Topoisomerases in Immune Cell Development and Function

The cellular processes such as transcription and replication are epitomized by the unwinding of two strands of DNA. However, this unwinding of DNA strands can promote intertwining of DNA molecules leading to the torsional stress. Topoisomerases (TOPs) are molecular machines designed by nature to resolve topological problems in DNA arising because of strand separation (1, 2). In 1971, the identification and extraction of Escherichia coli ω protein, capable of unwinding negative supercoiled covalent closed DNA, by James C. Wang (3, 4) marks the discovery of TOPs, “true magicians of the DNA world” as he called them. This was followed by the discovery of a “DNA untwisting enzyme” by James Champoux in 1972 from the nuclear extracts of murine embryo cells that was capable of resolving both negative and positive supercoils (5). In 1976, another protein with ATP-dependent DNA TOP activity was discovered in E. coli, and this enzyme was termed DNA gyrase (6). Eventually, the TOPs were divided into two categories: type I and type II TOPs, with ω protein isolated by James C. Wang and “DNA untwisting enzyme” isolated by James Champoux being type I, and DNA gyrase identified subsequently being type II.

The mammalian genome carries seven TOP-encoding genes: four that encode type I TOPs (TOP1, TOP1mt, TOP3A, and TOP3B) and three encoding type II TOPs (TOP2A, TOP2B, and SPO11) (4, 7, 8). Enzymatic activity of TOPs involves a highly reversible transesterification reaction through active-site tyrosine residue resulting in the formation of an enzyme–DNA intermediate, referred as cleavage complex. Type I TOPs unwind the DNA by introducing single-strand nick to allow another strand to pass through the gap. This enzymatic reaction does not require ATP, and type I TOPs use the energy stored in torsional stress of supercoiled DNA. The type I TOPs are further divided into two subgroups, types IA and IB, depending on whether the enzyme is covalently linked to the 5′ (type IA) or 3′ end (type IB) of the nicked DNA. TOP1 is a type IB enzyme that recognizes dsDNA and relaxes both positive and negative supercoils by a mechanism termed as “controlled rotation” (9). TOP1mt is another member of class type IB and is known to maintain the integrity and topology of mitochondrial DNA. TOP1 inhibitors such as camptothecin and its derivatives, topotecan and irinotecan, stabilize TOP1 cleavage complex (TOP1cc) and block the religation step in a reversible manner (10, 11). TOP3A and TOP3B, the two members of the type IA family, facilitate the relaxation of negatively supercoiled, but not positively supercoiled, DNA (Fig. 1A) (7, 12).

In contrast with type I TOPs, type II TOPs form only 5′ intermediates with DNA and require high-energy cofactor and magnesium to catalyze topological changes in DNA. Type II TOPs manifest passage of one DNA double helix through a transient enzyme-bridged break in another to manage DNA tangles and to relax positively or negatively supercoiled DNA (7, 13). Vertebrates and other multicellular eukaryotes have two bona fide type II TOP paralogs, TOP2A and TOP2B, often referred to as TOP2 collectively, which share ∼79% sequence identity in their catalytic core, whereas the C-terminal domain of the two enzymes differs significantly (∼31% identity) (14, 15). It is believed that the C-terminal domain of type II TOPs plays a role in sensing the geometry of DNA substrates with TOP2A preferentially relaxing positive supercoils but display of no such preference by TOP2B (14). Similar to camptothecin, drugs such as etoposide, teniposide, and anthracycline family drugs, including epirubicin, doxorubicin, and daunorubicin, function by stabilizing an intermediate wherein DNA is covalently linked to the enzyme, i.e., TOP2 cleavage complex (TOP2cc) (15, 16). The eukaryotic genome also encodes a third atypical type II TOP or more precisely TOP-like protein, SPO11, that is germ cell specific and is active during meiosis (Fig. 1A) (17).

