R-loop-induced DNA replication defects drive neuropathy in Koolen-de Vries syndrome

Koolen-de Vries syndrome (KdVS) is a neurodevelopmental disorder caused by KANSL1 haploinsufficiency. KANSL1 encodes a scaffold protein of the non-specific lethal (NSL) complex, which regulates transcription of constitutively expressed genes via acetylation of H4K5 and H4K8 around transcription start sites (TSS). How loss of KANSL1/NSL contributes to the neurodevelopmental defects in KdVS is unknown. Here, we find that KANSL1/NSL prevents the accumulation of R-loops (three-stranded DNA-RNA hybrid structures) at transcription start sites (TSSs) with low basal transcription and H4K5/H4K8 acetylation, where R-loops are typically not detected. If not suppressed, these R-loops delay DNA replication in early S-phase by stalling of the replisome. KdVS neural organoids also suffered from perturbed DNA replication concomitantly with neurodevelopmental defects, while KdVS neuronal cells showed reduced synaptic activity in a manner-dependent on R-loop accumulation. Our study offers insights into how KANSL1/NSL controls R-loop homeostasis to ensure faithful DNA replication during neuronal development, preventing neuropathy in KdVS patients. Overall design: U2OS WT cells treated with siLuciferase, siKANSL1 and siKANSL3 to a double pulse of EdU (10uM for 15 min, 60 minutes washout and 10uM for 15 min,). Next, cells were sorted in 384 well plates and subjected to scEdU-seq (van den Berg et al., 2024) .