Evolutive emergence and divergence of an Ig regulatory node: An environmental sensor getting cues from the aryl hydrocarbon receptor?

One gene, the immunoglobulin heavy chain (IgH) gene, is responsible for the expression of all the different antibody isotypes. Transcriptional regulation of the IgH gene is complex and involves several regulatory elements including a large element at the 3’ end of the IgH gene locus (3’RR). Animal models have demonstrated an essential role of the 3’RR in the ability of B cells to express high affinity antibodies and to express different antibody classes. Additionally, environmental chemicals such as aryl hydrocarbon receptor (AhR) ligands modulate mouse 3’RR activity that mirrors the effects of these chemicals on antibody production and immunocompetence in mouse models. Although first discovered as a mediator of the toxicity induced by the high affinity ligand 2,3,7,8-tetracholordibenzo-p-dioxin (dioxin), understanding of the AhR has expanded to a physiological role in preserving homeostasis and maintaining immunocompetence. We posit that the AhR also plays a role in human antibody production and that the 3’RR is not only an IgH regulatory node but also an environmental sensor receiving signals through intrinsic and extrinsic pathways, including the AhR. This review will 1) highlight the emerging role of the AhR as a key transducer between environmental signals and altered immune function; 2) examine the current state of knowledge regarding IgH gene regulation and the role of the AhR in modulation of Ig production; 3) describe the evolution of the IgH gene that resulted in species and population differences; and 4) explore the evidence supporting the environmental sensing capacity of the 3’RR and the AhR as a transducer of these cues. This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3’RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions, including emerging infections such as SARS-CoV-2.


Introduction
Decreased antibody (Ig) production significantly impacts human health by weakening the ability to maintain immunocompetence and survive infectious diseases (1)(2)(3)(4).Conversely, antibodies against selfproteins play a major role in autoimmune diseases (5)(6)(7).The immunoglobulin heavy chain (IgH) gene is responsible for the expression of all Ig classes/isotypes, i.e.IgM, IgD, IgG, IgE, and IgA.These isotypes have different effector functions in mediating immunity (8)(9)(10)(11).Therefore, environmental and/or genetic-induced alterations in processes that control IgH expression will significantly affect human health.However, the ability to directly determine the human health impact of environmental exposures is challenging due to the limited endpoints that can be evaluated in exposed populations and the difficulty in assessing altered immune function (12).However, epidemiology and clinical studies examining Ig levels as well as responses to vaccines are supportive of an increased risk of altered immunity to common infections following environmental exposures to chemicals and pollutants that induce low or moderate immune suppression (4,13,14).Studies have primarily relied on animal models (i.e.mouse and rat) to directly evaluate the immunomodulatory potential of chemicals and the underlying mechanisms (15).Several high-profile incidents of human exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and polychlorinated biphenyls (PCB) lead to the discovery of the aryl hydrocarbon receptor (AhR), which binds with varying affinity to a plethora of environmental chemicals and pollutants (16).In animal models, chemicals that bind the AhR are well-established inhibitors of IgH expression and Ig levels though the actual mechanistic role of the AhR is less clear (Table 1).Additionally, epidemiological studies and in vitro studies with human B cells support altered Ig levels following exposure to AhR ligands, though the studies are limited and suggest variation in sensitivity (Table 1).Interestingly, it is becoming increasingly evident that there are a number of endogenous, microbial-derived, and dietary AhR ligands and that the AhR is a regulator of various immune functions in response to the environment (70,71).This suggests a central physiological role of the AhR in preserving homeostasis and maintaining immunocompetence (72-75).Furthermore, the AhR is gaining attraction as a potential therapeutic target in inflammatory conditions with an AhR ligand currently in FDA review to treat psoriasis (76)(77)(78)(79).However, in the context of human IgH expression and Ig production, the effects of AhR or its mechanistic role is not well defined (Table 1).This is further complicated by the fact that our understanding of basic immune function has been largely based on mouse models.This is noteworthy because of the species differences in AhR ligand binding and signaling (73) as well as the significant differences in the IgH gene between rodents and humans (80).These differences will likely translate to functional differences.Given the importance of antibodies in defense and disease, this represents a significant knowledge gap in human B-cell function and impact of environmental exposures.This review will highlight the current state of knowledge regarding IgH gene regulation and role of the AhR in modulation of Ig production, the significant species and population differences in the IgH gene, and the evidence supporting the environmental sensing capacity of the 3′RR regulatory node with AhR signaling as a key transducer between environmental stressors and altered immune function.This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3′RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions.
The aryl hydrocarbon receptor signaling pathway: More than a protective mechanism against environmental chemicals The AhR signaling pathway was first characterized in the upregulation of Phase I and II metabolic enzymes upon exposure to halogenated aromatic hydrocarbons (HAH), such as 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD or dioxin).The AhR is a ligand-activated nuclear receptor that when activated by ligand translocates from the cytosol to the nucleus where it binds the AhR nuclear translocator (ARNT).The AhR/ARNT transcription factor complex binds to dioxin response elements (DRE) and through interactions between the AhR transactivation domain and coactivators or co-repressors modulates transcription of sensitive genes (81) (Figure 1, left side of dashed center line).A variety of environmental chemicals are ligands for the AhR, which first established AhR signaling as a protective pathway in clearing chemicals from the body through metabolism or conjugation to a more water-soluble chemical for excretion and elimination (82,83).However, increasingly, studies are pointing to physiological roles of the AhR in a variety of tissues and cell types, including the immune system (84-87).Although endogenous ligands such as lipoxin A4 and tryptophan metabolites have been identified to bind the AhR, no clear physiological role has been attributed to both the AhR and the endogenous ligands (70,88).Furthermore, AhR knockout studies in mice and human cell lines support differing biological effects on inflammatory responses and cytokine levels mediated by the AhR in the absence versus the presence of exogenous ligands (89)(90)(91)(92)(93)(94)(95)(96)(97)(98).
The AhR signaling pathway appears to have evolved to include metabolizing/detoxifying activities.Despite conservation of the AhR through evolution, the C. elegans (platelmint) AhR homolog does not bind dioxins and has low amino acid identity with mouse and human in the ligand binding domain, suggesting either binding to unknown endogenous ligands or ligand-independent effects (99)(100)(101)(102).This also suggests origins of a physiological function for the AhR distinct from its metabolizing/detoxifying activity (99)(100)(101).
In terms of ligand binding, the AhR, much like the estrogen receptor (ER), which is also a nuclear receptor, can bind a wide range of ligands.An increasing number of chemicals in the environment, including natural and industrial chemicals, as well as dietary and pharmaceutical chemicals have been shown to bind the AhR with varying affinity (71,103).Interspecies and intraspecies differences in ligand affinity have also been found (Table 2).For instance, dioxin binds the human AhR with ~10-fold lower affinity than the AhR from a dioxin-sensitive mouse strain, which appears to be due to a single amino acid difference (106,107).Additionally, the transactivation domain of the human and mouse AhR is only 58% homologous, which may result in interactions with different co-activators/corepressors and perhaps explain the differential effects on genes  (73,(109)(110)(111).Furthermore, a humanized AhR mouse model exhibited different dioxin-induced biological effects as compared to a wildtype mouse, which could not be explained by differences in AhR binding affinity for dioxin (112).Taken together these results suggest significant species differences in AhR ligand binding and signaling through the C-terminal transactivation domain.
While the human AhR has lower affinity for dioxin as compared to the mouse AhR, the reverse is true for some AhR ligands.Indirubin, which is naturally produced by plants and bacteria, binds the human AhR with much higher affinity than the mouse AhR (71,105).Indirubin has been detected in human urine at concentrations that induce AhR activity, indicating human exposure to biologically relevant concentrations of AhR ligands (113).Additionally, indole, a bacterial metabolite of tryptophan, is a human specific AhR ligand that does not bind the mouse AhR (72).In contrast to indole, the tryptophan-derived AhR ligand 6formylindolo [3,2-b]carbazole (FICZ) is more potent at inducing AhR activity in a rat hepatic cell line as compared to one from human (108).When comparing the potency of FICZ versus TCDD in different species, FICZ was far more potent than dioxin in a Xenopus laevis cell line (104), while dioxin demonstrated more potency than FICZ in mouse hepatic cell lines (105).Whereas, in human hepatic cell lines, dioxin and FICZ were equipotent (105).These studies support the existence of multiple species-specific differences in the AhR signaling pathway (Table 2), thus leading to uncertainty in translating the findings from murine studies to the physiological and ligand-mediated effects of the AhR in humans.
The number of AhR ligands from dietary sources such as indirubin and polyphenols as well as bacterial AhR ligands such as indole and tryptophan metabolites suggest a modulatory role of the AhR in reacting/ adapting to the environment (96,105,108,(113)(114)(115).Additionally, due to the number of bacterial metabolites that are AhR ligands, the AhR has been suggested to play a role in mucosal immunity as a sensor to control commensal bacteria (72, 84).C. elegans use bacteria as a food source and its primitive immune responses, termed "effector-triggered" immunity or surveillance immunity, are mainly directed against intrinsic bacterial attacks (116,117).Perhaps the AhR evolved from a common ancestral gene (as represented by the ligand independent AhR in C. elegans) to bind bacterial ligands and efficiently fine tune a more complex immune response to commensal and pathogenic bacteria.There is a growing body of literature supporting a role of the AhR in most aspects of the immune system (78,84,118,119).This review will focus on examining the role of the AhR in Ig production and the regulation of the IgH gene.