The dysfunction of TOPs in various eukaryotic model organisms has provided important insights into the essential nature of these enzymes and their exquisite role in tissue homeostasis. For example, Top1 is dispensable in unicellular eukaryotes such as yeast (18–20); however, it is essential for early development in multicellular eukaryotes, including fruit flies and mice (21, 22). On the contrary, Top1mt knockout mice are viable but display a phenotype under stress conditions (23–26). Similar to Top1, deletion of the Top3a gene leads to embryonic lethality in mice (27), whereas Top3b knockout mice do not show developmental defects but display aneuploidy in the spermatocytes and develop autoimmunity (28–30). Similarly, deletion of the Top2a gene results in lethality in mice during embryonic development, whereas Top2b-deficient murine embryos display neural and neuromuscular abnormalities resulting in perinatal death (31, 32). Spo11-null mice are viable but carry defective spermatocytes leading to infertility (Fig. 1B) (17, 33).

It is becoming increasingly clear that the purview of TOPs extends beyond the basic enzymatic function, and they have a role in regulation of tissue homeostasis. However, because of pleiotropic functions of TOPs and embryonic or perinatal death of gene-deficient animal models, it has been challenging to parse the tissue-specific function of TOPs. Studies on nonlethal mutations in TOP genes, mice carrying a conditional TOP allele deletion, and pharmacological intervention of TOP enzymes have shed some light on the tissue-specific role of these enzymes. Indeed, recent findings have indicated that both type I and II TOPs play a key role in postmitotic neurons and immune cells (8, 29, 32, 34–39). In this review, we focus on the immune system–specific requirements of TOPs and their importance in immunological disorders. First, we highlight the importance of TOPs in the development of diverse immune cell populations; second, we discuss the role of TOPs in immune responses.

An initial indication of the importance of TOP function in the immune system was the observation that T cell development is uniquely sensitive to loss of function of TOP3A in zebrafish. Boehm and colleagues (40), while performing a forward genetic screen to identify recessive mutants with thymopoiesis defects in zebrafish embryos, identified two Top3a alleles, HI064 and WW20/12, with abnormalities in thymopoiesis (Fig. 1C). HI064 is a null Top3a allele because it contains a nonsense mutation, and translated protein is predicted to be a truncated version of wild-type (WT) TOP3A, which lacks a significant portion of TOP IA homology domain containing the catalytic tyrosine residue and three zinc fingers. WW20/12 contains a missense mutation in the TOP IA homology domain replacing a hydrophobic residue with a hydrophilic and polar residue very close to three highly conserved amino acid residues critical for contact with the ssDNA substrate. The zebrafish with either HI064 or WW20/12 allele displays indistinguishable thymopoiesis abnormality phenotype, suggesting the missense mutation in WW20/12 allele probably produces nonfunctional TOP3A. Interestingly, in Top3a mutant embryos, analysis of neuronal development in the eye and brain showed little perturbation, indicating that loss of TOP3A did not strongly affect early neurogenesis. However, the mutant zebrafish embryos displayed compaction of the epithelial layer in thymus, which is critical for maturation of thymocytes. Most importantly, although lymphocyte progenitors develop normally and have normal thymic migration in the Top3a mutant embryos, deletion of Top3a significantly impacted the number of thymocytes (40).

TOP1 and TOP2A are also involved in the development of mature T cells through their interaction with a thymic regulator of immunological tolerance, transcription factor autoimmune regulator (AIRE). AIRE drives the expression of a battery of peripheral tissue Ags in medullary thymic epithelial cells (mTECs) and thereby regulates the deletion of self-reactive T cells during development. TOP1 and TOP2A were shown to serve as cardinal partners for AIRE, and these two TOPs regulated the AIRE-directed transcription program. Interestingly, two classes of TOPs had differential and nonredundant functions in AIRE-induced gene expression, where TOP1 primarily functioned with AIRE at superenhancers, while TOP2A interacted with AIRE at the promoter of AIRE-induced genes. The inoculation of mice either with topotecan (TOP1 inhibitor) or etoposide (TOP2 inhibitor) was able to establish autoimmune manifestations similar to those seen in AIRE-less disease, further supporting the role of two groups of TOPs in the development of T cells (Fig. 1D) (41, 42).

