key: cord-348159-v5hrcl5k
authors: Sang, Eric R.; Tian, Yun; Miller, Laura C.; Sang, Yongming
title: Epigenetic Evolution of ACE2 and IL-6 Genes as Non-Canonical Interferon-Stimulated Genes Correlate to COVID-19 Susceptibility in Vertebrates
date: 2020-09-10
journal: bioRxiv
DOI: 10.1101/2020.09.09.273268
sha: 
doc_id: 348159
cord_uid: v5hrcl5k

Current novel coronavirus disease (COVID-19) has spread globally within a matter of months. The virus establishes a success in balancing its deadliness and contagiousness, and causes substantial differences in susceptibility and disease progression in people of different ages, genders and pre-existing comorbidities. Since these host factors are subjected to epigenetic regulation, relevant analyses on some key genes underlying COVID-19 pathogenesis were performed to longitudinally decipher their epigenetic correlation to COVID-19 susceptibility. The genes of host angiotensin-converting enzyme 2 (ACE2, as the major virus receptor) and interleukin (IL)-6 (a key immune-pathological factor triggering cytokine storm) were shown to evince active epigenetic evolution via histone modification and cis/trans-factors interaction across different vertebrate species. Extensive analyses revealed that ACE2 ad IL-6 genes are among a subset of non-canonical interferon-stimulated genes (non-ISGs), which have been designated recently for their unconventional responses to interferons (IFNs) and inflammatory stimuli through an epigenetic cascade. Furthermore, significantly higher positive histone modification markers and position weight matrix (PWM) scores of key cis-elements corresponding to inflammatory and IFN signaling, were discovered in both ACE2 and IL6 gene promoters across representative COVID-19-susceptible species compared to unsusceptible ones. Findings characterize ACE2 and IL-6 genes as non-ISGs that respond differently to inflammatory and IFN signaling from the canonical ISGs and their epigenetic properties may serve as biomarkers to longitudinally predict COVID-19 susceptibility in vertebrates and partially explain COVID-19 inequality in people of different subgroups.

for cell attachment and entry [42, 43] . Several groups have reported that SARS-CoV2 exerts 95 higher receptor affinity to human ACE2 than other coronaviruses, which may contribute 96 to the high-contagiousness and rapid spread of SARS-CoV2 in humans [42, 43] . Being a key 97 enzyme in the body's renin-angiotensin-aldosterone system (RAAS), ACE2 catalyzes 98 angiotensinogen (AGT) to produce the active forms of hormonal angiotensin (Ang) 1-9, 99 which directly regulate the blood volume/pressure, body fluid balance, sodium and water 100 retention, as well as co-opt multiple effects on inflammation, apoptosis, and generation of [50] . These canonical ISGs are 129 mainly induced by type I and type III IFNs but overlap with those upregulated by type II 130 IFN (i.e. IFN-γ) [47] [48] [49] [50] . These ISGs comprise a frontline of antiviral immunity to restrict 131 virus spreading from the initial infection sites [50] . However, based on gene evolution and 132 epigenetic analyses, ACE2 may not be a member of these classical antiviral ISGs, and more ISGs expression (Figure 1 ) [51] [52] [53] [54] . To confirm that, we conducted cross-species 139 comparative analysis between IL-6 and ACE2 genes. First, annotation of ENCODE 140 epigenetic datasets discovered similarity of H3K4me3 and H3K27ac markers between IL-6 141 and ACE2 gene promoters in both humans and mice; however, significantly higher Z-142 scores and enrichment of H3K4me3 and H3K27ac in human IL-6 and ACE2 genes were 143 detected than in their mouse orthologs, respectively [55] . Second, detection of cis-144 regulatory elements (CREs) that bind core transcription factors of non-ISGs, including 145 PU.1, IRFs, and NF-κB, in ACE2 and IL-6 gene proximal promoter regions across 25 146 representative animal species [56] . Third, we found that the evolutionary increase of 147 ACE2, and especially the IL-6 genes response to inflammatory and IFN signaling may 148 serve as epigenetic marker for COVID-19 susceptibility in some animal species including 149 humans. Finally, using our non-biased RNA-Seq data, we further categorize some more 150 non-ISGs that resemble the expression pattern of either IL-6 or ACE2 [57] . Notably, we 151 detected two ACE2 isoforms, which differ in both proximal promoters and coding regions, 152 in some livestock species including pigs, dogs and cattle [30] . In pigs, the ACE2 short 153 isoform (ACE2S) has an expression pattern similar to IL-6 than the long isoform (ACE2L).

Collectively, our findings characterize ACE2 and IL-6 genes as non-ISGs responding lower PWM scores for these CREs than those for IL-6 genes, in particular the PWM scores 236 for NF-κB2 CRE in ACE2 genes were at 2-8 Log2units lower ( Figure 3D ). This indicates 237 that ACE2 genes were less responsive to non-canonical NF-κB signaling mediated by NF- 

Notably, only CRE matrices to IRF1 were shown in Figure 3C 

ACE2 group showed a mid-response to IFN-α but highest to IFN-γ ( Figure 8A-8D) . Figure   356 8D statistically demonstrates the stimulatory difference among three groups of IFN- 

