The 3D Genome Structure of a Blcap-Linked Silencer Loop Regulates Terminal Differentiation During Lens Fiber Cell...

Lens fiber cells (LFCs) remove their nuclei and organelles during terminal differentiation through a controlled process that resembles, but does not execute, apoptosis. The mechanisms that inhibit apoptotic pathways while permitting nuclear degradation remain unclear. Using Hi-C, CUT&Tag, and RNA-seq, we mapped chromatin and epigenetic changes during the LEC-to-LFC transition. We focused on a distal silencer region (DSR)-mediated chromatin loop near Blcap and its function in repressing apoptosis-related signaling to support proper nuclear clearance and lens transparency.

Hi-C sequencing mapped chromatin architecture in E16.5 mouse LECs and LFCs, focusing on apoptosis-related loci. CUT&Tag profiling for H3K27me3, CTCF, and SMC3, integrated with RNA-seq, identified a novel silencer element and an LFC-specific chromatin loop linking Blcap to the DSR. To assess function, an adeno-associated virus-delivered saCas9 dual-sgRNA system was used to delete loop anchor regions in vivo. Functional effects were validated through DNA-FISH, Western blotting, flow cytometry, and phenotypic analyses.

Multiomics analysis revealed extensive chromatin remodeling during LEC-to-LFC differentiation, including A/B compartment switching, silencer reprogramming, and transcriptional changes in differentiation- and apoptosis-related genes. A specific loop tethering Blcap to the DSR was identified in LFCs. Loop disruption abolished their spatial proximity, derepressed Blcap, reduced anti-apoptotic genes Bcl2 and Bcl2l1, increased apoptosis, and caused lens opacification.

The Blcap-DSR chromatin loop plays a key protective role by repressing Blcap and maintaining anti-apoptotic balance in LFCs. Through silencer-associated looping and epigenetic reprogramming, LFCs achieve controlled nuclear and organelle clearance without triggering cell death, ensuring proper differentiation and lens transparency.