Interestingly, although targeted mutation of Top1 and Top2a leads to embryonic lethality (22, 31), mice lacking the Top3b gene develop to maturity with no apparent defects (28). However, Top3b-null mice develop autoimmunity as they age with elevated levels of circulating autoantibodies, a phenotype peculiar of AIRE-less disease (29). Although incremental levels of autoantibodies in Top3b-null mice were attributed to apoptotic cells in thymus, an AIRE-dependent role of TOP3B in T cell development and maturation cannot be ruled out (Fig. 1E).

A clear demonstration of the need for TOP activity in B cell development has come from inherited human syndromes. Studies aiming to identify the genetic defects in the patients with reduced or absent B cells and hypogammaglobulinemia showed the role of TOP2B in B cell development. Papapietro et al. (43) and Broderick et al. (44) independently identified dominant loss-of-function mutations in Top2b in five families. They demonstrated that the mutations in Top2b cause syndromic B cell immunodeficiency, namely, Hoffman syndrome and BILU (B cell immunodeficiency, limb anomalies, and urogenital malformations), and they suggested that these mutations underlie the defects in B cell development and B cell activation in response to Ag stimulation (43, 44). Along similar lines, another clinical study recently reported absolute B cell deficiency in a 13-y-old patient with craniofacial and limb abnormalities, and the patient carried Top2b mutations (45). Patients with Top2b mutations have a distinct lack of CD19⁺ B cells in the bone marrow, suggesting a block in early B cell development and making this immunodeficiency different from other B cell immunodeficiencies (Fig. 1F). However, these patients have a minor fraction of B cells and detectable levels of Igs circulating in the peripheral blood, suggesting that B cells in the patients are not entirely lost and hinting toward a leaky block in the development of B cells. Most importantly, the defects in Top2b led to specific inhibition of B cell development, with peripheral T cells remaining unaffected despite the fact that both T and B cells originate from a common lymphocyte precursor. Interestingly, a significant number of patients with Hoffman syndrome exhibited a reduced number of NK cells in blood, and we will discuss this observation in the section NK cells. Most importantly, the immunodeficiency in patients with Top2b mutations was restricted to lymphocyte lineage with myeloid lineage not being affected (43, 45). Based on these observations, the International Union of Immunological Societies’ Inborn Errors of Immunity Committee (https://iuis.org/committees/iei/) has recently included mutations in the Top2b gene as one of the genetic causes for Ab deficiency in humans, and Top2b can be a candidate gene for screening mutations in patients demonstrating B cell deficiency (46).

The mutations in patients with Hoffman syndrome and BILU syndrome were reported at conserved residues in the TOPRIM (TOP-primase) domain of TOP2B. The TOPRIM domain of TOP2B is part of the DNA gate that catalyzes DNA cleavage and religation, and it is essential for catalytic activity of TOP2B. Interestingly, the Top2b mutations led to lower intrinsic enzymatic activity and reduced stability of the protein, potentially precipitating Top2b-null phenotype in the patients. Most importantly, these patient mutations create the dominant-negative form of TOP2B, which contributes to disease phenotype rather than Top2b haploinsufficiency (44).

Interestingly, although deletion of Top2b in murine B cell lineage affected B cell numbers in spleen and bone marrow, the extent of developmental deficiency was milder when compared with patients (Fig. 1F). The stronger effect on development of B cells in patients can be ascribed to the dominant-negative effect of the mutation on the functioning of TOP2B derived from the WT allele.

