An Analysis of the Implication of Estrogens and Steroid Receptor Coactivators in the Genetic Basis of Gender Incongruence

2.1 The genetic component

A genetic component should also be taken into account since different genes start to express before the formation of the testes [7, 8]. In fact, in mammals, sexual differentiation begins at the time of fertilization, through a different chromosomal complement in males and females, and will be driven by the SRYgene, which will guide the undifferentiated gonad towards the formation of the testes. Then, the testosterone will masculinize specific regions of the brain, either directly, or indirectly, through the action of the aromatase [9, 10].

Most studies about the genetic basis of GI analyzed the implication of some DNA polymorphisms related to ERs, α and β, the AR, the aromatase CYP19A1 or the CYP17A1 [11, 12, 13, 14, 15, 16, 17, 18, 19, 20] as well as the interaction effects (epistasis) among them [15, 21]. A summary of the principal studies about the genetic component of GI is shown in Table 1. This gene selection is based on the hypothesis that a small variation in the DNA sequence of these genes would imply a high variability in the sensitivity of the hormonal receptors to their ligands.

Henningsson et al. [11] were the first group to analyze three repeat polymorphisms, located in the estrogen receptor beta, the androgen receptor, and the aromatase genes in a trans female population. They found a relationship between the number of repetitions and gender incongruence. They found longer estrogen receptor and androgen receptor polymorphisms in the trans female population. Later, Hare et al. [12] replicated Henningsson’s study in a bigger population, finding longer androgen receptor polymorphisms. However, when Ujike et al., [13] analyzed the same polymorphisms in a Japanese population, they did not find any statistical difference. These and others polymorphisms were analyzed in a Spanish population by our group. Our results confirmed the involvement of both estrogen receptors (alpha and beta) in gender incongruence. Part of this data was confirmed by Foreman et al. [15].

2.2 The epigenetic component

An epigenetic component may also be involved since there is evidence that some environmental factors play a role in the sexual differentiation of the brain. For example, in mice, the sex difference in maternal anogenital licking of male compared with female pups produces a different methylation of the estrogen receptor α promoter in the preoptic area [22]. And in humans, certain environmental factors, such as a short crossover hormonal treatment (only 6 months), can modify the methylation profile of the estrogen receptor α promoter in a trans population [23, 24].

3.1 Estrogens

Estradiol (E2) exerts a wide variety of effects on growth, development, the function of reproductive systems and regulation in the central nervous system [25, 26]. The mechanism of action of the two ER isoforms α and β consists of binding with the E2 ligand to obtain the receptor’s dimerization (αα, αβ or ββ), originating the necessary conformational changes in the ligand binding domain (LBD) [27] and binding with high affinity to specific DNA sequences called estrogen response elements (EREs) [27] in the genes that are regulated by E2 (Figure 1). This conformational change in the LBD allows coactivators and other co-regulating proteins to be recruited. We must point out that this step is critical for the transcriptional regulation of genes induced by E2 [28].

Furthermore, estrogens are produced in many regions of the brain including the cortex, the hippocampus, the cerebellum, the hypothalamus and the amygdala, among others [29]. The actions of estrogens in the developing brain are generally permanent and range from the establishment of sexual differences to generalized trophic and neuroprotective effects [30]. In addition, estrogens are an important regulator of brain growth and differentiation, and ERs α and β are found in both the developing [31] and the adult human brain [32].

3.2 Estrogen and androgen receptors are transcription factors

We must point out that the packaging of DNA into chromatin causes a general repression of gene activity, and transcription factors function to relieve this chromatin-mediated repression [33].Thereby, once attached to their ligands, the receptors dimerize, enter the nucleus, and interact with the promoter regions of the target genes, modulating the expression of multiple genes in collaboration with some steroid coactivators (Figure 1). In the case of the AR, its ligand is androgen [34] while for the ERs, it is estrogen, 17β-Estradiol (E2) in particular [26].

3.3 Steroid receptor coactivators

Proteins called SRCs (Steroid Receptor Coactivators) serve as primary coactivators that interact with the complex formed by E2 and the hormonal receptor. Additionally, SRCs recruit multiple secondary coactivators such as p300 and the CREB-binding protein (also known as CREBBP) [35]. Both SRCs and p300 are the first coactivators that are coupled to the E2-ER complex [36] to activate the transcriptional process of the genes that are E2 targets (Figure 1).

Coactivators are proteins that influence the ability of the transcription factors to activate or inhibit expression of multiple genes in a cascade mode [37]. Given the intimate relationship between steroids and brain dimorphism, and coactivators and gene transcription, and since ERs α-β and the AR are hormonal receptors that act as transcription factors, it was clear that we should hypothesize the implication of DNA coactivators in the process of GI.

4.1 The characteristics of the study

Genomic DNA was extracted from 94 Spanish transgender individuals (47 transmen and 47 transwomen) versus94 Spanish cis gender individuals (44 cismen and 50 ciswomen). The transgender population was diagnosed and recruited through the Gender Unit of the Clínic Hospital of Barcelona (Spain) and the cisgender population was selected from a country census (Pizarra) matching by geographic origin and race.

