Chromatin disruptions in pathophysiologically relevant gene promoters underlie unique SARS-Cov-2 nucleocapsid-mediated gene regulation

SARS-CoV-2, the causative agent of COVID-19, is a positive-sense, single-stranded RNA virus that causes a spectrum of disease severity, from asymptomatic infection to severe illness to long-term sequelae. Similar to other human coronaviruses, SARS-CoV-2 proteins modulate host genomic responses through epigenomic modifications, facilitating viral replication and immune evasion. While the nucleocapsid protein is well known for its role in RNA stability and immune modulation, its impact on host chromatin organization remains unclear. To investigate this, we generated stable human alveolar cell lines expressing nucleocapsid proteins from endemic and pandemic human coronaviruses. Our analysis revealed that nucleocapsid proteins from all tested coronaviruses induced changes in nucleosome positioning and occupancy at specific gene promoters involved in coagulation pathways, hormone signaling, and innate immune responses. Additionally, SARS-CoV-2-specific alterations were identified in genes dysregulated in severe infections, such as suggesting a direct role for epigenomic modifications in disease pathophysiology. We also observed extensive changes in nucleosome susceptibility to nuclease digestion in SARS-CoV and SARS-CoV-2 samples that were not observed in common cold cell lines. Promoters with altered sensitivity and resistance to nuclease were linked to innate immune, metabolic, olfactory, and signaling pathways known to be dysregulated in severe COVID-19 and post-acute sequelae (PASC). These findings demonstrate that nucleocapsid protein expression alters chromatin structure at specific loci, implicating viral proteins in host genome dysregulation. Furthermore, we identified both shared and unique chromatin targets of SARS-CoV-2 and common cold coronaviruses, highlighting pathways for further investigation and potential therapeutic intervention. Overall design: To determine the role of CoV nucleocapsid proteins on nucleosome distribution and susceptibility to enzymatic digestion within promoters, we generated stable A549 human lung epithelial cell lines expressing the full length nucleocapsid proteins of seven human CoVs. Replicate cell lines were generated for each of the following nucleocapsids: HCoV-HKU1, HCoV-NL63, HCoVOC43, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2 which were compared to cells expressing an empty vector construct. The distribution of nucleosomes across promoters was determined by digesting crosslinked chromatin with Micrococcal nuclease (MNase), an enzyme which selectively cleaves linker DNA leaving nucleosomal DNA intact for high throughput sequencing (Shivaswamy et al. 2008; Schones et al. 2008). In addition to mapping the location of individual nucleosomes, a titration of MNase was used to determine the susceptibility of a given nucleosome to enzymatic digestion (Vera et al. 2014; Turpin et al. 2018; Cole and Dennis 2020; Cole et al. 2021; Mieczkowski et al. 2016; Brahma and Henikoff 2019). We chose to focus our analysis on promoter regions as nucleosomes within promoter regions have distinct roles in gene regulation, are highly occupied, and are especially well positioned compared to the rest of the genome.