Although TOP2B plays a key role in development of B cells, TOP1 has been demonstrated to play a role in Ag-dependent Ab maturation in B cells. On Ag stimulation, the Ig locus undergoes two distinct genetic modifications to generate Ab diversity: class-switch recombination (CSR) and somatic hypermutation (SHM). Activation-induced cytidine deaminase (AID) is an enzyme that mediates CSR and SHM by promoting DNA cleavage in the switch and variable regions of the Ig locus, respectively (47). It was shown that dynamic regulation of TOP1 expression is responsible for AID-dependent CSR and SHM, and increased expression of AID correlated with reduced TOP1 levels in the cell (48, 49). Small interfering RNA (siRNA)-mediated gene silencing of Top1 promoted AID-induced DNA cleavage, CSR, and SHM. Similarly, Top1 haploinsufficiency in mice led to increased frequency of SHM in B cells. Interestingly, SHM augmentation was dependent on transcription because inhibition of transcription had a negative impact on SHM, even in cells with Top1 knockdown (KD). It was proposed that reduced expression of TOP1 protein promotes transcription-induced non-B DNA structure formation, which offers sites for irreversible cleavage by TOP1, and this DNA cleavage augments CSR and SHM. Interestingly, poisoning of TOP1 by camptothecin, which stabilizes TOP1cc, inhibits both CSR and SHM, suggesting that the clearance of TOP1–DNA complexes is also critical for CSR and SHM (Fig. 1G).

Similar observations were made in a mouse B lymphocytic leukemia cell line P388/CPT45 that displays aberrant expression of Ig on the cell surface, and it was attributed to insufficient expression of TOP1 (49, 50). P388/CPT45 cells do not express endogenous AID, but it was observed that exogenous expression of AID in this TOP1-insufficient line led to increased SHM frequency, whereas overexpression of human TOP1 resulted in reduced SHM. Proteomic analysis of TOP1-associated proteins in this B cell line pointed toward a key role of SMARCA4, an ATP-dependent chromatin remodeler, in recruitment of TOP1 on chromatin because the B cell line with Smarca4-KD phenocopied the effects of Top1-KD on SHM (51). These results further highlight the regulatory role of TOP1 protein levels in the process of Ab maturation in B cells.

NK cells are at the crossroads of innate and adaptive immunity (52). Akin to B cells, the evidence for the role of TOPs in NK cell development and function has come from research on patients with immunodeficiency. Two recent studies reported that a significant proportion of patients suffering with Hoffman syndrome and BILU syndrome had lower frequencies of NK cells, and these patients carried loss-of-function mutations in the Top2b gene (Fig. 1H) (44, 53). However, unlike B cells, the effect of Top2b mutations on NK cell development was much more variegated, with a fraction of patients having normal NK cell frequencies and no patient displaying complete loss of NK cells. Most importantly, defects in NK cells were much more common in the patients with Hoffman syndrome in comparison with the patients with BILU syndrome, further highlighting the differential role of specific TOP2B residues in NK cell development and function. Interestingly, the study reported three patients with BILU syndrome, and all three patients carried A485P mutation in TOP2B; however, only one of the patients displayed significantly lower frequencies of NK cells. This observation suggests the contribution of additional factors in NK cell defects observed in patients with BILU syndrome.

Surprisingly, induced pluripotent stem cells (iPSCs) from healthy donors and patients with Hoffman syndrome displayed similar NK cell differentiation potential in vitro. However, iPSC-derived NK cells from Hoffman syndrome patients had impaired cell-mediated cytotoxicity toward cancer cells. These results suggest that Top2b mutant iPSCs have the potential to differentiate into NK cells, but the differentiated cells lack the functional attributes of NK cells (53).

As observed in patients with Hoffman syndrome, mice carrying a mutation in the Top2b gene showed fewer mature NK cells compared with WT counterparts, and these cells had limited cytotoxic killing activity toward cancer cells. Interestingly, TOP2B controlled the expression of transcription factors Nfil3, Ets1, and Id2 that are known to regulate the NK cell development (Fig. 1H) (53). Overall, these results highlight a previously unknown role of TOP2B in NK cell differentiation and function.

Although the role of TOPs in the development of innate immune cells still needs to be explored, TOPs are known to play key roles in the functioning of innate immune cells. Various lines of evidence suggest that TOPs regulate innate immune responses. However, there is significant granularity in their precise role in the regulation of immune responses across innate immune cell types.