Interestingly, most of them belong to IL-6, TNF and chemokine superfamilies, whose roles scores between ACE2 and IL-6 genes, except NF-κB2 that mediates non-canonical NF-κB response (D) has a significant lower mPWN score (2-6 Log2 units), indicating ACE2 genes are among different non-ISGs group other than IL-6. Canonical ISGs of human ISG15 and IRF1 are used as references. mPWM scores are calculated using tools at https://ccg.epfl.ch/pwmtools/pwmscore.php with CRE Matrices from MEME-derived HOCOMOCOv11 TF collection affiliated with the PWM tools. PWM, position weight matrix. Other abbreviations are as in Figure 4 . . This shows that ACE2 and especially IL-6 genes from CoV2(+) species contain CREs with significantly higher mPWM scores, indicating that in some vertebrate species, non-ISGs like ACE2 and especially IL-6 genes evolved to obtain high inductive propensity by inflammatory and IFN signaling, and may serve as epigenetic biomarkers (or triggers) for susceptibility prediction for COVID-19 and other ARD syndromes. Abbreviation: H_Bat, Great horseshoe bat, and other abbreviations are as in Figure 7 : Cross-species phylogenic and topological comparison of IL-6 and ACE2 gene promoters. Evolutionary analyses were conducted in MEGA X. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-52755.39) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. For topological comparison between phylogenic trees generated using IL-6 and ACE2 gene proximal promoters, the phylogenies of Newick strings were generated using the MEGA, and topological comparison between the Newick trees was performed with Compare2Trees at (http://www.mas.ncl.ac.uk/~ntmwn/compare2trees) to obtain the overall topological scores. Orange circle: COVID-19 susceptible species. Arrows: other tentative marker species to determine which group (IL-6 or ACE2) of non-ISGs are more determined for COVID-19 susceptibility. Abbreviations are as in Figure 4 .

COVID-19 Dashboard by the Center for Systems Science and Engineering

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Orange 724 circle: marking COVID-19 susceptible species. Arrows: other tentative marker species to determine 725 which group

Genome-wide categorizing non-ISGs based on the similarity of inductive pattern to IL-6

The non-biased genome-wide transcriptomic data was generated using a RNA-Seq 730 procedure in porcine lung macrophages stimulated with each of activation stimulator of IL-4

Significantly differentially expressed genes (DEGs) in renin-733 angiotensin system (RAS), interleukin (IL)-6, TNF and chemokine super-families were annotated and 734 grouped using heatmaps according to their inductive expression patterns similar to

C) Examples of canonical ISGs as reference; (D) Averaged transcriptomic expression levels 736 (normalized at Reads Per Kilobase of transcript per Million mapped reads, RPKM) of the grouped 737

ISGs or non-ISGs above. Indicated by arrows, pigs have two ACE2 isoforms

ACE2S similar to IL-6 was showing less responsive 739 to IFN-α but highly responsive to LPS and IFN-γ. In contrast, ACE2L and another key gene, AGT, in 740 RAS were categorized together with other non-ISGs (B)

Epigenetic regulation of non-ISGs such as IL-6 and ACE2 was sequentially regulated 745 by such as TNF, IFN and TLR signaling, which modify chromatin accessibility through activating 746 histone modification and recruitment of transcription factors including PU.1, IRF and NF-κB binding 747 on promoter regions of IL-6 and ACE2 genes. In turn, it will amplify inflammatory loop through IL-748 6-mediated response and inducing more ACE2 expression, which collectively contribute to the 749 occurrence of respiratory distress syndrome as in COVID-19. Therefore, high expression of non-ISGs 750 such as IL-6 and ACE2 could be biomarkers to determine COVID-19 susceptibility and disease 751 development in different animal species. Abbreviations: non-ISG, non-canonical interferon 752 stimulated genes

TLR, toll-753 like receptor

 Figure 8 : Genome-wide categorizing of non-ISGs based on the similarity of inductive pattern to IL-6 and ACE2 genes. The non-biased genome-wide transcriptomic data was generated using RNA-Seq of porcine lung macrophages activated with stimuli of IL-4, IL-10, LPS, IFNα or IFN-γ at 20 ng/ml and infected by porcine arterivirus virus for 5 h, using an Illumina procedure as previously described [57] . Significantly differentially expressed genes (DEGs) in renin-angiotensin system (RAS), interleukin (IL)-6, TNF and chemokine super-families were annotated and grouped using heatmaps according to their inductive expression patterns similar to: (A) IL-6, (B) ACE2; (C) Examples of canonical ISGs as reference; (D) Averaged transcriptomic expression levels (normalized at Reads Per Kilobase of transcript per Million mapped reads, RPKM) of the grouped ISGs or Non-ISGs above. Indicated by arrows, pigs have two ACE2 isoforms, namely ACE2L and ACE2S, which have different expression patterns, ACE2S similar to IL-6 was shown to be less responsive to IFN-α but highly responsive to LPS and IFN-γ. In contrast, ACE2L and another key gene, AGT, in RAS were categorized together with other non-ISGs (B), which is more like the expression pattern of canonical ISGs (C) than the IL-6 group (A). Figure 9 : Working summary for IL-6 and ACE2 as non-ISGs biomarkers and contribution to COVID-19 susceptibility. Epigenetic regulation of non-ISGs such as IL-6 and ACE2 was sequentially regulated by such as TNF, IFN and TLR signaling, which modify chromatin accessibility through activating histone modification and recruitment of transcription factors including PU.1, IRF and NF-κB binding on promoter regions of IL-6 and ACE2 genes. In turn, it will amplify the inflammatory loop through the IL-6mediated response and induce greater ACE2 expression, which collectively contributes to the occurrence of respiratory distress syndrome as in COVID-19. Therefore, high expression of non-ISGs such as IL-6 and ACE2 could be biomarkers to determine COVID-19 susceptibility and disease development in different animal species. Abbreviations: non-ISG, non-canonical interferon stimulated genes; GTF, sTF, or TF, general (G), tissue-specific (s) transcription factor (TF); TLR, toll-like receptor; TSS, transcription start site.