Sp100A isoform induces the histone chaperone, HIRA to localize 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 using CRISPR-Cas9 technology and shown to display normal keratinocyte differentiation and PML-NB formation. However, HIRA and its associated complex members (UBN1 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, with the SUMO interacting motif (SIM) playing an important role. These findings highlight the functional diversity of the Sp100 isoforms in modulating chromatin dynamics at PML-NBs. Overall design: RNA-seq analysis of Sp100 wildtype and knockout human Normal Immortalized Keratinocyte (NIKS) cell lines that were untreated (0hr IFN) or stimulated with 25 ng/mL Human Interferon Alpha 2a for 12 or 48 hours. Each condition has three biological replicates.