The use of pharmacological inhibitors to delineate the role of TOPs in these cells has been highly productive and has paved the way for discovery of their novel roles in the functionality of innate immune cells. Analysis of antineoplastic drugs, including a TOP2 poison doxorubicin, was found to have a marked effect on the maturation of human dendritic cells (DCs). Inhibition of TOP2 by doxorubicin, at ultra-low noncytotoxic concentrations (10 nM), led to a marked increase in the expression of costimulatory molecules such as CD40 and CD83 on human DCs (54). Interestingly, the ability of doxorubicin to induce maturation of human DCs did not enhance its T cell activation potential in mixed lymphocyte reaction assay (54). On similar lines, TOP1 inhibitor camptothecin and its analogs topotecan and irinotecan induced maturation of murine bone marrow–derived DCs with significantly elevated surface expression of MHC class II, CD40, and CD80 on these cells. Interestingly, as observed with TOP2 inhibitors, TOP1 inhibitors did not augment the capacity of bone marrow–derived DCs to activate naive T cells in mixed lymphocyte reaction assay (55). These results bring forward an important point that TOP1 and TOP2 inhibitors induce partial maturation of DCs with the functional hallmark of DC maturation, T cell activation potential, not being affected.

On the other end of the spectrum, chemical inhibition of TOP1 suppressed the proinflammatory immune response against pathogenic infections at cellular and organismal levels. Rialdi et al. (56) performed a chemical screen for innate immune system–intrinsic regulators of the transcriptional response to pathogens and observed an inhibitory activity of TOP1 inhibitor, camptothecin, on the expression of proinflammatory genes. Notably, inhibition of TOP1 activity compromised the inflammatory immune response against a variety of viral and bacterial pathogens and their products. Most importantly, the repression of proinflammatory gene expression was not due to camptothecin-mediated stabilization of TOP1cc or induced cell damage because siRNA-mediated depletion of Top1 phenocopied the attributes of TOP1 inhibition, and the effect of TOP1 inhibition on inflammatory genes was reversed by drug washout. Interestingly, in vivo administration of camptothecin alleviated lethality in a murine model of sepsis, highlighting the therapeutic potential of TOP1 inhibition for diseases characterized by exacerbated innate immune responses (56). In fact, a recent study by Ho et al. (57) proposed the need of evaluating the efficacy of a TOP1 inhibitor, topotecan, for severe coronavirus disease 2019 in humans as therapeutic treatment, with two doses of topotecan in animal models suppressing lethal inflammation induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Similarly, camptothecin was shown to inhibit inflammatory cytokine production by LPS- or IFN-γ–stimulated microglial cells, and TOP1 inhibition by topotecan mitigated neuroinflammation in mice and ameliorated disease pathology in a murine model for experimental autoimmune encephalomyelitis (58). On the contrary, deletion of TOP1 in excitatory neurons led to intense IBA1 immunostaining in the somatosensory cortex indicative of extensive neuroinflammation in these conditional Top1 knockout mice. These results suggest that TOP1 may control neuronal health to limit neuroinflammation (36). It is possible that TOP1 plays a different role in steady-state versus inflammatory conditions, and more studies toward this direction are needed to fully comprehend the function of TOP1 in health and immune-mediated diseases.

In contrast with TOP1, the role of type II TOPs, TOP2A and TOP2B, in innate immune responses has been debatable. The use of different infection and cellular models may explain the discrepancy in results across studies. Rialdi et al. (56) reported that loss of function of TOP2A and TOP2B by siRNA-mediated knockdown or by chemical inhibition does not phenocopy the effect of TOP1 inhibition on the expression of proinflammatory genes on viral infection in A549 cells. On the contrary, type II TOPs have been found to be important for proinflammatory cytokine production by macrophages, as well as for innate immune responses in murine models. Etoposide treatment led to a decrease of LPS- or IFN-γ–induced TNF-α production by IC-21 cells (macrophage cell line). However, it was also observed that perturbation in the expression of IL-6 on treatment with etoposide varied with type of stimulation. Whereas etoposide reduced LPS-stimulated production of IL-6, it augmented IFN-γ–induced expression of IL-6. These results highlight a differential role of type II TOPs in the transcriptional circuitry established on LPS or IFN-γ stimulation of macrophages. Interestingly, inhibition of type II TOPs with etoposide in NMRI mice led to an increase in numbers of monocytes and granulocytes, but when splenocytes from these etoposide-treated mice were stimulated with Toxic shock syndrome toxin-1 and Con A, they displayed reduced capacity to produce inflammatory cytokines (IL-6 and TNF-α) (59). Similarly, anthracycline family drugs, including epirubicin, doxorubicin, and daunorubicin, which are known to inhibit type II TOPs, alleviated proinflammatory cytokine production from human monocytic cell line THP1 in response to E. coli challenge. A similar disease-ameliorating effect of epirubicin was observed in the cecal ligation and puncture (CLP) model of sepsis (60).

