**4. Epigenetic regulation in relation to Covid19**

#### **4.1 Role of epigenetic mechanisms in the induction of cytokine storm**

As detailed in the previous section, epigenetic regulation plays a significant role during viral infections. Viruses of the *Coronaviridae* family that previously caused MERS (Middle East respiratory syndrome, MERS-CoV) and SARS (SARS-CoV) have previously been shown to dysregulate the host immune system by inducing epigenetic changes that antagonize antigen presentation or activate interferonstimulated genes (ISGs) [69, 70]. These viruses have also been implicated in blocking pathogen recognition and immune system signaling [71]. Due to this tight link with the host immune response, patients suffering from infections of coronaviruses, including SARS-CoV-2, are characterized by an abnormal induction of acute inflammation, namely cytokine storm. The excessive secretion of proinflammatory cytokines and recruitment of immune cells at the site of infection often leads to tissue damage and organ failure, which are hallmarks of Covid-19-related deaths [72].

The transcriptional regulation of cytokine production is under tight control of epigenetic mechanisms. Promoters of interferons (IFNs), tumor necrosis factors (TNFs) and ISGs that are drastically upregulated in Covid-19 patients are enriched by histone marks of open chromatin in activated macrophages and dendritic cells [71, 73, 74]. In addition to the common histone modifications, Covid-19 patients exhibit elevated levels of arginine citrullination on histone H3 [75]. Citrullination, which is a marker of a specific type of immune response to infection, namely neutrophil extracellular traps (NETs), is associated with chromatin decondensation and transcriptional activation [76]. Induction of NETosis is hypothesized to lead to sustained inflammation during SARS-CoV-2 infection and the subsequent cell death due to cytokine storm [77].

#### **4.2 Regulation of SARS-CoV-2 entry-associated factors by epigenetic mechanisms**

The novel coronavirus SARS-CoV-2 interacts with and requires the action of multiple host proteins for viral entry. Spike (S) protein, which is anchored into the viral envelope, binds to angiotensin converting enzyme 2 (ACE2) on the host cell surface [78]. ACE2 is a membrane protein found in a wide variety of cell types. The interaction between ACE2 and the receptor binding domain (RBD) within the S1 subunit of the spike protein initiates entry, while S2 subunit triggers direct membrane fusion or endocytosis upon cleavage and activation by host proteases FURIN and TMPRSS2 [79]. Two members of the cathepsin family, namely CTSB and CTSL are also involved in the viral glycoprotein processing and the fusion between viral and endosomal membranes [80].

Among all SARS-CoV-2 entry-associated host factors, ACE2 is the best characterized protein in terms of epigenetic regulation. ACE2 is located on the X-chromosome, which typically gets heterochromatinized and undergoes X-inactivation in females to achieve dosage compensation. In line with this, higher ACE2 expression was observed in males than in females, accompanied by marks of open chromatin [81]. The heterozygosity of ACE2 alleles, hence the lower levels of ACE2 expression in females is considered as a significant advantage in counteracting SARS-CoV-2 infection [82]. However, X-inactivation is often incomplete, and a significant proportion of X-linked genes, including ACE2, escape silencing [81]. Therefore, ACE2 seems to show a rather heterogeneous sex bias [83].

Several epigenetic factors such as DNA methyltransferase DNMT1, histone acetyltransferases p300 and HAT1, histone deacetylases HDAC2 and SIRT1, histone methyltransferase EZH2 and histone demethylase KDM5B have been reported as potential regulators of ACE2 expression [84, 85]. Accordingly, histone marks H3K27ac, H3K27me3, H3K4me1 and H3K4me3 were detected within the ACE2 locus. Furthermore, studies have shown that ACE2 is under tight regulation of DNA methylation. In all tissues tested, lung epithelial cells exhibited the lowest levels of DNA methylation in ACE2 promoter, which positively correlated with high expression [86]. It was also claimed that the CpG methylation pattern of ACE2 promoter is associated with age and gender, suggesting a possible explanation for increased mortality in elderly men during SARS-CoV-2 infection [84, 86].

Other SARS-CoV-2 entry-associated factors are subject to epigenetic regulation as well. A recent study identified a regulatory region upstream of FURIN gene that is heavily occupied by the histone acetyltransferase p300 in T cells [87]. Also, DNMT1-mediated hypermethylation of TMPRSS2 was associated with its downregulation [88]. Moreover, loss of DNA methylation was implicated in increased levels of CTSL/CTSB in pancreatic adenocarcinoma, which could cause greater susceptibility to SARS-CoV-2 infection [89]. In accordance with this finding, silencing of CTSL/CTSB was shown to inhibit SARS-CoV-2 replication and virally induced apoptosis [90]. Additionally, significant hypomethylation of CTSL promoter was observed in chronic myeloid leukemia [91].

