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Investigating how chromatin organization determines cell-type-specific gene expression remains challenging. Experimental methods for measuring three-dimensional chromatin organization, such as Hi-C, are costly and have technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. We present C.Origami, a multimodal deep neural network that performs de novo prediction of cell-type-specific chromatin organization using DNA sequence and two cell-type-specific genomic features-CTCF binding and chromatin accessibility. C.Origami enables in silico experiments to examine the impact of genetic changes on chromatin interactions. We further developed an in silico genetic screening approach to assess how individual DNA elements may contribute to chromatin organization and to identify putative cell-type-specific trans-acting regulators that collectively determine chromatin architecture. Applying this approach to leukemia cells and normal T cells, we demonstrate that cell-type-specific in silico genetic screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulation circuits in both normal and disease-related biological systems., (© 2023. The Author(s).)

DNA methylation is thought to regulate accessibility of chromatin and binding of regulatory elements; however, it is difficult to determine if chromatin accessibility or transcription factor (TF) binding overlap with methylated or unmethylated DNA if the assays are performed separately. In order to examine accessibility or TF binding simultaneously with methylation on the same DNA molecule, we developed EpiMethylTag which combines ATAC-Seq or ChIP-Seq (M-ATAC or M-ChIP) with bisulfite conversion. Our approach provides a fast, low-input, low sequencing depth method to determine whether DNAme and accessibility/TF binding are mutually exclusive or can coexist in certain locations., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The objective of this study is to determine how the epigenome is locally altered after DNA damage and investigate the impact of these changes on 3D chromatin organization, gene regulation and treatment resistance. In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome is largely unknown. Since epigenetic alterations such as DNA-methylation can impact the binding of factors involved in chromatin folding, and transcription factors that bind regulatory elements, the downstream impact on gene regulation could lead to the emergence of treatment resistance. Using newly developed tools to enable these studies, we hypothesize that genome-wide DNA damage leads to local alterations in DNA-methylation, genome organization, and results in persistent gene-expression alterations near sites of repaired damage. We use patient-derived human glioblastoma stem-like cells (GSCs) as a model. DNA breaks are induced using (i) irradiation or (ii) a novel "multi-cut" CRISPR-Cas9 DNA break system. Genomic profiling methods include HiC (genome-wide chromosome conformation capture), DNA-methylation profiling (bisulfite sequencing), targeting DNA-seq and RNA-seq. With radiation, we find significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. It is difficult to study local alterations around sites of radiation induced damage because breaks are introduced at different sites in each cell, resulting in stochastic DNA methylation alterations. To circumvent this issue, we developed a multi-cut CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. Induction of pre-mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation. To assess repair efficiency (i.e., deletions, translocations), we employed a custom panel of DNA probes that flank the 142 or 483 sites to allow for high coverage sequencing at each site following cut induction. To understand how DNA damage may lead to local epigenetic alterations and 3D chromatin organization changes, we performed HiC (genome-wide chromosome conformation capture), before and after cut induction, and subjected cultures to further downstream analysis. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites, enrichment of DNA methylation alterations and altered gene-expression. The multi-cut CRISPR-Cas9 DNA break system, which we develop and characterize here, can be rapidly employed in cell lines or animal models to study pre-mapped genome-wide DNA damage, and has widespread applicability in the radiobiology and DNA damage and repair fields. The findings here provide a mechanistic view of the interplay between genome-wide DNA damage, DNA methylation and genome re-organization, and have wide-ranging implications for the effect of DNA damage on the epigenome. [ABSTRACT FROM AUTHOR]