Humoral immunity in defense and disease
Igs are only produced by B lymphocytes and are critical mediators of the humoral immune response.They circulate through the blood, lymph, mucosa, tissue fluids and secretions to protect against  (120).Antigen recognition by antibodies is highly specific and the immune system has the capacity to produce different Igs specific for different antigens, which is remarkable considering that only two genes, the heavy and light chain Ig genes, encode for antibodies.This diversity is possible due to a process called random or somatic V(D)J recombination, which only occurs in developing B lymphocytes and produces an incredible amount of diversity starting from just a single pair of alleles for both the heavy chain gene and the two light chain genes (i.e.l and k) in each individual genome (Figure 2).Initial activation of B lymphocytes by a particular antigen results in production and secretion of IgM, which is pentameric.Since IgM affinity for antigen is generally low during a primary or initial immune response, a pentameric structure with 10 antigen binding pockets enhances the ability of IgM to interact with the antigen (120).
As activation of the humoral response progresses, antigen-activated B lymphocytes undergo somatic hypermutation and class switch recombination.Somatic hypermutation (SHM) is a process that induces random point mutations in the antigen binding pocket with the objective of increasing Ig affinity for the initial antigen, which may or may not happen.Those B cells with mutations that do increase the affinity for antigen will be preferentially activated by the antigen due to their increased affinity, a process called affinity maturation.Class switch recombination (CSR) induces a DNA recombination event that changes the Ig isotype from the large pentameric IgM to a smaller monomeric (IgG, IgE, IgA) or dimeric (IgA) Ig isotype (Figure 3).These processes allow for antigen-specific, high affinity antibodies that are smaller and have different effector functions.
Because of the central role of Ig in humoral immunity, any impairment in Ig production can lead to increased infections and higher morbidity (1)(2)(3).Primary immunodeficiency diseases (PID) that are mediated by antibody deficiencies are clinically relevant (121).PIDs were considered rare diseases but are more common than originally thought with an estimated prevalence of approximately 1 in 1,200 persons in the US with approximately greater than 80% considered to be due to an antibody deficiency (122).The prevalence in the European Union appears to range from 0.8 to 18.8 in 100,000 (123).The most common PIDs are selective IgA deficiency and common variable immunodeficiency disease, which presents with low serum IgG, IgA, IgM and poor to absent specific antibody production (122)(123)(124).Selective deficiencies in IgM as well as the inability to make IgG antibodies against specific pathogens (i.e.specific antibody deficiency) have been identified and are clinically relevant PIDs.These diseases have been associated with increased susceptibility to recurrent infections as well as autoimmune and allergic diseases and malignancies due to disrupted immune regulation (2,122,125,126).Although several gene mutations (either gain of function or loss of function) have been associated with PIDs, the genetic component for common variable immunodeficiency disease and other selective Ig deficiencies is unknown (127).A combination of genetic, environmental, and epigenetic factors likely contributes to these Ig deficiencies.We propose that exposures to AhR ligands and genetic differences within the 3′RR of the IgH gene are contributing factors.We will first review studies supporting altered Ig production by chemicals that have been shown to bind the AhR.