Other interesting evidence for the role of type II TOP, TOP2A, in innate inflammatory responses comes from a murine model for sepsis-induced acute lung injury (ALI). Mice with ALI had elevated expression of TOP2A, whereas levels of miR-125b-5p were downregulated. It was demonstrated that miR-125b-5p acts as an inhibitor of TOP2A expression. Interestingly, augmented miR-125b-5p expression led to a reduction in levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 and improved lung pathology in a mouse model with ALI. However, TOP2A overexpression deflated miR-125b-5p–mediated health benefits, suggestive of a critical role of TOP2A in inflammatory disease attributes of sepsis (61).

Furthermore, TOP1 and TOP2 are involved in the maintenance of macrophage identity and functioning of these cells through their interaction with Speckled Protein 140 (SP140). SP140 is an immune-restricted speckled protein with major activity in macrophages and mature B cells, and mutations in SP140 have been implicated in Crohn’s disease (62–65). Perturbation of interaction between SP140 and TOP1/TOP2 led to de-repression of TOP activity at developmentally silenced genes in macrophages. Interestingly, functional dysregulation of macrophages because of loss of SP140 could be rescued by inhibition of TOP1 and TOP2, hinting toward the role of TOPs in maintaining the fidelity of macrophage cell lineage and promoting innate immune responses (66). Altogether, these observations bring forward the potential of TOP1 and TOP2 inhibition as an effective host-directed therapy to limit lethal systemic inflammation during broad-spectrum pathogenic infections and other inflammatory disorders.

TOP1 and TOP2 have also been implicated in invasive cytosolic DNA sensing, an integral component of innate immunity. Zhao et al. (67) and Wang et al. (68) independently demonstrated the key role of TOP1 and type II TOPs in cyclic cGMP-AMP synthase (cGAS)-mediated sensing of cytoplasmic chromatin. cGAS is a pattern recognition receptor that recognizes cytoplasmic chromatin fragments during senescence to mount an inflammatory immune response. It was shown that stabilization of TOP1cc or TOP2cc in tumor cells enhanced the binding of cGAS to dsDNA and induced innate immune signaling to promote expression of chemokines such as CCL5 and CXCL10 and type I IFNs including IFN-β (67, 68). This in turn led to activation of DCs and T cell recruitment at the site of tumor. Inhibition of type II TOPs by teniposide also promoted upregulation of the Ag presentation machinery on tumor cells, including enhanced expression of MHC class I, MHC class II, β2-microglobulin, Transporter associated with antigen processing-1 and -2 that further promoted T cell activation against tumor and helped in tumor clearance (68). These results highlight an indirect role of TOP inhibitors in tumor regression via stimulatory activity toward innate immune sensing, enhancing Ag presentation capabilities, and setting up the events for tumor infiltration of other immune cells.

In summary, both type I and II TOPs play diverse roles in innate immune responses. On one hand, TOP inhibitors drive partial maturation of DCs; on the other hand, inhibition of TOPs restrains inflammatory cytokine production in various cellular and animal models. However, current studies also highlight a challenge- and gene-dependent role of TOPs during inflammatory response. This is exemplified by the differences in perturbation of IL-6 levels on inhibition of type II TOPs when cells are challenged with either LPS or IFN-γ. Similarly, TOP1 also appears to regulate innate immune responses differently in steady-state versus inflammatory conditions. Table I summarizes various roles of TOPs in the functioning of the innate immune system.

