Hemin-driven chromatin remodelling by atherosclerotic risk gene SMARCA4 switches human blood-derived macrophages from leukocyte disposal to erythrocyte disposal

Background Putative genetic risk loci for atherosclerotic vascular disease include SMARCA4 , a chromatin remodeling gene important for gene activation. Its causal role in atherosclerosis has been uncertain. Intraplaque hemorrhage (IPH) is a late event in atherosclerosis that is linked to plaque destabilisation and increased inflammation. IPH is countered by Mhem macrophages, which are directed by hemin-mediated induction of Heme Oxygenase 1 ( HMOX1 ) via Activating Transcription Factor 1 (ATF1). Atf1 deficiency in vivo impairs hematoma clearance, promoting inflammation and oxidative stress. Like its homologue cyclic-adenosine monophosphate response element binding protein 1 (CREB1), ATF1 is normally cyclic-AMP activated. Hypothesis Hemin-directed chromatin remodelling by SMARCA4 regulates specificity of ATF1 gene-binding, thereby switching between leukocyte disposal and erythrocyte disposal, contributing to its role in atherosclerosis. Results We here show that SMARCA4 is genetically independent of the adjacent LDLR locus (p<0.05). In human blood-derived macrophages, hemin triggered histone acetylation (H3K9Ac) and SMARCA4 recruitment in advance of p-ATF1 recruitment at the HMOX1 enhancer. si-RNA-mediated SMARCA4 -knockdown suppressed p-ATF1 binding to HMOX1 but increased its binding to cyclic-AMP responsive genes FOS and NR4A2 , with corresponding changes in mRNA levels. This functionally correlated with SMARCA4 -knockdown switching hemin to mimic prostacyclin (PGI2), for induced genes and phagocytic disposal of leukocytes rather than erythrocytes. Conclusions These data establish SMARCA4 as an independent atherosclerosis risk gene and reveal a novel mechanism in which it switches between disposal of leukocytes or erythrocytes, with important clinical implications for atherosclerotic inflammation and intraplaque hemorrhage including treatment by histone deacetylase inhibitors. ### Competing Interest Statement JJB holds part of the IP on a novel class of HO-1 real-time fluorescence activity probes (WO2022101635). JJB was funded by a BHF Senior Clinical Research Fellowship FS13/12/30037, BHF Project Grants PG/15/57/31580, PG/17/71/33242, PG/21/10422). Some of the imaging was carried out with instruments in FILM facility, Imperial College London. The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is part supported by funding from the Wellcome Trust (grant 104931/Z/14/Z) and Biotechnology and Biological Sciences Research Council (BBSRC) (grant BB/L015129/1). This work was supported by the NIHR Imperial Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. SEH acknowledges funding from the BHF (PG08/008) and the UCL Biomedical Research Centre.