TET Proteins in the Spotlight: Emerging Concepts of Epigenetic Regulation in T Cell Biology

The ten-eleven translocation (TET) family of proteins consists of three proteins: TET1, TET2, and TET3. TET proteins are Fe²⁺- and O₂-dependent dioxygenases that recognize 5-methylcytosine (5mC) and oxidize it to generate 5-hydroxymethylcytosine (5hmC) (1). TET proteins can further catalyze the oxidation of 5hmC to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (2, 3). The discoveries of these oxidized forms of cytosine took place relatively recently, from 2009 to 2011. However, during the past decade, multiple research articles have placed TET proteins in the spotlight, as critical epigenetic regulators that shape development of various cell lineages (4). Among the cell types that have been shown to be regulated by TET proteins are the T cells.

The process of T cell lineage commitment and specification is tractable, well defined, and takes place in the thymus. There, double-positive (DP) cells, which comprise the vast majority of thymocytes, will be stringently selected to give rise to functionally specialized subsets that will migrate to the periphery to execute their immunologically complex functions (5). Specifically, DP cells that too strongly recognize Ags will be eliminated by negative selection (6). The vast majority of DP cells do not recognize Ags and will be eliminated by death by neglect (7). Only those DP cells that exhibit optimal recognition and response after Ag presentation will be positively selected (8). The DP cells that recognize peptide Ags presented by MHC class I molecules will upregulate the transcription factor RUNX3 and will differentiate to become CD8 single-positive (SP) cells. DP cells that recognize peptide Ags presented by MHC class II molecules will give rise to CD4 SP cells. The commitment to the CD4 cell lineage is governed by GATA-3, whereas the maturation process of CD4 SP cells is defined by the lineage-specifying factor ThPOK (9). Upon reception of TCR signals of intermediate strength, some CD4 cells will undergo further differentiation to give rise to regulatory T cells (Tregs) (6, 10). The transcription factor FOXP3 orchestrates Treg development and immunosuppressive function (11, 12).

In addition, some DP cells will be selected through lipid Ag recognition presented by an MHC class I–like molecule known as CD1d (13). These positively selected cells will upregulate PLZF and will differentiate to invariant NKT cells, also known as iNKT cells (14). iNKT cells are also known as unconventional T cells as they recognize lipid Ags, and they express an invariant TCRa chain and an oligovariant TCRβ-chain (13). Importantly, the iNKT cells develop in a preactivated state in the thymus, and they can potently secrete cytokines (15, 16).

In the periphery, CD4 cells differentiate to helper lineages such as Th1, Th2, Th17, and Th9 cells that express distinct lineage-specifying transcription factors and secrete different cytokines (17). Besides the thymically derived Tregs, CD4 cells can differentiate in the periphery to give rise to inducible regulatory T cells (iTregs) (18). Similarly, naive CD8 cells will migrate in the periphery and upon Ag encounter will differentiate to give rise to short-lived effector CD8 cells and memory CD8 cells (19). The advent of genomics and single-cell technologies revealed an unexpected diversity among peripheral populations of T cells (20). Specifically, some of these cells exhibit stem cell–like properties, allowing them to differentiate and give rise to new cells fit to battle Ags (viruses or altered self, such as tumors), whereas other cells upon continuous Ag exposure lose this potency and become terminally exhausted/dysfunctional, unable to combat deleterious threats for the organism (19).

The process of lineage specification has been extensively studied, and the major transcription factors involved in the process have been defined (21). However, the advent of genomics and, lately, single-cell analysis have revealed an unprecedent complexity in the regulation of gene expression (22–29). The systems biology approach demonstrates complex interactions and cross-talk among epigenetic modifications, chromatin accessibility, and conformation that cooperatively affect transcription factor binding and ultimately transcription to regulate gene expression (30, 31).

In this study, we will focus on the TET family of proteins to investigate how epigenetic regulation can impact T cell lineage specification, function, and proliferation. We will summarize the current knowledge in the field, define open questions and next steps, and reflect on how these findings can be translated to shape T cell biology.