We analyzed 247 polymorphisms distributed in the coactivators NCOA-1 (63 SNPs), NCOA-2 (64 SNPs), NCOA-3 (30 SNPs), NCOA-4 (4 SNPs), NCOA-5 (8 SNPs), p300 (9 SNPs) and CREBBP (69 SNPs) (Table 2), in a population of 94 Spanish transgender individuals versus94 Spanish cisgender individuals, with the same geographic origin, race and biological sex. All the polymorphisms were in Hardy–Weinberg equilibrium.

4.2 The findings

4.3 Concordance of our findings with the literature about receptor coactivators

To our knowledge, no studies have been published about the role of steroid receptor coactivators in the genetic basis of GI. Our data are in concordance with a recent work that showed that the nuclear receptor coactivators, NCOA-1, NCOA-2 and p300, are essential for efficient ER transcriptional activity in the brain [33, 38]. Furthermore, NCOA-1 and NCOA-2 are distributed in several specific areas of the brain in different proportions, such as the hypothalamus and the hippocampus, showing at the same time, difference in the coupling with the ERs [38, 39]. These differential interactions between NCOA-1 and NCOA-2 with the ER subtypes α and β suggest that these brain regions have distinct expression pattern of co-regulators, and understanding how nuclear receptor coactivators function with various steroid receptors is critical to understanding how hormones act in different brain regions.

Moreover, our results are also in concordance with the study of the functional significance of the nuclear receptor coactivator NCOA-1 in the developing brain [40]. The authors, Auger et al., investigated the consequence of reducing NCOA-1 protein during sexual differentiation of the brain, and reported that reducing this protein interferes with the defeminizing actions of estrogen in neonatal rat brains. Their data indicated that NCOA-1 expression is critically involved in the hormone-dependent development of normal male reproductive behavior and brain morphology. Thus, our data are in agreement with the results of Auger et al., [40] since the polymorphic analysis of this coactivator showed significant differences when allelic and genotypic frequencies and haplotypes analyses were carried out.

Our data are also in concordance with other studies about the critical role of p300 and CREBBP in ERα transcription. p300 and CREBBP are two of multiple secondary coactivators recruited by NCOA1, NCOA2 or NCOA3 to form a receptor-coactivator complex that can promote chromatin remodeling and facilitate transcriptional activation [35]. In our work, we found statistical significances in p300, CREBBP, NCOA1 and NCOA2, but not in NCOA3.

Transcription by RNA polymerase II requires the coordinated action of multiple factors such as DNA-binding factors, coactivators, chromatin remodeling, with the basal transcriptional machinery. Futhermore, p300 and CREBBP, do not bind DNA on their own, but they play an essential role in the transcription process mediated by E2 [33, 41]. Thus, ERα functions cooperatively with p300 and CREBBP to increase transcription [42]. Yi et al. [35] demonstrated the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator, and p300 as secondary coactivator. The structural model suggests that the ER binds the ERE-DNA as a dimer and then recruits two NCOAs; these two coactivators, in turn, secure one molecule of p300 to the complex through multiple contacts.

It is very important to maintain the nucleotide sequence of the genes encoding the coactivators in order to maintain the interactions of the ER-E2 -NCOA -p300-CREBBP complex and thus perform the genomic function of estrogens. Therefore, our data are in concordance because finding significant polymorphisms in the sample analyzed may result in ineffective or low effective interactions affecting the E2 target genes involved in brain dimorphism.

In our work, we found 2/9 polymorphisms with statistically significant differences (P9 and P10) in p300 in the interaction analysis with covariate “sex”. This implied differences in haplotype distribution according to sex, and thus, the haplotype 2 (T-A) (Table 8) only showed significant differences in the population assigned as females at birth. The other haplotypes did not show differences in the distribution between cis and trans population, nor in males or females.

Based on experiments in rodents, it is believed that male sexual differentiation of the brain is caused by androgens, after conversion to estrogens by the aromatase. Moreover, observations in human subjects show that the direct effects of testosterone on the developing fetal brain and also during puberty, are of great importance for the development of male gender identity [43]. However, the analysis of the androgen coactivator NCOA-4 did not show any significant data.

Currently, it is still very difficult to interconnect molecular, brain, and behavioral findings [44] due to the complex interactions among behavior, genes, hormones, receptors and enzymes. But we must point out that MRI studies in people with GI, show characteristic brain profiles [5]. Both trans populations (females and males) share some common features: firstly, the involvement of the two ERs in neurobiological origin [21] and, secondly, their cortex, in some regions, is thicker than in cismen [5]. These observations support the hypothesis that transmen and transwomen undergo an atypical developmental process with respect to the sexual differentiation of their cortex [5], hypothetically, under the influence of brain estrogens, androgens, their receptors and some of their coactivators.

4.4 Consistency of the findings with the current hypothesis of a multiplicity of mechanisms involved in the complex “mosaic” model of the mammalian brain

Finally, our data are also consistent with the current hypothesis about the existence of a complex “mosaic” model of the mammalian brain [45], with a multiplicity of mechanisms involved, allowing a variable degree of masculinization/feminization within the brain. The simple model according to which testosterone masculinizes the brain of men away from a predetermined female profile, has been replaced by a complex model, according to which sexual effects on the brains of women and men are exerted by a complex combination of behavior, genetic, epigenetic and hormonal factors [45].