Since their discovery in 1971, TOPs have garnered tremendous interest, and evidence gathered over the past two decades underscores the tissue-specific roles of these enzymes. In this review, we aimed to bring attention to the possible involvement of these enzymes in the development and function of immune cells. TOPs are associated with immunodeficiencies, autoimmune disorders, and inflammatory diseases (29, 40, 56, 58, 61), highlighting their unappreciated role in the maintenance of immune homeostasis and the regulation of immune responses. We have just started to learn about their role in the adaptive immune system with studies pointing toward their function in thymopoiesis and the development of B and NK cells (40, 43, 44, 53). However, insights into their role in the development of innate immune cells are still lacking. Nonetheless, we have begun to appreciate the role of TOPs in innate immune responses, and these enzymes may regulate transcriptional programming of innate immune cells, such as macrophages and DCs, during pathogenic challenge (54–56).

Although the immunological underpinnings of TOPs are becoming increasingly evident, we still have a lot to learn. An important aspect that is largely unexplored is the mechanism of TOPs in the regulation of immune cell differentiation and function. Existing reports on the role of TOPs in neurons indicate gene length–dependent activity (38). Similar observations were made by Broderick et al. (44) for TOP2B in B cells, where ablation of the Top2b gene in murine B cells led to reduced expression of key B cell–specific transcription factors that are notably longer (Ebf, 390 kb; Pax5, 178 kb; and Foxo1, 85 kb). Interestingly, recent studies also reveal a dynamic interplay between TOPs and chromatin in immune cells. Rialdi et al. (56) found that 75% of TOP1-dependent immune response genes required switch/sucrose nonfermenter–dependent nucleosome remodeling for transcriptional activation. In parallel, TOP1 was found to interact with SMARCA4 (also referred as BRG1), which is the ATPase subunit of switch/sucrose nonfermenter complex in B cells (51). We previously demonstrated the occupancy of TOP1 at H3K27ac-marked superenhancers in mTECs (42). These observations are suggestive of an epigenetic layer in the molecular mechanism of TOPs. It is possible that there is a dysregulation of cross-talk between TOPs and chromatin remodeling machinery in immunological disorders, and this axis can be harnessed to design new putative therapies for these diseases.

Another important mechanistic facet of TOPs is their role in genome organization. TOP2B was recently demonstrated to interact with chromatin architectural proteins CCCTC-binding factor and the subunits of cohesin complex that regulate enhancer–promoter interaction and the formation of chromatin loops (69). Interestingly, chromatin loop anchors bound by CCCTC-binding factor and cohesin are vulnerable to TOP2B-mediated DNA double-stranded breaks in murine B cells, and these DNA breaks may help in clearing up intertwined DNA that builds up during chromatin loop formation (70). Thus, TOP2B might control the genome organization, and this regulatory axis may drive the cellular phenotypes observed in immune cells. Because the toolkit of TOPs is expanding, it will be important to know whether different tools are used across diverse immune cells, and this variegation can be used to therapeutically target TOPs in a specific immune cell type.

The versatility of TOPs and their ease for targeting through drugs makes them a viable molecule for translational research. Several TOP poisons are used in cancer chemotherapy. TOPs have also recently gained traction as the “go to” target molecule for diseases such as rheumatoid arthritis, SARS-CoV-2–induced lethality, and sepsis. Thus, understanding the role of TOPs in immune cell development and immune responses becomes essential to design drug regimens with the least possible adverse consequences. This knowledge will also aid in developing better therapeutic regimens for regression of cancers because TOP-directed chemotherapy may impact gene expression programs in immune cell populations and can have an indirect impact on cancer. Overall, further insights into the immunological role of TOPs will likely have a significant impact on the well-being of the human race.

We could not include the exciting work of many scientists working in the field of TOP biology because of space constraints, and we apologize for that. (Fig. 1 in this review was created with BioRender.com. We thank Harshdeep Kaur, Pallawi Choubey, and Vanshika Sood for critical comments on the manuscript.

The authors have no financial conflicts of interest.