Role of CTCF Binding Site Mutations in Oncogene Activation via Enhancer Hijacking
DOI:
https://doi.org/10.5281/zenodo.22756849Keywords:
CTCF, enhancer hijacking, TAD boundary, oncogene activation, T-cell acute lymphoblastic leukemia, base editing, Hi-CAbstract
Somatic mutation of CTCF binding motifs at topologically associating domain (TAD) boundaries has emerged as a
recurrent, non-coding mechanism of oncogene activation in cancer, in which boundary erosion permits otherwise
insulated enhancers to contact and inappropriately activate a neighbouring proto-oncogene, yet how individual
patient-derived CTCF motif mutations quantitatively affect boundary strength and downstream oncogene
expression has rarely been tested by direct genetic reconstruction. Here we used CRISPR base editing to introduce
four recurrent, patient-derived CTCF motif point mutations found near the TAL1 and LMO2 oncogenes in T-cell
acute lymphoblastic leukemia (T-ALL) into an isogenic human T-cell progenitor line, and profiled the consequences
using in situ Hi-C, CTCF ChIP-seq, ATAC-seq and RNA-sequencing. Base editing of the strongest of the four
motifs, located at a boundary immediately downstream of TAL1, reduced CTCF occupancy at that site by 71% and
produced a marked increase in inter-domain Hi-C contact frequency spanning the boundary, consistent with partial
TAD fusion; the three weaker motifs produced smaller but still significant reductions in local boundary insulation.
RNA-sequencing showed that TAL1 expression increased 8.3-fold following the strongest motif's disruption, with
smaller but significant increases following disruption of the weaker motifs, and that this increase was accompanied
by de novo ATAC-seq accessibility gain at TAL1's promoter matching the accessibility signature of a nearby stem
cell-specific enhancer normally insulated from it. Combinatorial disruption of two motifs produced TAL1 induction
greater than either single mutation alone but less than additive, indicating partial redundancy between boundary
elements. A matching CTCF motif mutation near LMO2 similarly increased LMO2 expression and induced ectopic
enhancer contact, reproducing the two most common enhancer-hijacking events reported in primary T-ALL. These
results establish a direct, quantitative relationship between individual CTCF motif mutations, boundary insulation
strength, and oncogene induction, with direct relevance for interpreting non-coding mutation burden in cancer
genomes.
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