Sp100A isoform promotes localization of the histone chaperone HIRA to PML nuclear bodies

PML nuclear bodies (PML-NBs) are dynamic subnuclear structures important for chromatin dynamics and anti-viral defense. In this study we investigate the role of Sp100 isoforms in promoting localization of the H3.3 histone chaperone HIRA to PML-NBs in human keratinocytes. Sp100 knockout (KO) cell lines were generated in normal keratinocytes using CRISPR-Cas9 technology and shown to display typical skin development, normal PML-NB formation, and a normal interferon response as determined by RNAseq. However, the histone chaperone HIRA and its associated complex members (Ubinuclein-1 and ASF1A) failed to localize to PML-NBs in the absence of Sp100, even after interferon stimulation. Exogenous expression of the four main isoforms of Sp100 showed that the Sp100A isoform is the primary driver of HIRA localization to PML-NBs; predictive structural analysis of the various Sp100 isoforms using several AI methods (AlphaFold 3, Chai-1, and Boltz-2) plus molecular dynamics simulation of Sp100B highlighted how interspersed structural domains and intrinsically disordered regions (IDRs) could influence this localization. Analysis of Sp100A proteins with mutations in seven distinct functions showed that the SUMO-interacting motif (SIM) plays an important role in HIRA localization to PML-NBs. These findings highlight the functional diversity of the Sp100 isoforms in modulating chromatin dynamics at PML-NBs and further discern the molecular features underpinning the relationship between histone chaperones and PML-NBs. We conclude that HIRA recruitment to PML-NBs is not required for immediate interferon signaling but as suggested by others, is potentially required for maintaining epigenetic memory by delayed H3.3 incorporation at interferon-stimulated genes.