AhR and Ig production
Dioxin markedly inhibits Ig expression and secretion in an AhRdependent manner in both in vivo and in vitro rodent models (Table 1) (21, 31-33, 35, 128).Similarly, studies evaluating human primary lymphocytes have generally identified an inhibitory effect of AhR ligands including dioxin and benzo[a]pyrene on plasma cell differentiation and IgM secretion (Table 1) (28,30,(38)(39)(40)(41)(42).However, sensitivity of IgM secretion to dioxin varied in B lymphocytes from different human donors.Though the majority tended toward inhibition of IgM secretion, some exhibited no effect and others an increase in IgM secretion (30,39,40).Additionally, the inhibitory effect of dioxin on IgM secretion was correlated with a specific Blymphocyte subtype: CD5 + B cells expressing a high level of lymphocyte-specific protein tyrosine kinase (LCK) (17,41,42).CD5 + B cells represent a more innate-like B-cell population (129).Further mechanistic analysis identified an inhibition of Ig secretory processes rather than altered Ig gene expression in the inhibition of IgM secretion from human CD5 + cells exposed to dioxin (17).Additionally, dioxin was shown to increase LCK expression in an AhR-dependent manner, which appears to play a role in the Ig secretory process (41).
These results with high LCK-expressing CD5 + B cells contrast with mouse studies, which support decreased Ig secretion due to an AhR-mediated transcriptional inhibition of Ig genes (Table 1).
However, other studies evaluating the effect of AhR ligands on human Ig production suggest other mechanistic pathways besides the inhibition of secretory processes seen in high LCK-expressing CD5 + B cells.This may indicate interplay between the AhR and different cellular signaling pathways at different stages of Blymphocyte maturation and function.For instance, dioxin increased spontaneous IgE secretion in B lymphocytes isolated from patients with atopic dermatitis that was not due to effects on B-lymphocyte proliferation and appeared to be limited to post-switched IgE + B-cells (47).A similar increase in IgE was demonstrated when human B lymphocytes or peripheral blood mononuclear cells were first stimulated to induce a CSR to IgE and then treated with dioxin or an extract of polyaromatic hydrocarbons from diesel exhaust particles, which include AhR ligands (58).Epidemiological studies also support differential Ig responses associated with exposure to dioxin or polychlorinated biphenyls (PCBs) (Table 1).Evaluation of Korean Veterans (Vietnam War) suspected of being exposed to the dioxin-contaminated herbicide, Agent Orange, demonstrated an increase in plasma IgE and a decrease in plasma IgG1 (52).Additionally, a correlation between increased dioxin plasma concentrations and decreased IgG levels was revealed in a population exposed to dioxin in Seveso, Italy (46).Furthermore, studies evaluating vaccine-responsiveness in children suggest a clinically relevant decrease in Ig titers (i.e.concentrations) due to perinatal exposure to dioxins or PCBs, many of which are AhR agonists (Table 1).However, there were differences in the response, which may relate to differences in toxicant exposure.Higher concentrations of PCBs associated with decreased Ig titers against tetanus and diphtheria that correlated with higher concentrations of PCBs in a Faroe Islands cohort (59,60).Whereas evaluation of a Norway cohort identified an association between maternal dietary exposure to PCBs and dioxins with decreased specific Ig titers to the measles vaccine but not to the rubella, tetanus or influenzae type b vaccines (61).Additionally, perinatal exposure of a Slovakian cohort to PCB-153, which can antagonize the AhR (20,69), correlated with a decrease in antigen-specific IgA and IgG titers (62).
It is difficult to impossible to directly evaluate the role of the AhR in Ig secretion in human primary B lymphocytes or in epidemiology studies.Instead, analysis is limited to evaluating AhR levels and activity.Stimulation with toll-like receptor ligands or CD40 ligand and IL-4 increased AhR expression and activity, suggesting an   increased sensitivity of stimulated human primary B cells to AhR ligands (66)(67)(68).Additionally, primary human CD5 + B cells exhibited higher basal and stimulation-induced AhR levels and activity compared to the CD5 -B cells, which correlated with increased sensitivity of the CD5 + B cells to inhibition of IgM secretion by dioxin (42).Furthermore, a study using a human B lymphoma cell line directly demonstrated dioxin-induced inhibition of IgM secretion that was dependent on AhR expression (43).Although these studies support a role of the AhR in B-lymphocyte differentiation and Ig secretion, the mechanism and impact of the AhR on the production and secretion of other Ig isotypes and subtypes (i.e.IgG1-4, IgA1,2 and IgE) in human is unknown.
In mouse, a key regulator of CSRactivation-induced cytidine deaminase (AID)was identified as a direct target of the AhR (51).In these studies, B-cell stimulation resulted in greater mRNA and protein levels of AID in AhR knockout mice as compared to AhR wildtype mice.This increase in AID correlated with an increased percentage of IgA + and IgG3 + B cells.Additionally, chromatin immunoprecipitation analysis identified AhR binding to a silencer element in the first intron (region 2a) of the gene that encodes AID (i.e.aicda).These results suggest that the AhR, independent of exogenous ligand, is a physiological negative regulator of AID (51).Additionally, treatment with dioxin enhanced the inhibitory effect of AhR on aicda mRNA and AID protein (51).In contrast, two different groups demonstrated no effect of AhR ligands (dioxin and ITE [2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester]) on stimulation-induced aicda expression (36, 49).How these results will translate to human is uncertain.Our in-silico analysis of the human AICDA gene did not identify AhR binding motifs (data not shown).However, this does not preclude the possibility of the AhR binding to DNA through a non-canonical signaling pathway (Figure 1, right side of dashed center line).
As summarized above and in Table 1, studies demonstrate differential and varied effects of AhR ligands within and among Ig isotypes in rodent and human studies.Perhaps these differential effects are mediated by different molecular interactions between the AhR and other signaling pathways or proteins/transcription factors that are specific to B-lymphocyte subtype and/or external and internal modulators (i.e.antigen and environmental exposures, inflammation, hormones, disease conditions, etc.).Additionally, based on mouse studies, the AhR has been implicated as a determinant of B-cell differentiation with or without exogenous ligand (51,130).These studies have focused on the role of the AhR in the activity of specific proteins (i.e.AP-1, Bcl6, Prdm1, Bach2, XBP-1, Blimp-1 and Pax5) involved in an all-or-none bistable switch to either activate or inhibit B-cell differentiation and Ig expression in individual B lymphocytes; AhR activation flips the bistable switch to inhibition (18,31,(130)(131)(132).This inhibitory effect has been speculated to result in a more permissive cellular state for memory cell generation (51) or CSR, which may account for reports identifying an increase in certain Ig isotypes by AhR ligands (130) (Table 1).However, this bistable switch dictating cell fate may not translate directly to human B lymphocytes.Studies with primary B lymphocytes isolated from different human donors suggest a different mechanism or at least set of protein targets.Like mouse, dioxin increased Bcl-6 and inhibited Prdm1 and XBP-1 expression in human primary B-lymphocytes but unlike mouse, dioxin had no effect on Pax5 or Blimp-1 expression (133)(134)(135)(136).
Additionally, comparative analysis of dioxin-induced changes in gene expression in mouse, rat and human primary B lymphocytes using RNA-seq supported dioxin-induced inhibition of IgM secretion via species-specific pathways (137).
A differential effect of AhR activation on Ig isotypic profiles could lead to disease states such as decreased immune competence (i.e.decreased IgM and IgG) and increased hypersensitivity (i.e.nonspecific increase in IgE) as supported by animal models and epidemiology studies (Table 1).However, different AhR ligands may also produce different effects.For example, dioxin is not readily metabolized resulting in a prolonged activation of the AhR; whereas dietary and endogenous AhR ligands are more readily metabolized, which has been shown to induce differential effects likely mediated by altered signaling (138).Indeed, in mouse models, the AhR has been shown to directly interact (i.e.proteinprotein interactions) with other cytosolic proteins and signaling cascades (e.g.ubiquitin and c-src) and transcription factors (e.g.NF-kB/Rel proteins, AP-1, POU/Oct, NF-1, SP-1, KLF-6, and ER) resulting in alternative signaling from the canonical AhR/ARNT/DRE signaling pathway first discovered in the induction of metabolic enzymes (115,(139)(140)(141)(142)(143)(144)(145)(146)(147) (Figure 1).The convergence of differential AhR ligands and signaling pathways in B-lymphocyte function and dysregulation is largely unknown.This represents a clinically relevant and significant gap in our knowledge as it impedes our ability to critically assess the risks linked to environmental exposures.Furthermore, understanding the mechanistic links between the AhR and human Ig production would identify sensitive populations and could lead to a novel therapeutic target in controlling Ig-mediated disease states such as PIDs and hypersensitivity/autoimmune diseases.We propose that the human IgH gene has evolved to respond to internal and environmental cues and that the AhR is an environmental sensor that adapted, for good or bad, during evolution to relay environmental cues to the IgH gene.

Transcriptional regulation of the IgH gene locus, the master gene for all antibody isotypes
Although expression of both a light chain gene and a heavy chain gene is necessary to produce antibodies, the heavy chain gene is responsible for expressing all of the functional Ig isotypes and subtypes.The current understanding regarding transcriptional regulation of the Ig heavy chain gene is largely based on mouse models.In the mouse, regulation of the IgH gene is governed through a complex interaction of several regulatory elements, whose activity is B-lymphocyte specific and depends on the cellular maturation state.The most 5' regulatory element is the variable heavy chain (V H ) promoter, which lies immediately upstream of the V region and contributes to B-lymphocyte specific activity of the IgH locus (148) (Figure 3).Regulatory elements with pivotal impact in IgH remodeling via DNA recombination are the 5′ m enhancer (Em) and the 3′ IgH regulatory region (3′RR), an enhancer complex mapped downstream of the cluster of C region genes (Figure 3).Activation of the 5′ Em promotes VDJ recombination during the earliest steps of Bcell ontogeny and is temporally followed by the activity of the 3′RR, which controls differentiation processes such as CSR and SHM (149-159).AID mediates both SHM and CSR by converting cytosines into uracils and inducing DNA repair mechanisms.Unfaithful repair leads either to incorporation of point mutations in the VDJ region (altering the affinity of the antigen-binding pocket, i.e.SHM) or to DNA recombination at switch sequences (linking a different C-region to the VDJ region, i.e.CSR).Interleukin signaling and a network of transcriptional regulation promote CSR within the IgH gene to produce specific Ig isotypes (160,161).As discussed above, both internal and external factors likely influence these processes, perhaps because of selective pressure to allow more complex organisms to adapt to their environment.

