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HiChIP is a novel method for the analysis of chromatin interactions based on in situ Hi-C that adds an immuno-precipitation (ChIP) step for the investigation of chromatin structures driven by specific proteins. This approach has been shown to be very efficient as it reliably reproduces Hi-C results and displays a higher rate of informative reads with a required lower amount of input cells when compared with other ChIP-based techniques (as ChIA-PET). Although HiChIP data preprocessing can be performed with the same methods developed for other Hi-C techniques, the identification of chromatin interactions needs to take into account specific biases introduced by the ChIP step. In this chapter we describe a computational pipeline for the analysis of HiChIP data obtained with the immuno-precipitation of Rad21 (part of the cohesin complex) in human embryonic stem cells before and after heat-shock treatment. We provide a detailed description of the preprocessing of raw data, the identification of chromatin interactions, the evaluation of the alterations induced by treatment, and, finally, the visualization of differential loops., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The chromatin organization in the 3D nuclear space is essential for genome functionality. This spatial organization encompasses different topologies at diverse scale lengths with chromosomes occupying distinct volumes and individual chromosomes folding into compartments, inside which the chromatin fiber is packed in large domains (as the topologically associating domains, TADs) and forms short-range interactions (as enhancer-promoter loops). The widespread adoption of high-throughput techniques derived from chromosome conformation capture (3C) has been instrumental in investigating the nuclear organization of chromatin. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D structures, as in principle it can detect any pair of restriction fragments connected as a result of ligation by proximity. However, the analysis of the enormous amount of genomic data produced by Hi-C techniques requires the application of complex, multistep computational procedures that may constitute a difficult task also for expert computational biologists. In this chapter, we describe the computational analysis of Hi-C data obtained from the lymphoblastoid cell line GM12878, detailing the processing of raw data, the generation and normalization of the Hi-C contact map, the detection of TADs and chromatin interactions, and their visualization and annotation.

In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. Here we show that global conformational differences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, by leveraging custom data analysis solutions. We find the male chromosome X has more mid-/long-range interactions. We also identify differences at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding of the dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, our data strongly indicate that chromosome X dosage compensation affects global chromosome structure.

MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation., (Copyright © 2019 Elsevier Inc. All rights reserved.)