Background: SP140 is an epigenetic reader with immune-restricted expression and single nucleotide polymorphisms (SNPs) within SP140 associate with Crohn’s Disease. Our lab previously found SP140 to be a master orchestrator of macrophage identity and function through occupancy and repression of lineage-inappropriate genes such as HOX genes1. SP140 depletion in mouse or human macrophages resulted in severely compromised microbe-induced activation1. Importantly a loss of SP140 is causative of disease since a hematopoietic-specific depletion of Sp140 in mice resulted in exacerbated dextran sulfate sodium (DSS)–induced colitis1. The specific mechanism by which SP140 imparts its epigenetic function to modulate macrophage transcriptional programs for identity and function is unknown.
Methods: We overexpressed FLAG-SP140 and FLAG-Empty Vector constructs in HEK293 cells, immunoprecipitated SP140, and performed Mass Spectrometry (MS) to reveal SP140 binding partners. Co-immunoprecipitation (IP) experiments were conducted to validate the top SP140 interacting hits. We performed CRISPR-mediated deletion of SP140 in human and mouse macrophages and assessed expression and function of SP140 interacting proteins.
Results: The top SP140 interacting proteins are involved in DNA unwinding (Topoisomerase I (Topo1), II alpha (Top2A) and II beta (Top2B); DNA-PK and SUPT16H (FACT complex 160 subunit) which were all confirmed by co-IP experiments. Deletion of SP140 resulted in increased DNA double stranded breaks (phospho-H2AX) and activity of Topoisomerases Top2 and Top1.
Conclusions: The epigenetic reader SP140 interacts with topoisomerase and other proteins involving chromatin remodeling. Thus, SP140 links histone modifications to transcriptional regulation through control of DNA unwinding mechanisms. SP140 suppresses these DNA unwinding mechanisms at loci of lineage inappropriate genes to maintain cell identity. Furthermore, we are exploring if this function of SP140 is lost or switched in Crohn’s Disease patients harboring SP140 SNPs.