Evolution of the IgH region and emergence of a regulatory node
Multicellular organisms need to defend themselves against infection by pathogens, but only vertebrates mount sophisticated defenses including specialized cells for an acquired humoral immune response (162).Specific to humoral immunity, the IgH gene is expressed in all Gnathostomata (jawed vertebrates) (162).Despite common mechanisms, somatic diversification, the number of antibody isotypes, and tertiary antibody structure vary in individual species, demonstrating substantial divergence and propensity of segmental rearrangements during evolution (163)(164)(165)(166)(167)(168)(169).However, the location and order of the heavy chain C-region genes does not vary as could be expected.In Tetrapods (terrestrial vertebrates) and bony fish, a single IgH gene locus evolved to contain multiple Cregions, which are mainly expressed by CSR (166,170).Additionally, most species have homologs of the most 5′C-region genes, i.e. m and d, which are the only two C-regions that are expressed via alternative mRNA splicing rather than CSR.Cartilaginous fish on the other hand have multiple IgH genes in different chromosomal locations (166,170).
The genome and gene duplications that occurred in early vertebrates (171) may have given rise to an interesting coincidence of multiple IgH and AhR genes in cartilaginous fish.Unlike the multiple IgH genes that are not conserved beyond cartilaginous fish, multiple AhR genes appear to have been maintained in many species; however, xenopus, mouse, gorilla, and human appear to be limited to having only one AhR gene (171).Additionally, in line with the development of adaptive immunity in Gnathostomes, the AhR, which is a far more ancient gene, also adapted to bind environmental chemicals and induce metabolic enzymes as an additional protective mechanism (171,172).It has also been previously proposed that a regulatory role of the AhR in the immune system may have co-evolved with the development of adaptive immunity (171).In support of this, dioxin treatment of a zebrafish autoimmune model (i.e.Teleost bony fish) increased FoxP3 expression and decreased IL-17, indirectly supporting AhR involvement in developing peripheral tolerance to the generation of autoantibodies (173).Other studies with different bony fish species demonstrated a dependence on the AhR for an effective immune response against bacterial pathogens (174,175).Ig and B cells were not directly evaluated in these studies.
Evaluation of the IgH region across Tetrapods (terrestrial vertebrates) demonstrates an increase in C-region genes as well as duplications within specific heavy chain classes (170,176,177).This expansion in C-regions gave rise to some pseudo, nonfunctional Cregions but also to an expanded repertoire of functional C-regions depending on the species and presumably the advantage of the adaptation (165).Interestingly, the order of the C-region homologs is fairly conserved, i.e. 5′ V-D-J variable segments followed by Cregions of m, d, g, e, a classes.Additionally, substantial species-specific variations in the number of subclasses are evident.For example, mice and humans carry four g genes whereas horse has 7 g genes (178)(179)(180).Rabbits carry 13 a genes compared to one in mice and two in humans (179-181).It is worth noting that the C-region remains prone to segmental variations of both loss and gain that results in divergence within the same species (182).Genetic drift or selection appears to have resulted in segmental variations that are not sporadic but are a characteristic that may be reflected in a specific allelic frequency in different populations (183).It appears that, at least in human, the IgH region can still evolve toward loss or gain of C-regions and regulatory regions (182)(183)(184).
Our understanding of the structure and function of the IgH gene relies heavily on mouse models.However, there are significant structural differences between rodent and human IgH loci that is worth considering when translating results from the mouse model to humans.Evolutionarily this difference arose from a marked divergence in the IgH region between the Platyrrhini Parvorder (New World monkeys, e.g.squirrel monkeys, wooly monkeys, marmoset) and the Catarrhini Parvorder (apes, humans and Old World monkeys, e.g.rhesus monkeys) because of a large duplication of a portion of the IgH region.This divergence allowed for the emergence of additional antibody subclasses (i.e.IgG3, IgG4 and IgA2) in the Catarrhini Parvorder (185).Moreover, the same segmental structural variation involved the 3′RR region, giving rise to the 3′RR1 and 3′RR2 that are present in all Catarrhini (186).We found that species quite far in phylogenesis, like grey seal, dromedary camel, North American beaver, and lesser Egyptian jerboa also show the presence of a duplicated 3′RR region (Figure 4).Since these duplications are not uniform in size or breakpoints, they are likely a result of independent events during genome rearrangement that happened after divergence of the specific lineages for each species.These duplications resulted in redundance and retention of functional sequences in the 3′RR and therefore may provide additional mechanisms for controlling antibody production (187)(188)(189)(190).
The current reliance on non-primate models and non-Hominoidea primates may not entirely translate to human B lymphocyte biology and antibody production because of the genetic divergence originating from the segmental duplication of the IgH locus.This is of concern considering that toxicological studies and preclinical therapeutic and safety evaluations utilize mainly rodent models, specifically mouse and rat, as well as marmoset or other monkey species that do not have this large duplication and therefore may not accurately reflect human antibody regulation and production (168,185).Moreover, the human relevance of non-Hominoidea models may also be limited because of divergence among members of the Catarrhini Parvorder, e.g. the pseudogenization of some IgH gene segments or the large number of detected variants within the 3′ RR that seem species-specific (186,191).Similarly, in examining the role of the AhR in influencing immune function, not only species differences in the IgH gene but as mentioned above species differences in AhR ligand binding and specificity as well as the number of AhR genes are also factors to consider when translating non-human studies to human.For example, although there is only one AhR gene in the mouse as in human, there are differences in AhR ligand binding affinity and specificity between the two species (Table 2).In terms of primates, marmoset (New World Monkey) and green monkey (Old World Monkey, Chlorocebus Family) differ from humans in that they express two AhR genes (171).The number of AhR genes in other primates as well as the binding affinity/specificity for ligand in primates in general has yet to be determined.
The presence in the human-lineage of the two 3′RR copies, perhaps via transposition of the primordial m enhancer along with duplications, could be the preparatory steps that provided the necessary regulatory elements to mediate CSR and enable the diversification of functional roles and timing of different Ig isotypes [reviewed by (192)].The 3′RR demonstrates both remarkable conservation and divergence among species (Figure 5).The number of enhancers within the 3′RR varies, e.g. the mouse and rat have 7 enhancers (hs3A, hs3B, hs1.2, hs4, hs5, hs6, hs7), while humans have 3 enhancers (hs3, hs1.2, hs4) (Figures 4, 5).Orthologues of the human hs3, hs1.2, and hs4 enhancers were found in mammals belonging to non-primate species, including rat, mouse, cat, dog, panda, rabbit, and bat (Figure 5) (186).Additionally, a large area internal to the 3′ RRs showed a conserved presence of an inverted duplication (i.e.palindromic region) surrounding the hs1.2 enhancer, which was found in all of the analyzed species, suggesting a conserved characteristic, though the actual sequence of the palindrome varies (186).The palindromic region could form a hairpin loop, suggesting that the orientation of the hs1.2 enhancer would be irrelevant for enhancer function (186).Indeed, the hs1.2 enhancer is in different orientations in different species and even between the two human 3′ RRs (193).The latter finding may also suppress the enhancer conversion between the two copies and favor the divergence between the 3′RRs (194).Strikingly, a specific evaluation of the sequence conservation among distant species in the hs1.2 consensus site for transcription factor binding showed conservation in the core of the enhancer, but high variability in the GC-rich regions that harbor most of the transcription factor binding sites (data not shown).Hypothesizing the palindromic sequences to be the stem of a hairpin, the hs1.2 enhancer region would be in a loop protruding from the chromatin, thus easily accessible to transcription factors that can bind to single-stranded DNA.This protruding three-dimensional structure could also facilitate long-distance cis-interactions with other IgH gene regulatory elements (e.g.V H promoter, Em, and intronic promoters associated with each constant region) to promote IgH expression and CSR (152,(195)(196)(197)(198)(199) as well as SHM within the IgH gene (197,200,201).The finding in mammalian genomes of syntenic regions of palindromes having sequences unmatching when compared among different species suggests that mutations on one branch of the palindrome were often compensated by mutations on the other branch, preserving the complementarity and consequently the stem of the hairpin (186).
The 3′RRs and their unique structural features are likely controlled by binding of several cis-acting transcription factors (186,201,202).Furthermore, there is evidence of the formation of a G-quadraplex structure within the hs1.2 enhancer that seems a maintained feature due to the persistent presence of GC-rich short sequences inside the hs1.2 enhancer (i.e.GC-rich region in Figure 6) (186,197).The human hs1.2 enhancer has putative binding sites for several transcription factors (SP-1, AP-1, NF-1, NF-kB, AP-1.ETS, POU/Oct, AhR, ER), some of which have been confirmed to actually bind transcription factors.Additionally, mouse studies have identified binding of the ER and the AhR, both of which may directly transduce internal and environmental signals through the 3'RR regulatory node (146,187,193,(203)(204)(205)(206)(207)(208)(209)(210)(211)(212)(213)(214) (Figures 7, S2).The binding dynamics and interplay of the various transcription factors that could bind within the native hs1.2 enhancer remains to be elucidated, particularly in human.Some transcription factors could bind the hs1.2 region when in a double-stranded DNA conformation, thus opening the chromatin and allowing for the formation of the hairpin loop or the G-quadruplex.At least for Sp-1 and AP-1, their high affinity binding to G-quadruplexes has been demonstrated (201,215,216).Alternatively, the three-dimensional structure could prevent binding of transcription factors, carrying out a structural regulation of the protein-DNA binding.These DNA structural changes may not be replicated in reporter studies due to the lack of chromatin and the inability to include the whole native form of the 3′RR due to its large size (~17 kb).Additionally, polymorphisms within a putative quadruplex locus have been associated with altered gene expression further supporting the role of these three-dimensional structures in transcriptionally regulating genes (217)(218)(219).Notably, the human hs1.2 enhancer exhibits genetic polymorphisms, not present in the mouse, that involve the quadruplex site and appear to influence the transcriptional activity of the 3′RR (186,187,197,220,221).