Initially, the focus was to understand how 5hmC is distributed across the genome. Studies revealed that 5hmC is highly enriched across the gene body of very highly expressed genes, including genes that encode for T cell lineage–specifying factors such as Zbtb7b that encodes for ThPOK, Runx3 that encodes for RUNX3, Tbx21 that encodes for T-bet, and Gata3 as well as important regulators of conventional and unconventional T cell biology such as Bcl11b and Satb1 that encode, respectively, for BCL11B and SATB1 (32). In addition, 5hmC marks active enhancers in T cells (32, 33) and coexists with open chromatin marks and marks of active transcription (32, 34, 35). These early studies made clear that enrichment of 5hmC frequently correlates with increased gene expression. Moving forward, the next big question was to decipher the functional implications of 5hmC. To this end, various groups have used mouse genetics to dissect the contribution of TET proteins and 5hmC in T cell biology. Further work focused on regulation of specific genes in distinct T cell subsets has revealed critical roles of TET proteins and 5hmC in regulating gene expression and function.

It has been shown that DNA demethylation and deposition of 5hmC at enhancers of genes during thymic development can impact the stability of gene expression at later developmental stages (36–40). Indeed, extensive work from various groups has established that TET-mediated DNA demethylation at the CNS2 locus of the Foxp3 gene is fundamental to maintain stable FOXP3 expression and suppressive function of Tregs (Fig. 1). Deletion of either Tet2 or both Tet2 and Tet3 at the DP stage using CD4-cre (41) mice showed that although Tregs were developed, they were unstable. This finding was attributed to reduced expression of Foxp3 due to aberrant cytosine methylation of the CNS2 locus (36, 37). Importantly, another study revealed that hydrogen sulfide (H₂S) deficiency resulted in reduced expression of Tet1 and Tet2 and subsequent defective cytosine demethylation of the CNS2 locus of Foxp3. This led to unstable FOXP3 expression, defects in Treg maintenance and function, and, ultimately, autoimmunity (38). Collectively, these studies showcased how TET proteins, by demethylating the CNS2 locus, maintain the stability of Foxp3 gene expression and the optimal function of Tregs.

Similar findings have been reported for the expression of the Cd4 gene (39, 40). Specifically, concomitant deletion of Tet1 and Tet3 using RORc-cre mice resulted in hypermethylation in cytosines of a newly identified enhancer, E4m. As a consequence, Tet1/3-deficient CD4 cells gradually showed reduced CD4 expression in culture (39). Deletion of another cis-regulatory element, E4a, established a critical role of this element for maintaining Cd4 expression in vivo in effector CD4 cells (40). Mechanistically, DNA demethylation during thymic development by TET proteins is required for proper licensing of E4a via E4m. The licensing of E4a maintains Cd4 expression in dividing effector CD4 cells in the periphery.

Genetic studies using mouse models have shown that deletion of TET proteins in many cases does not result in a simple “on/off” effect on gene expression. Instead, it affects the magnitude of gene expression and impairs the optimal levels of the expression of genes (34, 42). An example is the Zbtb7b gene (42).

Overall, these studies pinpoint that TET proteins, by mediating DNA demethylation of regulatory elements at early developmental stages, safeguard the activity of these elements at later developmental stages to preserve stable and optimal gene expression.

Ablation of one or more members of the TET family of proteins impact the development and function of various T cell subsets. Specifically, deletion of Tet2 impairs Th1 and Th17 cell differentiation (33) and compromises cytokine secretion due to aberrant methylation of enhancer elements (Fig. 1). In addition, TET2 is suggested to regulate DNA demethylation of Th1 and Th17 regulatory elements by cooperating with lineage-specific transcription factors (33). Moreover, deletion of Tet2 revealed skewing of cells toward follicular Th (Tfh) cells in a model of lymphocytic choriomeningitis virus infection (43) (Fig. 1). The authors proposed that TET2 is recruited by FOXO1 and RUNX1 at specific genomic loci to mediate DNA demethylation and induce expression of genes involved in suppressing Tfh fate (43).

Extensive research has revealed a critical and pleiotropic role of TET proteins in shaping Treg identity and function (Fig. 1). Besides the well-established role of TET proteins in regulating DNA demethylation at the CNS2 locus of Foxp3, thus maintaining the stability of its expression, TET proteins have additional roles after CD4 cells differentiate to Tregs. Specific ablation of both Tet2 and Tet3 at Tregs using Foxp3-cre conditional mice revealed that the Tet2/3-deficient Tregs progressively exhibit an inflammatory phenotype characterized by skewing toward helper lineages, secretion of IL-17, and abnormal proliferation (44, 45).

