Integrative Multi-Omics Analysis of IFNγ-induced Macrophages and Atherosclerotic Plaques Reveals Macrophage-dependent STAT1-Driven Transcription in Atherosclerosis Short title: STAT1-Dependent genomic changes in Atherosclerosis

Mahdi Eskandarian Boroujeni¹Natalia Lopacinska¹Aleksandra Antonczyk¹Katarzyna Kluzek¹Joanna Wesoly²Hans AR Bluyssen^1*

• ¹Human Molecular Genetics Research Unit, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland., Poznan, Poland
• ²Laboratory of High-Throughput Technologies, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland., Poznan, Poland

Atherosclerosis is a chronic inflammatory disease of blood vessels, characterized by atherosclerotic lesions in largeand medium-sized arteries. IFNγ is a crucial mediator of atherosclerosis through activation of signal transducer and activator of transcription (STAT)1. Macrophages (MØ), in different subtypes, play a central role in atherosclerosis, from early foam cell formation to advanced plaque development and potential rupture. Recent evidence in MØ supports a collaborative role of STAT1 with PU.1, in association with histone acetylation and methylation marks, in MØ-specific IFNactivated transcriptional responses.This study investigated the role of MØ STAT1-mediated signaling in atherosclerosis progression through multi-omics integration of IFNγ-induced MØ and expression analysis in human and mouse atherosclerotic lesions. First, by integrating ATAC-seq, ChIP-seq, and RNA-seq data from IFNγtreated and untreated bone marrow-derived MØ, we identified 1139 STAT1-dependent integrative genes. Active transcription of these genes was characterized by prominent promoter STAT1-PU.1 co-binding, increased histone methylation and acetylation and chromatin accessibility. Moreover, KEGG-analysis unraveled a strong connection to lipid metabolism and atherosclerosis-related pathways, whereas STARNET analysis identified high association with LDL cholesterol and diseased vessel traits. Using scRNA-seq data analysis of human carotid and coronary atherosclerotic lesions revealed dynamic changes of STAT1-dependent integrated genes in MØ subtypes, including foamy MØmacrophages, monocytes, inflammatory MØmacrophages, tissue resident MØmacrophages and conventional dendritic cells. Comparative MØ-dependent expression analysis in aortic lesions from LDLr-/-and ApoE-/-high fat diet mouse models substantiated overlap between human and mouse atherosclerosis and identified 24 MØ-specific commonly expressed STAT1-dependent integrated genes.Collectively, we provide detailed insights into MØ-specific IFN-activated transcriptional changes, mediated by STAT1-PU.1 co-binding and associated epigenetic changes, and offer the identification of new biomarkers and therapeutic targets in atherosclerosis. Moreover, we present a novel STAT1-dependent gene signature that could potentially serve to monitor "MØ macrophage-dependent" plaque progression during human atherosclerotic disease.