Genetic variation in the 3'RR correlates with antibody levels and disease
The presence of specific 3′RR1 hs1.2 enhancer alleles have been associated with various disease states and antibody levels.For instance, celiac disease, dermatitis herpetiformis and psoriasis correlated with a higher frequency of hs1.2 allele *2 (227,229).Compared to controls, a significant increase in IgM levels in patients with lupus erythematosus and systemic sclerosis was associated with the presence of the hs1.2 allele *2 (214,228).In the above pathologies, more severe symptoms associated with homozygosity for allele *2, which was also the case in the severity of IgA nephropathy towards renal failure (225).However, it should be noted that the frequency of alleles *3 and *4 was very low in the patient population evaluated.Therefore, the analysis was primarily limited to evaluating alleles *1 and *2.In other studies, analysis of single nucleotide polymorphisms (SNPs) in the 5kb region that connects the human hs1.2 and hs3 enhancers identified a correlation between psoriasis and the presence of specific 3′RR haplotypes.The SNPs correspond to potential methylation sites that could influence conformational changes and may be a target of environmental factors (235).Interestingly, the SNPs involved specific 3′RR haplotypes with a very low presence in the African population as evidenced by GnomAD loci mapping (235), corresponding to a low rate of both allele *2 and psoriasis in West African populations (221,236).A recent study also found that specific 3′RR haplotypes within the 5kb region connecting the hs1.2 and hs3 enhancers preferentially associated with allele *1 or with allele *2 (237).
A human population study examined the linkage of the 3′RR haplotypes with allotypes of the g1 and g3 constant regions in different continents (238).Allotypes are polymorphisms within the Ig heavy and light chains in the same species (239).As seen in previous studies (221), the highest frequency of hs1.2 allele *2 is in Europe with the lowest in Africa (238).Interestingly, a linkage between 3′RR1 haplotypes and g1 and g3 constant region allotypes was demonstrated in populations outside of Africa but not within Africa, suggesting that this large region of DNA underwent positive natural selection as humans dispersed out of Africa (238, 240).Analysis of cell lines from the 1000 Genomes Project identified higher expression of g1 and g3 constant regions in cells homozygous for the hs1.2 allele *1 vs. the other hs1.2 alleles (238).However, the expression of functional transcripts and antibody levels were not determined.
Evidence of a correlation between antibody levels and hs1.2 alleles was identified in a longitudinal study of healthy children before and after 5 years of age, which appears to be divergent from the increase in g1 and g3 expression with hs1.2 allele *1 mentioned above.Before 5 years of age, children with high blood concentration of IgM, IgG and IgA had a frequency of hs1.2 allele *2 around 80% with an increase in frequency of approximately 30% above the control population of that geographical area.Surprisingly, the same children two years later did Species comparison of the IgH gene locus.The schematic illustrates the similarities and differences in the number of IgH constant regions and composition of the 3'RR among six different species (i.e.human, mouse, gray seal, North American beaver, dromedary camel, and lesser Egyptian jerboa rodent).The NCBI refseq number and location of the sequence depicted are identified under the species name.TMEM121 was used as a reference of synteny because it is the gene closest to the IgH region.Duplication of or within the 3'RR occurred in both human and mouse.However, the murine 3'RR has an internal duplication that produces two copies of the hs3 enhancer (hs3A and hs3B); whereas the human genome contains two 3'RRs (i.e.3'RR1 and 3'RR2) due to a large duplication of constant region genes and the hs3, hs1.2 and hs4 enhancers.Analysis of species quite far in phylogenesis identified the presence of distinct and varied duplications in the IgH gene that include duplications of constant regions and the 3'RR enhancers.This wide range of arrangements within the IgH gene among different species suggests that many of these duplications happened independently in various branches of the evolutionary tree.
not have this correlation between allele *2 and high blood concentrations of IgM, IgG, and IgA and no such correlation was present after 7 years of age; a large cohort of healthy students (aged 19 to 33 years) also exhibited no correlation between allele *2 and high Ig titers (234).Serone and co-workers (234) theorize a selective and limited age-dependent advantage of allele *2 in populations that migrated out of Africa and were exposed to a change in the environment and infectious pathogens.This may have resulted in more protection during childhood but with increased life expectancy a potential dysregulation of this age-dependent regulation could lead to increased risk of autoimmune disease (234).Due to the different hs1.2 alleles primarily varying in the number of transcription factor binding sites (Figure 7), it is tempting to speculate that environmental factors such as AhR ligands can lead to this dysregulation and increased risk of altered antibody levels and disease.