Deletion of Tet3 at the DP stage using CD4-cre conditional mice results in skewing of iNKT cells toward the NKT17 lineage as assessed by RORγt upregulation (34, 46). Concomitant ablation of Tet2 and Tet3 results in an increase of iNKT cells in the thymus as well as in peripheral organs (34). A significant increase of NKT17 cells was observed. Mechanistically, the observed NKT17 skewing was attributed to reduced expression of ThPOK and T-bet, due to aberrant maintenance of cytosine methylation. In wild-type iNKTs, ThPOK and T-bet suppress RORγt expression to shut down the differentiation toward NKT17 lineage (34, 47) (Fig. 1). Moreover, it was shown that the double-deficient Tet2/3 double-knockout (DKO) iNKT cells exhibited abnormal proliferation that was dependent on CD1d-mediated Ag presentation (34).

In addition, loss of TET2 was shown to promote the formation of central memory CD8 T cells postinfection with lymphocytic choriomeningitis virus (48). It was suggested that TET2 may regulate the expression of transcription factors involved in shaping memory versus effector fate (48, 49) (Fig. 1). Interestingly, upon secondary challenge, Tet2-deficient CD8 cells could more efficiently control the viral load. Along these lines, in a mouse model of melanoma, Tet2-deficient CD8 T cells exhibited enhanced tumor-suppressive properties (50). Molecular analysis demonstrated that loss of TET2 alters the chromatin accessibility landscape of CD8 cells (50). Overall, these studies suggest that deletion of Tet2 in CD8 cells favors the establishment of central memory fate and thus renders CD8 cells more effective in antiviral and antitumor response. Importantly, Tet2-deficient chimeric Ag receptor (CAR) T cells exhibit superior efficacy in a patient with chronic lymphocytic leukemia (51). Multiomic studies demonstrated that Tet2-deficient CAR T cells exhibited an epigenetic landscape that favors establishment of memory fate. Although the above studies suggest that inhibition of TET2 expression could have important therapeutic implications in cancer immunotherapy, the contribution of other members of the TET family in shaping memory fate has not been investigated in detail. However, a recent study reported that deletion of Tet3 delays the emergence of tissue-resident CD8 T cells (52).

A common theme that has emerged from the in vivo studies of Tet loss was that Tet-deficient cells failed to properly mature (34) and demonstrated aberrant proliferation that could not be contained by an intact immune system (34, 44). As TET proteins are frequently mutated in hematological cancers (53), including T cell lymphomas such as angioimmunoblastic T cell lymphoma and peripheral T cell lymphoma not otherwise specified (54–56), it is critical to understand the mechanisms by which these proteins exert their tumor-suppressive function. Moreover, studies of genetically modified mice that lack TET proteins demonstrated a vast array of phenotypes (53, 57), indicating the multifaceted roles of TET proteins in regulating gene expression and, ultimately, T cell function (58, 59). In the following sections, we discuss emerging concepts regarding the mechanisms of TET function.

An emerging question in the field is to dissect the catalytic-dependent versus the catalytic-independent roles of TET proteins in T cell biology. The importance of the TET-mediated DNA demethylation activity is now well established. But can TET proteins, by interacting with other proteins and independently of their enzymatic activity, regulate gene expression? For instance, in embryonic stem cells (ESCs), it has been shown that TET1 can interact with SIN3A to suppress gene expression in a catalytic-independent manner (60, 61). Intriguingly, mice that express full-length TET2 with impaired enzymatic function develop myeloid leukemias but do not develop lymphoid malignancies, suggesting that TET2 nonenzymatic functions may exert tumor-suppressive functions in lymphoid cells (62).

However, recent studies using mice that express full-length TET2 with compromised enzymatic activity showed that catalytic dead TET2 (TET2^CD) could not rescue the aberrant proliferation and the skewing toward the NKT17 lineage of Tet2^CDTet3 KO iNKT cells (42). Moreover, Tet2^CDTet3 KO iNKT cells and Tet2^CDTet3 KO CD4 cells exhibited reduced expression of Zbtb7b comparably to the Tet2/3 DKO-deficient subsets (42). In addition, Tet2^CDTet3 KO CD8 SP cells showed innate-like characteristics, such as upregulation of CD44, CXC3R1, CD122, and Eomes, similarly to the Tet2/3 DKO CD8 SP cells (34, 42). Along these lines, Tet2^CD heterozygous mice develop clonal hematopoiesis when treated with LPS to trigger inflammation (63). Further work in additional T cell subsets will decipher catalytic-dependent versus catalytic-independent roles of TET proteins in regulating T cell differentiation, proliferation, and function.

