Direction and modality of transcription changes caused by TAD boundary disruption in Slc29a3/Unc5b locus depends on tissue-specific epigenetic context

Topologically associated domains (TADs) are believed to be involved in the regulation of gene expression. While the impact of TAD perturbations is usually studied in developmental genes with highly cell-type-specific expression patterns, this study examines genes with broad expression profiles divided by a strong insulatory boundary. We focused on mouse Slc29a3/Unc5b locus, which encompasses two distinct TADs. Our analysis demonstrates that deletions of CTCF binding sites within this locus lead to alterations in local chromatin architecture, disrupting existing loops and forming novel long-range interactions. We evaluated the transcription changes of Unc5b, Slc29a3, Psap, Vsir, Cdh23, and Sgpl1 genes across various organs, finding that TAD boundary disruption results in variable transcriptional responses, where not only magnitude, but also direction of gene expression changes are tissue-specific. Current models of genome architecture, including enhancer competition and hijacking, only partially account for these transcriptional changes, indicating the need for further investigation into the mechanisms underlying TAD function and gene regulation. This work is the first trial to simultaneously induce multiple adjacent deletions og CTCF binding sites, guided by ssODNs, in murine zygotes by CRISPR/Cas9 system. We analyzed the repertoire of generated deletions and confirmed lack of CTCF binding in modifyed sites. In this work we first used novel and simple approach for monoallelic expression measurements by NGS sequencing of targeted RNA-seq with UMI.