Role of the 3'RR in transcriptional regulation of the IgH gene
Transcriptional regulation of the IgH gene has been largely defined in rodents.The mouse 3′RR consists of a set of enhancers, either rodent-specific (hs5, hs6, hs7) or conserved in vertebrates (hs1.2, hs3, hs4) (Figure 5) and has been functionally evaluated by many in vitro and in vivo approaches (198,210,(241)(242)(243)(244).For the mouse 3′RR, the hs1.2, the two copies of hs3, and hs4 are individually weak enhancers, but when all four are linked together they become strong co-activators (154,241,245,246).Deletion of these four enhancers in mice results in a dramatic decrease in CSR and in secretion of all antibody isotypes (149-154, 156-159).Studies have also demonstrated a long-range intrachromosomic interaction between the 3′RR and the V H and intronic promoters (152,198,211,247).The V H promoter is essential to producing functional IgH transcripts, i.e. transcripts that can be translated into Ig heavy chain proteins.The intronic promoters located just upstream of each IgH constant region produce nonfunctional or germline transcripts that include the switch sequences and do not lead to the production of functional proteins (Figure 3).Germline transcription at the intronic promoters is thought to open the chromatin to allow for and direct CSR to a specific IgH constant region and expression of a specific antibody isotype (248, 249).Regulation of intronic promoter activity by the 3′RR appears to involve either co-activation or competition depending on the activating stimuli, which may have evolved to respond to T-independent vs. T-dependent immune responses (250).Germline transcripts have been shown to form R-loops at their corresponding switch sequences resulting in a G-rich DNA single strand that is accessible to AID, which the 3′RR plays a role in recruiting (151,(251)(252)(253). Additionally, specific enhancers within the 3′RR may have greater influence on the expression of specific constant regions (154, 254).Taken together these studies indicate an essential role of the mouse 3′RR in IgH expression, CSR and therefore final antibody production by plasma cells (255).
The mouse 3′RR is also sensitive to modulation by environmental factors (Table 3).AhR ligands have been shown to markedly inhibit 3′ RR activity, which mirrored their inhibitory effect on IgH expression and antibody secretion (19,33,57,146).Besides AhR ligands, other chemicals altered 3′RR activity that directly correlated with their effects on IgH expression and antibody secretion.The reactive oxygen species hydrogen peroxide exhibited a biphasic effect, with low concentrations inducing and higher concentrations inhibiting 3′RR activity and antibody secretion (256).Gold nanoparticles and the badrenergic receptor agonist terbutaline induced 3′RR activity and antibody secretion (57,257).These environmental factors likely influence distinct signaling cascades that alter binding of transcription factors within the 3′RR resulting in changes in IgH expression.Additionally, the nature of the cellular stimulation initiated by a response to antigen (i.e.TLR, co-stimulation through CD40 and/or CD86 or cytokine receptors) is expected to be a factor on the transcription factor milieu and the effects of environmental factors.Multiple transcription factors such as the AhR, NFkB/Rel, POU/Oct, Pax5 have been identified as positive and/or negative regulators of the mouse 3′RR, which may be dependent on the maturation stage of the B cell and the specific enhancer.Coactivation of b-adrenergic receptors (environmental factor) and of Mouse 3'RR and syntenic region in Vertebrata.The schematic is focused on a comparative analysis of the 3'RR and a constant region (Ca) within the IgH gene locus.Green boxes highlight the position of the 7 enhancers and Ca in the murine chromosome 12 (mm39 genome assembly).The rows below show alignments of mm39 to other genomes using a gap scoring system (see UCSC Net track for details); detected orthologous regions are indicated by black boxes.Three of the enhancers (i.e.hs5, hs6, and hs7) are specific to mouse and rat, while the other enhancers (i.e.hs3A, hs1.2, and hs3B) appear shared among more species, almost to the same level as Ca.The 'Repeats' row indicates segments of the murine IgH region that are detected as 'repeats' (e.g.SINEs, LINEs, satellites) by RepeatMasker software (https://www.repeatmasker.org) and could be ignored in a synteny comparisons because of their characteristics.
Furthermore, studies have identified binding of ER to various areas of the mouse IgH gene including Em, switch sequences and the hs1.2 enhancer (212,263).Mutation of the ER binding sites within the Em or hs1.2 enhancer decreased CSR to IgA in a mouse B-cell line model (264).However, LPS stimulation with exogenous estrogen reduced IgG3, IgG1 and IgA secretion in purified B cells from female mouse spleens, but over an age span of 6 weeks to over 5 months, female mice had higher serum titers of most antibody isotypes compared to male mice (265).A regulatory role of ER in the IgH gene may be particularly relevant to the gender differences seen in incidence of autoimmune disease and effectiveness in fighting infections.
These studies evaluating the transcriptional regulation of the IgH gene have primarily utilized mouse models.The significant genetic differences between the mouse and human IgH genes will likely result in differences in regulation and responses to external stimuli (Table 4).As described earlier, the two most striking differences are that 1) the human 3′RR is duplicated along with several constant regions and 2) the 3′RRs contain three instead of seven enhancer elements (i.e.hs3, hs1.2, hs4) (Figure 4 and S1) (80,186,187,220,222). Like the mouse 3′RR, the human 3′RRs are thought to physically interact with intronic promoters (266).Presumably each 3′RR regulates CSR and gene expression of a specific set of C regions.However, analysis of CSR in humans with a homozygous deletion of the 3′RR1 suggested that the 3′RR2 could compensate for this loss (248, 267).Furthermore, the mouse 3′RR was shown to be capable of bidirectional activation of intronic promotors using a mouse model with an insertion downstream of the 3′RR of a b-globin gene regulated by an intronic promoter (250).Taken together, these observations suggest that the human 3′RR1 is capable of regulating transcription of upstream and downstream constant regions and perhaps the 3′RR2 only comes into play when the 3′ RR1 is deleted by CSR to constant regions downstream of 3′RR1.
Reporter plasmids have been used to evaluate the transcriptional activity of the human 3′RR enhancers in isolation or linked together.Like the mouse 3′RR enhancers (hs3A, hs1.2, hs3B, hs4), these studies suggest a strong cooperative activation when all of the human 3′RR enhancers (hs3, hs1.2, hs4) are linked together (266).However, the majority of these studies have only evaluated the basal activity of the individual or linked 3′ RR enhancers and none of these studies have evaluated the native 3′RR (i.e.enhancers with intervening sequences) mostly due to the large size of the native human 3′RR (~17 kb) (Figure S1) (187,188,208,220,222,223,266) (Table 3).This limitation of reporter plasmids is particularly important when considering the evolutionarily conserved palindromic sequences flanking the hs1.2 enhancer, which, as discussed earlier, likely play a role in the three-dimensional structure of the 3′RR and promoting long-range interactions with other IgH gene regulatory elements (152,186,(195)(196)(197)(198)(199)201).However, in contrast to the in vivo mouse models, there are limited human cellular models to study the role of the 3′RRs on molecular processes (i.e.IgH expression, antibody secretion, CSR and SHM) involved in antibody production.An additional consideration is the genetic polymorphisms within the human hs1.2 enhancer, which, as discussed in the previous section, has been associated with Ig expression levels and several diseases (186,193,220,(222)(223)(224).Therefore, the genetic profile of the human hs1.2 enhancer may be a key influencer on the sensitivity of the 3′RR and Ig expression, as well as risk for developing antibody-mediated disease from exposure to environmental stressors.