Cell-specific deletion of TET proteins does not result in massive gain of DNA methylation across the genome (34, 58, 64). Instead, we observe focal gain of cytosine methylation that impacts cell-specific gene expression (34). This specificity suggests that interaction with lineage-specific transcription factors mediates the recruitment of TET proteins at specific loci (Fig. 2). For instance, lineage-specifying factors during B cell differentiation, such as Pu.1 and E2A (65), and also during reprogramming (66) have been shown to interact with TET2 and recruit it across the genome. TET2 was shown to interact with p65 in dendritic cells to mediate DNA demethylation at NF-κB binding sites in dendritic cells upon vitamin C activation (67). However, the transcription factors that mediate TET recruitment across the genome in thymic subsets remain elusive. In peripheral T cells, it was shown that activated SMAD3 and STAT5 mediate recruitment of TET1 and TET2 in Tregs (38). In addition, it was hypothesized that FOXO1 may interact with TET2 to recruit it at genomic loci and mediate DNA demethylation. Similarly, it has been suggested that lineage-specific transcription factors mediate recruitment of TET2 across enhancers to regulate DNA demethylation and expression of cytokines, such as IFN-γ in Th1 cells and IL-17 in Th17 cells (33). Although there are technical challenges, it will be instrumental to decipher the T cell–specific TET interactome to gain insight into the mechanisms by which TET proteins shape T cell identity and function.

In addition, whole-genome bisulfite sequencing studies to assess cytosine modification at the single-nucleotide level revealed that regions that were gaining methylation during iNKT cell development showed enhanced methylation upon deletion of TET2 and TET3 (34). This observation suggests that at least in some loci, TET proteins act in competition with DNA methyltransferases (DNMTs) to maintain optimal methylation levels. Per this analogy, this is reminiscent of the poised status of gene expression maintained by concomitant deposition of permissive (such as H3K4me3) and repressive (such as H3K27me3) histone marks (68). Presumably, the dynamic regulation of methylation ensures keeping gene expression off while enabling rapid upregulation of gene expression when needed. Detailed mechanistic work in ESCs has demonstrated competitive activity of TET proteins and DNMTs to regulate enhancers in both human and mouse ESCs (69). Along these lines, it has been reported that DNMT3A and TET1 compete to regulate promoter epigenetic landscapes in mouse ESCs (70). Mechanistically, it was suggested that TET1 may protect its genomic targets from DNMT3a-mediated methylation (70). This type of detailed analysis is needed in T cell studies. It will be important to establish the competition between TET proteins and DNMTs to tightly regulate levels of cytosine modification across the genome of different T cell subsets and ultimately understand the biological significance of this interplay among proteins with opposing functions.

Importantly, oxidized cytosines are stable modifications (71, 72) that can be recognized by readers and mediate their recruitment to the DNA (73). For instance, 5fC and 5caC can be preferentially recognized by DNA repair proteins (73). In addition, enrichment of 5hmC has been reported at double-strand breaks upon DNA damage and colocalizes with proteins involved in the DNA damage response, such as 53BP1 and γH2AX (74). Loss of TET1 in B cells (64), TET2 and TET3 in iNKT cells (34), and TET1, TET2, and TET3 in hematopoietic stem cells (HSCs) (75) has been reported to result in increase of double-strand breaks. In addition, loss of TET1, TET2, and TET3 results in increased numbers of chromosomal aneuploidies in ESCs (76). Acute deletion of TET proteins in ESCs resulted in reduced expression and increased cytosine methylation of the gene Khdc3 (76). Although Khdc3 expression is restricted at early stages of embryonic development, we cannot preclude that TET proteins exert critical roles in maintaining proper chromosome segregation in hematopoietic populations as well to prevent aneuploidies. Thus, further research is required to decipher the precise contribution of TET proteins in safeguarding genomic stability in T cells.

CTCF binding across the genome is regulated by the cytosine methylation status. Specifically, increased cytosine methylation prohibits its binding, whereas reduced cytosine methylation permits CTCF association with the DNA. Interestingly, increased presence of 5caC can create new CTCF binding cites across the genome (77).

