851. A mechanism for PHF21B recognition of H3K36me3

Abstract

Abstract Background We have recently identified the epigenetic reader PHF21B as a regulator of synaptic plasticity and social memory in mice. The PHF21B gene is located on chromosome 22q13.3, a region that is deleted in Phelan-McDermid syndrome, a neurodevelopmental disorder characterized by intellectual disability, impaired social communication, and features overlapping with autism spectrum disorder. We have previously shown that PHF21B interacts with H3K36me3, an epigenetic mark associated with active gene expression and critical roles in transcription, splicing, and genomic stability. Deficiencies in H3K36me3 are associated with various cancers and developmental disorders. However, the structural domain that mediates the interaction between PHF21B and H3K36me3 has yet to be characterized. Aims & Objectives The identification of the specific protein domain in PHF21B that is required for H3K36me3 recognition could provide mechanistic insights relevant to understanding and treating PHF21B-associated neurodevelopmental features. Method Myc-tagged wild-type PHF21B and mutants PHF21BΔPHDZnf and PHF21B ΔCc, which respectively lack the PHD-zinc finger domain in residues 352–465 and the C-terminal region in residues 423–531, were generated and expressed in HEK293T cells. The interaction between PHF21B and H3K36me3 was assessed by bidirectional co-immunoprecipitation and anti-Myc and anti-H3K36me3 antibodies, followed by western blotting. GFP-transfected cells were used as negative controls. Results H3K36me3 was immunoprecipitated by wild-type PHF21B and PHF21B ΔCc, but not by PHF21B ΔPHDZnf (P < 0.001, one-way ANOVA with Tukey's post-hoc test). These findings were confirmed by reciprocal co-immunoprecipitation, where Anti-H3K36me3 antibodies pulled down wild-type PHF21B and PHF21B ΔCc, but not PHF21B ΔPHDZnf. Therefore, H3K36me3 binding was abolished in the ΔPHDZnf mutant. Discussion & Conclusions The PHD-zinc finger domain of PHF21B is necessary for its interaction with H3K36me3, and the C-terminal region is dispensable. Given that PHF21B reads active chromatin marks at synaptic plasticity genes, disruption of this PHD-zinc finger–H3K36me3 interface may underlie transcriptional dysregulation in neurodevelopmental conditions associated with 22q13.3 deletions. We have defined a specific protein domain that could serve as a therapeutic target for Phelan-McDermid syndrome. Future studies will need to examine whether the PHD-zinc finger domain is sufficient to rescue the neuronal functions of PHF21B.