Environmental factors and transcriptional regulation by the human hs1.2 enhancer
Limited studies have evaluated the impact of genetic differences within the human hs1.2 enhancer on transcriptional activity or the role of specific transcription factors in mediating hs1.2 enhancer activity.In vitro analysis of human hs1.2 enhancer activity using reporter plasmids demonstrated that the hs1.2 alleles harboring a higher copy number of the 40 bp monomer have increased transcriptional activity (i.e.allele *1 < allele *2 < allele *3 < allele *4, see Figure 7) (223,225).The monomer(s) is flanked by SP-1 sites and contains possible binding sites for the transcription factors NF-kB/ Rel, AP-1, and NF-1 (187,193,220,222,223).As mentioned previously, we identified a potential AhR binding site, i.e.DRE, within the repeated monomer sequence of the human hs1.2 enhancer.This DRE motif is similar to the functional DRE we previously evaluated in the mouse hs1.2 enhancer (19,34,146).Furthermore, Jones and coworkers (265) identified a potential ER binding site within the monomer of the human hs1.2 enhancer, which overlaps with the suggested AP-1 binding site (223).As discussed earlier, the AhR binds a plethora of ligands from environmental, dietary, and pharmaceutical sources.The ER is also known to be promiscuous in ligand binding, which has generated considerable public concern regarding the potential of chemicals that bind and Sequence and notable regions of the hs1.2 enhancer.Allele *1 and *2 of the human hs1.2 enhancer are illustrated to highlight the presence of the internal 40 bp monomer (40mer), which has the potential to form quadruplex DNA (i.e. a stable secondary DNA structure).A "core" (magenta) and a "tail" (light blue) sequence are common to all the variants, while the "40mer" repeat (yellow box) may be present from one to four times in human.Transcription factor consensus binding sites within the human hs1.2 enhancer alleles.Depicted are the four known alleles of the human hs1.2 enhancer, which differ mainly in the copy number ("repeat") of the internal 40 bp monomer.A "core" (magenta) and a "tail" (light blue) sequence are common to all the variants, while the yellow box ("repeat") may be present from one to four times in human.However, at present, the highest copy number has been detected in the chimpanzee hs1.2 enhancer, which has twelve repeats.The green boxes are GC-rich regions.The red "cap" box is a GC-rich region longer than the ones indicated by the green boxes.The alleles were analyzed in silico for potential transcription factor binding sites using TFBIND (https://tfbind.hgc.jp/).The consensus binding sites for several transcription factors were identified and reported below each allele.Multiple partially overlapping sites for the same transcription factor were merged and depicted as a single larger box (See Supplementary File S2 for unabridged depiction of consensus binding sites of the allele *4).Consensus sites mapping in the hs1.2 "repeat" generally change in copy number according to the number of monomers, thus potentially making the enhancer alleles differentially receptive to transcription factors.Moreover, each copy of the monomer harbors a region that could form a quadruplex DNA 3-D structure (see Figure 6).
activate the ER (e.g.bisphenol A) to disrupt endocrine function (268,269).In the context of antibody production, the AhR and ER could transduce a variety of signals through the hs1.2 enhancer to affect 3′ RR activity and IgH regulation.What may have originally evolved as an internal and environmental sensor for adaptation and host protection, may now be a target for dysregulation by environmental and industrial chemicals and pollutants.
Consistent with a functional impact of transcription factor binding within the 40 bp monomer, deletion of the monomer in allele *1 or deletion of one of the repeated monomers in allele *2 of the hs1.2 enhancer resulted in decreased transcriptional activity (19).Limited studies have evaluated the effect of cellular stimulation or chemical exposures on the activity of the human hs1.2 enhancer alleles.Unlike the inhibitory effect of dioxin on the mouse hs1.2 enhancer, dioxin induced a significant increase in overall human hs1.2 transcriptional activity that was dependent on the AhR and the allelic state of the hs1.2 enhancer (Tables 3, 4); however, the AhR does not appear to bind the DRE-like site within the monomer (19,213).Mutational analysis suggested a partial role of the NF-1 binding site and not the DRE-like site in the AhR-mediated induction of hs1.2 activity (213).It is likely that the AhR mediates its effects through protein-protein interactions (e.g.ER, NF-kB, AP-1, POU/Oct, NF-1, SP-1, and KLF-6) rather than directly binding to the DNA (see Figure 1, right side of the dashed center line) (115,(139)(140)(141)(142)(143)(144)(145)(146)(147).These results are contrary to the demonstrated binding of the AhR to the DRE within the mouse hs1.2 enhancer and the inhibitory effect of dioxin on enhancer activation following cellular stimulation, which further supports species differences in the regulation and activity of the 3′RR (19) (Tables 3, 4).Additionally, analysis of the hs1.2 sequence suggests that there is no site-specific pressure towards the conservation of the DRE consensus (i.e.GCGTG), but the characteristic sequence of the hs1.2 enhancer (modularity and repeated GC-rich regions) easily give rise to a DRE-like consensus.Thus, DRE consensus sites appear to vary in the hs1.2 from different species in both position and number and whether it is even present (data not shown).Our analysis suggests a similar effect on ER binding sites.Additionally, in contrast to Jones and co-workers (265) who reported a relatively conserved ER binding element in the 40 bp monomer of the hs1.2 enhancer when considering mouse, Callicebus moloch (New World Monkey), gorilla, chimpanzee, and human alleles *1 and *2, our in-silico analysis using TFBIND (https://tfbind.hgc.jp/)only identified ER consensus sites outside of the monomer repeat (Figure 7).It is worth noting that actual binding of the ER has only been evaluated in mice (212,263,265).These results support species differences in transcription factor binding and activity of the hs1.2 enhancer, whose regulation may have evolved through slightly divergent pathways to adapt to environmental signals.How these species differences relate to an overall effect on antibody production is yet to be determined.
Mutational analysis studies of specific binding sites within or outside of the sequence repeats identified positive and negative regulators of human hs1.2 reporter activity; however, the different reporter studies demonstrated contradictory effects on hs1.2 activity likely due to variations in the flanking regions and number of sequence repeats incorporated into the reporter, differences in cellular models, and the inability of a gene reporter to accurately reflect the interactions of different transcription factors within and among the different enhancer regions of the 3′RR (187,208,213).Additionally, our insilico evaluation of the human hs1.2 alleles for consensus transcription factor binding sites identified multiple potential binding sites for the AhR, NF-kB, AP-1, NF-1, and ER, as well as other transcription factors (Figures 7, S2).The transcriptional effect of these sites has not been fully evaluated and our analysis did not identify the previously suggested sites for NF-1 and ER within the monomer repeat (213,223,265).Further studies are needed to fully characterize the transcription factors regulating human hs1.2 activity, including the impact of allelic variants and the response to internal and environmental factors, with the goal of elucidating how these factors impact IgH expression, SHM, and CSR.
Snyder and coworkers (213) suggested that the polymorphic region of the hs1.2 enhancer plays a fine-tuning role in the regulation of the hs1.2 enhancer because of the strong activating role of the AP-1.ETS site vs. the inhibitory role of the POU/Oct site, which are both outside of the polymorphic region (i.e., 40 bp monomer repeat).The fact that the polymorphic region can expand the number of transcription factor binding sites (e.g.AhR, NF-kB, AP-1) may produce more of a regulatory role on overall hs1.2 activity and lead it to be more sensitive to environmental cues.This would seem to be a favorable feature considering that the humoral immune response should be adaptive and responsive to external insults that are able to evade the body's first line of defense.Additionally, antibody production should also be responsive to the signals produced by the microbiota because of the role antibodies play in maintaining a healthy balance of commensal microorganisms in the mucosal tissues.Furthermore, it is not surprising that B lymphocytes would be responsive to internal signals from the endocrine system as seen with adrenergic receptor agonists and estrogen (57,206,212,263,265).In line with this premise, a recent study of COVID-19 patients associated the presence of hs1.2 allele *2 to decreased disease severity (based on the need of oxygen supplementation and fraction of inspired oxygen) and a decreased likelihood of developing pneumonia but only in women, leading the authors to hypothesize that this protective effect is estrogen-dependent and mediated by the ER binding sites within the repeated monomer (237).Since our in-silico analysis only identified ER binding sites outside of the monomer, another possibility is an enhanced interaction between the ER and the increased number of transcription factors binding within the two monomers of allele *2.(Figure 7).In regard to external factors that influence B-cell function, the AhR may play an important environmental sensor role because of the diverse array of ligands produced from the diet and microbiota and even light exposure (72,84,96,105,108,(113)(114)(115).Rannug (270) proposed a diurnal oscillation in the concentration of AhR ligands, which is particularly interesting when considering that the AhR is part of a family of circadian rhythm proteins (271).The breakdown of tryptophan in the skin by sunlight exposure to the AhR ligand FICZ may be an important factor in skin homeostasis through a balance between immune suppression (high FICZ concentrations) and inflammation (low FICZ concentrations) (270).This is further supported by the improvement of psoriasis with sun exposure and the effectiveness of the AhR ligand tapinarof in resolving mild-to-severe plague psoriasis in Phase 3 trials (272).In conclusion, varying intrinsic and extrinsic signals could influence the overall transcription factor binding profiles and transcriptional control of the 3′RR and therefore antibody production, SHM, and CSR.As discussed above, evidence from mouse models supports the sensitivity of the 3′RR and antibody production to environmental chemicals (Table 3).However, the significant differences in the IgH gene between rodents and humans likely translates to functional differences as supported by the studies evaluating hs1.2 enhancer activity.Given the importance of antibodies in defense and disease, this represents a significant knowledge gap in human B-cell function.Based on the evidence to date, we propose that the genetic polymorphisms (i.e.hs1.2 alleles and 3′RR haplotypes) in human evolved to adapt to internal stimuli and changing environmental conditions, and that xenobiotics, such as chemicals in pollution and forest fires, and disease conditions significantly disrupt 3′ RR activity by high jacking its environmental sensing function and tipping the balance towards a pathological response.Understanding genetic differences and the signaling pathways that converge at the 3′RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions.

