Hypothesis

Progressive telomere shortening disrupts long-range chromatin loops that sequester splicing regulators at the nuclear periphery, leading to increased splicing entropy and the emergence of oncogenic isoform variants. Restoring telomere length rescues loop integrity, reduces splicing noise, and suppresses cancer‑associated isoform switches.

Mechanistic Basis

Telomeres organize the 3D genome through TPE‑OLD, forming repressive loops that tether Alu‑rich regions and associated splicing factors (e.g., SRSF2, HNRNPU) to the lamina【Robin et al., 2014](https://pubmed.ncbi.nlm.nih.gov/25403178/)】. As telomeres erode, these loops dissolve (Chevalier et al., 2025)[https://pubmed.ncbi.nlm.nih.gov/40065531/], releasing splicing regulators into the nucleoplasm. The resulting alteration in nuclear speckle composition raises the stochasticity of exon inclusion, measurable as a rise in Shannon entropy of the transcriptome. This entropy increase coincides with activation of subtelomeric oncogenes (e.g., ISG15, DSP) and promotes isoform shifts that favor proliferation and invasion—consistent with observations that telomere dysfunction drives transcriptional plasticity without necessarily changing total gene expression levels【Sullivan et al., 2021](https://pmc.ncbi.nlm.nih.gov/articles/PMC7902064/)】.

Testable Predictions

1. Isoform entropy (calculated from RNA‑seq using tools such as SUPPA2) will remain low in cells with telomeres >8 kb, rise sharply after telomeres fall below ~5 kb, and correlate with the appearance of cancer‑specific splice variants.
2. Forced telomere elongation via CRISPR‑dCas9‑TERT or transient TERT expression will re‑establish chromatin loops, decrease splicing entropy, and revert oncogenic isoform patterns to a non‑transformed state.
3. Acute disruption of RBPJ‑mediated looping (using degron‑tagged RBPJ) will phenocopy telomere shortening: increased splicing entropy and oncogenic isoform switching despite unchanged telomere length.

Experimental Approach

• Generate isogenic human fibroblast lines with graded telomere lengths using CRISPR‑Cas9 telomere editing (telomere truncation or elongation).
• Perform deep RNA‑seq (≥100 M reads per sample) and compute isoform‑level Shannon entropy; validate with long‑read sequencing (PacBio Iso‑Seq) to capture full‑length transcripts.
• Assess chromatin looping by Capture‑HiC targeting telomere‑proximal Alu elements and splicing factor ChIP‑seq (SRSF2, HNRNPU) to quantify peripheral retention.
• Measure oncogenic phenotypes (soft‑agar colony formation, invasion) alongside entropy metrics to establish causal links.
• Include controls: telomerase‑inert cells, RBPJ degron with/without telomere length manipulation, and rescue experiments with loop‑stabilizing peptides.

If predictions hold, telomeres emerge not merely as division counters but as structural regulators of splicing fidelity, offering a mechanistic bridge between aging‑associated genome disorganization and the isoform dysregulation that fuels tumorigenesis.