But what are the functional implications? Mechanistically, it has been shown that TET-mediated increased CTCF binding promotes alternative splicing of pre-mRNA (78) by transiently preventing elongation of polymerase II and enabling assembly of spliceosome at weak upstream splicing sites, resulting in the inclusion of alternative exons. TET1 and TET2 by oxidizing 5mC to 5hmC and other oxidized modified cytosines (oxi-mCs) are instrumental for CTCF-mediated alternative splicing (79).

As CTCF is involved in genome topology, it is critical to understand how TET proteins and 5hmC might affect chromatin architecture. For instance, during heart development, it has been shown that YY1 binding is altered due to gain of methylation and loss of chromatin accessibility, resulting in impaired enhancer–promoter interactions that impair expression of genes that are critical for heart development (80). Thus, it is possible that TET proteins and 5hmC (as well as 5fC and 5caC), by regulating recruitment of architectural proteins, can impact high-order chromatin organization and gene expression of lineage-specific genes (Fig. 2). Studies in T cells during development and immune response are required to elucidate the precise contribution of TET proteins and oxi-mCs in shaping the chromatin architecture.

In recent years, it has become evident that transposable elements (TE) exert important regulatory roles (81). For instance, in CD8 cells, specific TE are enriched in immune cell–specific enhancers. These TE contain transcription factor binding motifs (82). In addition, deletion of SETDB1 impairs Th2 lineage stability due to derepression of TE (specifically endogenous retroviruses), as lack of SETDB1 results in loss of the suppressive mark H3K9me3 that represses Th1 enhancers (83). Interestingly, perturbation of Tet gene expression has been correlated with derepression of retroviral element expression in Tet2/3 DKO iNKT cells (84). This observation could be attributed either to indirect effects or to catalytic-independent roles of TET proteins. Interestingly, a recent publication indicates that, in mouse ESCs, TET1 regulates H3K9me3 and H4K20me3 deposition at retroviral elements in a catalytic-independent manner (85). In this context, the interaction of TET1 with SIN3A plays a pivotal role in regulating histone modifications that suppress endogenous retroviral elements (85). Further research is needed to decipher the precise impact of TE derepression in TET-deficient T cells.

Another critical question in the field is to understand how the enzymatic activity of TET proteins is controlled. The nutrient environment and the availability of metabolites, such as vitamin C (35, 36, 86, 87) and α-ketoglutarate (aKG), which is a cofactor for TET proteins (1), impact the TET-mediated DNA demethylation. For instance, vitamin C was shown to enhance stability of Tregs and promote their immune-suppressive function (36). Mechanistically, this was attributed to enhanced TET enzymatic activity and DNA demethylation of the CNS2 locus of FOXP3, maintaining stable FOXP3 expression (36). The precise roles of vitamin C in other T cell subsets remain unknown. However, extensive research regarding the role of vitamin C in promoting TET activity in other cell types emphasizes the translational potential of leveraging the nutrient environment to enhance TET activity and improve immune response. Strikingly, in Tet2-deficient HSCs, treatment with vitamin C by enhancing the activity of TET3 results in increased hydroxymethylation and can delay/control the emergence of hematological malignancies (88). In addition, vitamin C (ascorbate) depletion in HSCs accelerates leukemogenesis due to reduced TET activity (89). In B cells, vitamin C promotes plasma cell differentiation and Ab production both in vivo and in vitro by enhancing TET2- and TET3-mediated DNA demethylation of critical transcription factors, such as BLIMP1 (90, 91). Thus, it is critical to dissect in further detail how vitamin C might control TET-mediated DNA demethylation in effector and memory T cell subsets to impact immune response. Another exciting research avenue is to investigate whether vitamin C treatment might have therapeutic implications in TET2-deficient T cell lymphomas, as it has been suggested for myeloid leukemias (88).