SUPPLEMENTARY FIGURE 1
Chromosomal location of the segmental features of the human IgH gene.The top image represents the ideogram of human chromosome 14.Dark boxes depict the approximate location of bands, as seen on the Giemsa-stained chromosome.The constriction site (brown triangles) indicates the centromeric region, thus the p arm is on the right and the longer q arm on TABLE 3 Internal and external modulators of mouse and human 3'RR and its enhancers.The gene annotation of the IgH region comes from the V39 release of GENCODE on the hg38 assembly of the human genome.The faded background colors highlight the different size of the genomic areas hosting V, D, J or C regions.Enhancers of the whole IgH region are also positioned, as reported in GeneHancer database, but we integrated these data with the position of the well-known enhancers belonging to the human 3'RRs.

FILE S2
Unabridged consensus transcription factor binding sites within the *4 allele of the hs1.2 enhancer.Allele *4 of the human hs1.2 enhancer is depicted as a detailed overview of the schema in .A general schematic of allele *4 is represented on page 1.A "core" (magenta) and a "tail" (light blue) sequence are common to all the hs1.2 allelic variants.The core sequence is also highly conserved in vertebrates.The "repeat" (yellow box) may be present from one to four times in human (see Figure 7) (alleles with more copies were found in primates).The allele *4 was analyzed in silico for potential transcription factor binding sites using TFBIND (tfbind.hgc.jp) and the results were depicted using SnapGene Viewer (https://www.snapgene.com).One transcription factor is reported on each page, starting from page 2. Codes refer to the sequence consensus motif in each transcription factor binding site, as listed in the "Gene Set Enrichment Analysis" database (GSEA -https://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp).

FIGURE 1
FIGURE 1 Schematic of the canonical and non-canonical aryl hydrocarbon receptor signaling pathways.Depending on the ligand and perhaps other unknown factors, aryl hydrocarbon receptor (AhR) signaling could occur through the canonical and/or the non-canonical pathway.In the cytosol, the AhR is complexed with Src kinase and several chaperone proteins, including the 90 kDa heat shock protein (hsp90), p23 and XAP2 (or AhR-interacting protein, AIP).The chaperone proteins protect the AhR from ubiquitination and keep it in a conformation that can bind ligand.Ligand binding causes a conformational change resulting in the disassociation of XAP2 and Src, exposure of the AhR nuclear localization signal, and AhR translocation into the nucleus.Once in the nucleus the other chaperone proteins dissociate from the AhR leading to canonical or non-canonical signaling.Canonical signaling (left side of the center, dashed line) involves dimerization of the AhR and the AhR nuclear translocator (ARNT) and binding to dioxin response elements in sensitive genes.The AhR/ARNT nuclear complex interacts with coregulators to either positively or negatively (indicated by the "+/-") modulate gene transcription (bent arrow indicates the transcription start site).Non-canonical signaling (right side of the dashed center line) involves genomic and nongenomic signaling.The non-genomic signaling could be mediated through the release of Src or interactions with other kinases (1) and the E3 ubiquitin ligase activity of the AhR (2).The genomic signaling could be indirect (3) or direct (4) through interactions with transcription factors other than ARNT, such as NFkB/Rel proteins, AP-1, POU, NF-1, SP-1, and KLF-6.Figure was created with BioRender.com.

FIGURE 2
FIGURE 2Schematic of somatic recombination of the Ig heavy and light chain genes.The Ig genes are inherited in a fragmented, non-functional state and require somatic recombination during B-lymphocyte development to produce Ig protein.There are two light chain genes (l and k) and one heavy chain gene and two alleles of each gene.Only one allele of the light chain and heavy chain gene can be functionally expressed in each B lymphocyte.Expression requires DNA recombination to select one variable (V) and one joining (J) segment for the light chain gene and one V, one diversity (D), and one J segment for the heavy chain gene.The recombined VJ (light chain) and VDJ (heavy chain) exons form the antigen binding pocket of the Ig.This random recombination of V(D)J segments allows for the incredible diversity in the antigen binding pocket of Igs.In humans, there are 34-38 V segments, five J segments and one constant (C) segment for the k light chain; 29-33 V, 4-5 J, and 4-5 C segments for the l light chain; and 38-46 V, 23 D, 6 J, and 9 C segments for the heavy chain.The schematic illustrates a hypothetical somatic recombination of one allele for each of the l and k light chains and the heavy chain.However, each B lymphocyte will only successfully recombine one light chain and one heavy chain allele.Recombination of the light chain genes requires one recombination of V to J; whereas the heavy chain requires two recombination events, first D to J and then V to DJ. Dotted lines represent the location of the double strand cut (top) and the recombined segments forming the coding exon (bottom joining of lines).Transcription is initiated at the 5′ end of the recombined exon and terminated at the 3′ end of the C exon. Figure was created with BioRender.com.

FIGURE 3
FIGURE 3Schematic of the human IgH gene locus and class switch recombination.The human recombined IgH gene contains a variable heavy chain promoter (V H ), variable region (recombined VDJ), intronic enhancer (Em), constant regions for all antibody isotypes (Cm, Cd, Cg 1-4 , Ce, Ca 1,2 ; Cye is a nonfunctional pseudogene) and two 3'IgH regulatory regions (3'RR1 and 3'RR2).Immediately upstream of each C H , except d, is an intronic promoter (I H ) and a cytidinerich switch sequence (S H ). Following successful V(D)J recombination, the first antibody isotypes expressed are IgM and IgD.Class switch recombination (CSR) involves an irreversible DNA recombination of the IgH gene that allows a different constant region to be expressed without changing the antigen recognition.Switch to another isotype is dependent on specific cellular stimuli that targets a specific intronic promoter and induces transcription through the switch region of the targeted constant region.The schematic illustrates CSR to Ce to produce IgE.Transcription initiated through the Ie produces nonfunctional germline transcripts and opens the chromatin for targeting by activation-induced cytidine deaminase (AID)(1).AID targets the switch sequences upstream of Cµ and the constant region Ce, causing nonhomologous recombination between the switch regions (i.e.Sµ and Se)(2).CSR results in removal of the intervening DNA between the recombined switch sequences (Se-Sµ DNA Switch Circle) (3) and a recombined IgH gene with VDJ linked to Ce (4).Transcription of the IgH gene results in functional VDJ-Ce transcripts (5) that will be translated into the e heavy chain protein to form IgE antibodies(6).Figure was created with BioRender.com.

FIGURE 5
FIGURE 5 2 and hs4 enhancers (206).Additionally, AhR activation in the presence of LPS stimulation altered NFkB/Rel protein binding within the hs1.2 and hs4 enhancers by reducing RelB binding and increasing RelA binding, which corresponded to altered enhancer activity (146).Other studies evaluated the effect of AhR activation on Pax5 protein levels.Pax5 is a negative regulator of mouse 3′RR activity and antibody production but B-cell activation and differentiation into plasma cells results in decreased Pax5 expression, thus lifting the repressive effects of Pax5 on 3′RR activity and antibody production (204, 207, 258-262).AhR activation in combination with cellular stimulation reversed the stimulation-mediated decrease in Pax5 expression, which correlated with the inhibitory effect on IgH expression by AhR ligands

1 For the mouse 3 '
RR, the AhR ligands analyzed included dioxin, indolo[3,2-b]carbazole, omeprazole, and primaquine.Only dioxin was evaluated for the mouse and human hs1.2 enhancer and the mouse hs4 enhancer.2Low concentration increased 3'RR activity and high concentration decreased 3'RR.*, AhR-dependent based on AhR KO or AhR antagonist.^mutation of ER binding site decreased CSR but estrogen treatment also decreased CSR.ND, not determined.

TABLE 1
Effects of dioxin or dioxin-like chemicals on antibody production and role of the AhR.

TABLE 1 Continued
Exposure to Dioxin or Dioxin-like Chemicals1

TABLE 2
Species differences in AhR ligand specificity and activation of the AhR.

TABLE 4
Comparison of mouse versus human 3'RR characteristics and effect of AhR activation.Effect of AhR activation on 3′RRInhibit ND the left of the ideogram.The red box highlights the 14q32.33band harboring the IgH region that is annotated in detail in the lower part of the figure.