Structural Variants at the Anchors of Intron-Intron CTCF Loops Drive Transcriptional Change

CCCTCF binding factor (CTCF) is involved in numerous cellular functions. CTCF can organize chromatin into large domain structures but can also regulate gene transcription by binding to insulator sequences and/or by mediating chromatin interactions. Targeted genome conformation assays like ChIA-PET have allowed the assessment of CTCF-bound chromatin interactions genome-wide. Since 22-29% of CTCF chromatin binding sites are intronic, it is highly likely there are uncharacterized regulatory regions in introns relating to CTCF-mediated gene transcriptional control. To study this, we leveraged structural variation (SV) in the 129S with ChIA-PET and RNA-seq datasets from C57BL/6J (B6) and 129S1/SvImJ (129S) mouse embryonic stem cells (ESC). In this study we propose correlating genetic variation at intronic CTCF interaction hubs with ESC transcriptional changes. We hypothesize that SVs at the hubs of CTCF chromatin interactions can regulate transcription within the same genes. Our integrative analysis attributed 624 SVs (290 insertions; 334 deletions) to CTCF-mediated chromatin interactions differences between the B6 and 129S ESC. Characterization of the SVs revealed that 30% of the SVs overlapping CTCF interaction hubs contain the transposable element, B2 SINE, while 41.8% showed no repetitive element features. 15% and 11% of the same CTCF interaction hubs showed either significant transcriptional change or significant differential exon usage, respectively. We functionally validated a subset of these intronic SVs using CRISPR/Cas9 and assessed changes in CTCF chromatin binding, gene transcription, and chromatin looping using ChIP-qPCR, qPCR, and Region Capture MicroC. Our findings have identified novel intronic regulatory elements at hubs of CTCF chromatin interactions that can regulate gene transcription.