Bromodomain and PHD finger containing protein 1 (BRPF1) is a chromatin reader which complexes with histone acetyltransferases KAT6A and KAT6B. The BRPF1 complex plays essential roles in neurodevelopment, with heterozygous BRPF1 loss leading to neurodevelopmental disorders (NDDs), primarily the BRPF1 haploinsufficiency disorder Intellectual Developmental Disorder with Dysmorphic Facies and Ptosis (IDDDFP). Indeed, heterozygous loss of KAT6A and KAT6B also leads to NDD’s. These disorders are characterised by intellectual disability and global developmental delay.
The molecular mechanism of BRPF1, KAT6A and KAT6B has not been thoroughly investigated owing to issues in reliable detection methods and low endogenous expression. Using CRISPR-Cas9 editing via ribonucleoprotein nucleofection, we have successfully HiBiT tagged BRPF1, KAT6A and KAT6B, as well as BRPF family members BRPF2/BRD1 and BRPF3 in human neuroblastoma SH-SY5Y cells. Furthermore, we have introduced IDDDFP patient pathogenic variants into HiBiT-BRPF1 to directly investigate the molecular consequences of different variants.
For the first time, we have identified the genomic binding targets of BRPF1 through Cut&Run using the HiBiT antibody, and will extend this to the other targets. Additionally, using the HiBiT toolkit, we have detected BRPF1 and complex members via immunofluorescence microscopy, on-blot luciferase assay and luminescence plate-based methods. Furthermore, experiments carried out in BRPF1 patient pathogenic variant lines support that IDDDFP is a BRPF1 haploinsufficiency disorder, with patient variants resulting in a loss of function.