#### **4.3 Interaction between the host epigenetic factors and viral proteins**

Interactome analysis of SARS-CoV-2 proteins has provided experimental evidence of physical interaction between several viral proteins and human factors, implicating them in a variety of cellular processes such as epigenetic regulation of gene expression, RNA processing, DNA replication, trafficking and transport of proteins, mitochondrial function, cellular structure, and cell signaling pathways [92]. Viral envelope protein E interacts with bromodomain proteins BRD2 and BRD4 via its C-terminal end that mimics the N-terminal tail of histone H3. As specific binders and readers of histone acetylation, bromodomain-containing proteins are associated with transcriptional activity [93]. By disrupting BRD2/4 binding to histone H3, protein E can induce genomic alterations that affect host gene expression. Another inhibitory link with histone acetylation was established between

*Epigenetic Regulation Mechanisms in Viral Infections: A Special Focus on COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.98866*

Nsp5 and HDAC2, which could potentially influence the host immune response against SARS-CoV-2. HDACs are commonly classified as transcriptional repressors since their main task is the removal of histone acetylation, a mark of active chromatin. However, HDAC2 plays an activating role during the transcriptional elongation of ISG expression via regulating BRD4 availability at newly activated promoters [94]. Similarly, Nsp8 was identified as a binding partner of histone lysine methyltransferase NSD2, which sets H3K36me3 at the gene bodies of actively transcribed genes [95]. H3K36me3 is suggested as an epigenetic mark of transcriptional memory in ISGs, indicating another layer of innate immune response regulation [96]. Viral proteins Nsp13 and Orf10 interact with ubiquitin specific peptidase USP13 and the components of the Cullin-RING E3 ubiquitin ligase complex, respectively. USP13 has previously been attributed significant immune response-related roles in interferon-induced signaling by STAT1 targeting and deubiquitination [97] and increased immune cell infiltration in several types of cancers [98]. Interestingly, USP13 antagonizes antiviral response via ubiquitination of STING, an important effector of innate immune signaling in response to viral infections [99]. Likewise, Cullin-RING E3 ubiquitin ligase complex members are often hijacked by viruses, inducing the proteasomal degradation of host restriction factors, and promoting viral replication [100]. Nsp13 also interacts with TLEs and TBK1/TBKBP1 proteins which are modulators of NF-kB-dependent inflammatory response and IFN signaling [101].

The list of interactions between SARS-CoV-2 and the epigenetic factors of the host cell that are based on experimental evidence has also been extended by in silico approaches that identified p53 as a binding partner of spike (S) protein [102] and several human miRNAs targeting SARS-CoV-2 transcripts [103]. Conversely, an interplay between SARS-CoV-2 miRNAs and the immune signaling pathways of the host was suggested, which could contribute to the prolonged latency of the virus leading to asymptomatic individuals.

#### **4.4 (Epi)genetic susceptibility to Covid-19**

Certain risk groups have been associated with increased susceptibility and disease severity since the emergence of the SARS-CoV-2 outbreak. Age is one of the main risk factors for Covid-19, as evident by its high occurrence and mortality rates in elderly patients [104]. Epigenetic machineries often become defective during the process of aging as well, which results in increased genomic instability, altered gene expression profiles and loss of resilience [105]. These age-related epigenetic changes could hamper the activation of innate and adaptive immune responses, which could also be manipulated by viruses to evade host repression. Coronaviruses have previously been linked with accelerated rate of host immune system aging through epigenetic mechanisms such as DNA methylation and transcriptional silencing that impede with host antigen presentation and the expression of major histocompatibility complexes [70]. Moreover, age-dependent fluctuations in the levels of glycosylation and NAD+, which have epigenetic associations, are implicated in predisposition to SARS-CoV-2 infection [106, 107].

There is a growing body of evidence pointing towards the role of DNA methylation in Covid-19 severity. Analysis of genome-wide DNA methylation profiles of severe COVID-19 cases revealed increased methylation of IFN-related genes while inflammatory genes were hypomethylated [108]. Likewise, a genome-wide association study identified a total of 44 CpG sites, most of which were located to coding genes including the components of the inflammasome complex and the major histocompatibility complex HLA-C as potential markers of COVID-19 severity and respiratory failure [109]. Furthermore, in lupus patients, loss of DNA methylation

in ACE2 and interferon/cytokine-regulated genes, together with enhanced NF-kB expression were defined as contributors of severe COVID-19 [110]. Lastly, SARS-CoV-2 can demethylate and activate the expression of Syncytin-1 and Syncytin-2 genes of the host that are required for the creation of giant multinucleated cells, a process known as syncytium formation [111]. Syncytin genes are normally methylated and silenced during development, except for the mammalian placenta, where induction of multinucleated cells provides tissue impermeability in aid of immune tolerance between mother and child [112]. Syncytium formation followed by extensive cell death is suggested as an underlying cause of the detrimental effects of cytokine storm in COVID-19 patients [113].
