whereas directional recognition of DNA motifs directly by ZNF143 itself regulates promoter activity via chromatin looping.
Project description:Regulatory DNA elements can control expression of distant genes via physical interactions. Here, we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using Chromosome Conformation Capture combined with high-throughput sequencing (4C-seq) data. Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation. A high resolution 4C-seq protocol involving two restriction digests and a revised analysis pipeline was applied to several viewpoints in four genomic loci (the well-characterized alpha-globin and beta-globin loci, and the novel Oct4 and Satb1 loci), allowing robust detection of physical interactions between regulatory DNA elements.