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

Abstract

Background: Topologically associating domains (TADs) are believed to play a role in the regulation of gene expression by constraining or guiding interactions between the regulatory elements. While the impact of TAD perturbations is typically studied in developmental genes with highly cell-type-specific expression patterns, this study examines genes with broad expression profiles separated by a strong insulator boundary. We focused on the mouse Slc29a3/Unc5b locus, which encompasses two distinct TADs containing ubiquitously expressed and essential for viability genes. We disrupted the CTCF-boundary between these TADs and analyzed the resulting changes in gene expression.

Results: Deletion of four CTCF binding sites at the TAD boundary altered local chromatin architecture, abolishing pre‑existing loops and creating novel long‑range interactions that spanned the original TAD boundary. Using UMI-assisted targeted RNA-seq we evaluated transcriptional changes of Unc5b, Slc29a3, Psap, Vsir, Cdh23, and Sgpl1 across various organs. We found that TAD boundary disruption led to variable transcriptional responses, where not only the magnitude but also the direction of gene expression changes were tissue-specific. Current hypotheses on genome architecture function, such as enhancer competition and hijacking, as well as genomic deep learning models, only partially explain these transcriptional changes, highlighting the need for further investigation into the mechanisms underlying TAD function and gene regulation.

Conclusions: Disrupting the insulator element between broadly expressed genes resulted in moderate, tissue-dependent transcriptional alterations, rather than uniformly activating or silencing the target genes. These findings show that TAD boundaries contribute to context‑specific regulation even at housekeeping loci and underscore the need for refined models to predict the effects of non‑coding structural variants.

Fig. 1

Spatial organization of the Unc5b/Slc29a3 locus and its gene editing. A - Hi-C map and gene localisation of mouse Unc5b/Slc29a3 locus. From [58]. B - Hi-C map and gene localisation of human UNC5B/SLC29A3 locus. From [59]. C - Hi-C map and gene localisation of western clawed frog Unc5b/Slc29a3 locus. From [60]. D - CTCF-bs cluster at the mouse Slc29a3/Unc5b locus and CTCF ChIP-Seq tracks from mouse liver tissue (Wild-type and Mutant variant inherited from mouse #16). Mutations coordinates are shown by red rectangles, and CTCF-bs orientations shown by blue triangles. E - Gene editing design employed for deleting the genomic region with two CTCF-bs. F - Sanger sequencing of the obtained deletion breakpoint regions. Obtained sequence aligned to the expected sequence of deletion. G - gene editing design for telomeric CTCF-binding sites knock-out

Fig. 2

A-C - NGS genotyping results for Left (A), Middle (B), and Right (C) sites. Potential microhomology motifs at deletion borders are highlighted by magenta. Variants chosen for homozygous line derivation (mouse #16) are highlighted in bold

Fig. 3

Alterations in Spatial Chromatin Architecture at the Slc29a3/Unc5b locus. A - Gene locations and their activity status are indicated by a color scale from green (active expression) to red (repressed state). B - Location and orientation of CTCF binding sites. C, D - cHi-C Interaction Maps, displaying spatial chromatin interactions within the Slc29a3/Unc5b locus (chr10:60,103,000–61,356,000, mm10) in the liver, cerebellum, and kidney tissues for obtained model mice and wild-type controls. Maps above the main diagonal represent mice with deletions of two (C) or four (D) CTCF-bs, while maps below the diagonal show data from wild-type mice. Black-filled arrowheads indicate chromatin loops that were disturbed in mutation genotype, white-filled arrowhead indicate differing between wild-type and CTCF-bs deletion conditions. E– Insulation score for wild-type (black), two (blue) or four (red) CTCF-bs deleted. F– Chip-seq profiles of the Unc5b/Slc29a3 locus according to ENCODE data. Each column shows data for one of three organs: Liver, Cerebellum, and Kidney. Red vertical line represent 5-kb deletion of centromeric CTCF-bs. G - Bar plots representing expression of genes within Unc5b/Slc29a3 TADs across tissues. Data is shown in TPM units, whiskers represent standard error. H, I - statistically significant changes in gene expression relative to the wild-type levels for mice with deletions of two (H) or four (I) CTCF binding sites

Fig. 4

Transcription changes at the Slc29a3/Unc5b locus. A - graphical scheme illustrating the allele imbalance quantification in hybrids using UMI-assisted targeted RNA-seq. B, C - Heatmap representations of locus gene expression changes observed via UMI-assisted targeted RNA-seq for two (B) or four (C) CTCF-bs deletion versus wild-type alleles. D - Heatmap representations of locus gene expression changes measured via digital PCR for four CTCF-bs deletion mice versus wild-type mice. E, F - Enformer in silico predictions of the transcriptional effect of deletions of two (D) or four (E) CTCF-bs versus wild-type. G, H– AlphaGenome in silico predictions of the transcriptional effect of deletions of two (F) or four (G) CTCF-bs versus wild-type