It is known that distinct T cell subsets exhibit different metabolic traits. Naive cells are metabolically quiescent, relying on oxidative phosphorylation for their metabolic needs, whereas effector cells are metabolically active to support their increased proliferative needs (92). Memory cells are metabolically less active compared with effector cells (93). The dynamic metabolic state of T cell subsets can impact the availability of aKG, a product of the Krebs cycle, thus affecting the activity of TET proteins. However, aKG can also impact the activity of jumonji domain-containing histone demethylases (94). Thus, detailed research is required to illuminate the precise mechanisms by which aKG impacts gene expression and function of T cell subsets. Notably, it has been reported that aKG and IL-2 can enhance recruitment of CTCF at specific loci across the genome of Th1 cells and CD8 cytotoxic cells to regulate expression of genes (95–98). Specifically, the authors demonstrate that high levels of IL-2 promote glycolysis and increase levels of aKG to promote expression of genes involved in effector function, such as Ifng, Cxcr4, and Prdm1 (95). Strikingly, exogenous, cell-permeable aKG in low levels of IL-2 was sufficient to induce this gene expression program, which was not observed under low levels of IL-2 without exogenous aKG (95). Collectively, these findings suggest that the nutrient microenvironment can impact TET DNA demethylase activity and control the genome-wide distribution of oxi-mCs. As a result, gene expression of specific genes as well as recruitment of specific readers such as CTCF may be affected. Thus, it is critical to understand how the interplay of metabolic events and TET activity can shape the gene expression and, ultimately, function of distinct T cell subsets.

On one hand, TET proteins exert tumor-suppressive roles and loss of TET expression at early stages of immune cell differentiation compromises cell maturation, resulting in aberrant proliferation and, ultimately, in malignant transformation (58). On the other hand, loss of TET2 promotes the formation of memory CD8 cells with superior function in the context of viral infection and cancer (48, 50, 51). An emerging question is how can we specifically modulate TET activity to achieve favorable outcomes in a context-specific manner? It has been shown that either restoring Tet gene expression or enhancing TET enzymatic activity by vitamin C can have favorable outcomes in the context of leukemia or, for instance, in the context of Treg-suppressive function (36, 88). Identifying small molecules to effectively and specifically inhibit TET function and/or expression might be useful in the context of tumor immunotherapy to shift the balance of T cells toward memory cells. In addition, in the tumor microenvironment, rendering Tregs more fragile and unstable can increase the efficiency of the effector T cells.

Another intriguing question is to what extent TET proteins might be involved in the maintenance or the reversion of T cell dysfunction. Epigenetic regulators have been involved in T cell dysfunction (99, 100). It was demonstrated that T cell dysfunction is characterized by two distinct chromatin states (101). One of these chromatin signatures characterizes dysfunctional cells at early stages of their differentiation in the tumor microenvironment. Importantly, at this stage, the cells retain the potential to be reprogrammed and regain effector function after checkpoint blockade (101). However, tumor-infiltrating lymphocytes at later stages of their presence in the tumor microenvironment establish a chromatin signature that is refractory to reprogramming, and thus, they cannot regain effector function after checkpoint blockade, rendering these cells terminally dysfunctional (101). Interestingly, deleting DNMT3a in CAR T cells has been reported to prevent dysfunction and increase antitumor response (102). Thus, it is imperative to investigate the contribution of TET proteins in the epigenetic landscape of dysfunctional T cells.

In conclusion, the past 12 y, we have come to appreciate the versatile functions of TET proteins in regulating gene expression and immune function. However, although their enzymatic-dependent roles are well established, their catalytic-independent functions, especially in T cells, remain elusive. In addition, technical challenges have limited our understanding of the unique versus the shared functions of TET proteins in immune cell types. Specifically, lack of Abs suitable for chromatin immunoprecipitation assays in combination with a limited number of available cell types compromised our capacity to interrogate the binding of each TET protein. However, the advent of techniques that allow investigators to assess genome-wide binding/recruitment of factors using limited amounts of starting cells, such as CUT&RUN (103), may improve our ability to address these questions. In addition, deciphering the interacting partners of TET proteins in various T cell subsets will elucidate the mechanisms by which TET proteins shape T cell biology. Ultimately, delineating the mechanisms by which TET proteins control T cell lineage specification, function, and proliferation has the potential to transform treatments against cancer. Broadly, research in the field of TET proteins demonstrates how pursuing and investing in fundamental research to better understand mechanisms that regulate gene expression can have profound, transforming translational impact.

The author has no financial conflicts of interests.

I thank the members of the Tsagaratou laboratory and Dr. Al Baldwin at the University of North Carolina at Chapel Hill for inspiring discussions relevant to topics covered in this perspective.