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Transcription Factors in the Regulation of Leydig Cell Gene Expression and Function

Cell differentiation and acquisition of specialized functions are inherent steps in events that lead to normal tissue development and function. These processes require accurate temporal, tissue, and cell-specific activation or repression of gene transcription. This is achieved by complex interactions between transcription factors that form a unique combinatorial code in each specialized cell type and in response to different physiological signals. Transcription factors typically act by binding to short, nucleotide-specific DNA sequences located in the promoter region of target genes. In males, Leydig cells play a crucial role in sex differentiation, health, and reproductive function from embryonic life to adulthood. To better understand the molecular mechanisms regulating Leydig cell differentiation and function, several transcription factors important to Leydig cells have been identified, including some previously unknown to this specialized cell type. This mini review summarizes the current knowledge on transcription factors in fetal and adult Leydig cells, describing their roles and mechanisms of action.


INTRODUCTION
Localized in the testicular interstitium, Leydig cells are the principal source of testosterone and insulin-like 3 (INSL3), two hormones that regulate male reproductive development and function. In mammals, there are at least two distinct populations of Leydig cells, fetal Leydig cells (FLC) and adult Leydig cells (ALC), which are responsible for the synthesis of steroid hormones in the prenatal and postnatal testes, respectively [reviewed in (1,2)]. Steroidogenesis is a multi-step process requiring various transporters and enzymes to convert cholesterol into a steroid hormone [reviewed in (3)]. The expression of the genes coding for these steroidogenic proteins is finely regulated to avoid steroid hormone insufficiency or excess across the lifespan.
Transcription factors (TFs) are fundamental to the regulation of gene expression. They are specialized proteins that recognize and bind to regulatory DNA sequences, modulating the rate of gene transcription [reviewed in (4)]. TFs typically recruit or interact with other TFs forming a unique molecular code that is key for specifying temporal-and tissue-specific gene expression as well as hormone responsiveness in hormone-sensitive target tissues. Moreover, TFs exhibit a dynamic behaviour that is characterized by their ability to interact with various partner proteins and to regulate different target genes according to many determinants such as cell type, development stage, and signal stimulus, among others.
In recent years, the development of novel and powerful methodological approaches in molecular genetics has led to the emergence of new information regarding the role of TFs in the regulation of Leydig cell differentiation and function, and by extension, in male fertility and reproductive health. In this mini review, we provide a brief overview of the roles and mechanisms of action of some of the most characterized TFs in Leydig cells. We have adopted the most recent classification of TFs, which is based both on amino acid sequence homology and the tertiary structure of their DNA-binding domains (5). Using this classification, TFs that have been identified in Leydig cells are presented in Table 1; Table 2 lists the target genes for these TFs in Leydig cells.

AP-1 Factors
The activator protein 1 (AP-1) is a dimeric complex that includes members of the JUN, FOS, activating transcription factor (ATF), and musculoaponeurotic fibrosarcoma (MAF) families of TFs (54). Among the AP-1 members, JUN and FOS are the best characterized. The JUN subfamily comprises three members (cJUN, JUNB, and JUND) while four members compose the FOS subfamily [cFOS, FOSB, Fos-related antigens 1 (FRA-1, FOSL1), and Fos-related antigens 2 (FRA-2, FOSL2)]. Members of the JUN family can homodimerize or heterodimerize, whereas FOS family members only form heterodimers. The DNA sequence recognized by AP-1 members differs according to the dimer involved. JUN : JUN and FOS : JUN dimers recognize the TPA-response element (TRE; TGA(C/G)TCA) and the cAMP-responsive element (CRE; TGACGTCA), whereas ATF dimers preferentially recognize the CRE motif, and MAF dimers bind to MAF recognition elements (MAREs), a long palindromic sequence that contains TRE or CRE motifs (55) [reviewed in (56)].
AP-1 members were first described in Leydig cells in the late 1990s (57). AP-1 factors regulate several genes in Leydig cells such as the steroidogenic acute regulatory protein (Star) gene, which is activated by cJUN (6,7,12). In addition, cJUN cooperates with other TFs, including GATA4, STAT5B, and NUR77 leading to a stronger activation of the Star promoter (7)(8)(9). Both cJUN and cFOS regulate Star promoter activity by recruiting CREB and CBP (10). Transcription of the gap junction protein alpha1 [Gja1, also known as connexin43 (Cx43)] gene, involved in the initiation and maintenance of sperm production, is also controlled by cJUN, JUNB, and FOSL2, and by a cJUN/cFOS cooperation (11,58). Furthermore, the ferredoxin 1 (Fdx1) promoter is activated by a cJUN/SF1 cooperation (12). Fdx1 is a partner of Cyp11a1, participating in the conversion of cholesterol into pregnenolone, the first and rate-limiting step in steroidogenesis. It is important to note that the nature of the cJUN dimerization partner influences its role in gene regulation. For example, the combination of either FOSL2 or cFOS with cJUN inhibits the stimulatory effect of cJUN on the Star promoter (6,10,59). AP-1 factors in Leydig cells have been reviewed elsewhere (56).

CREB-Related Factors
CREB-related factors include three members: CRE-binding protein (CREB), cAMP response element modulator (CREM), and CRE-activating transcription factor (ATF-1). CREB factors homodimerize and heterodimerize with other CREB members and with other bZIP TFs, such as AP-1 members (60). CREB factors regulate transcription by binding to a CRE motif (TGACGTCA) similar to that recognized by AP-1 members, leading to overlap and redundancy in their activities (61). Although CREM is the most abundant member in MA-10 Leydig cells, all CREB members activate Star transcription through CRE elements located in the proximal promoter region (13,14). Moreover, CREB factors cooperate with SF1 (NR5A1, Ad4BP) to enhance Star transcription (15). CREB also stimulates CKLFSF2B promoter activity in response to LH/cAMP (16). Cklfsf2b codes for a protein that inhibits steroidogenesis in Leydig cells (16). Therefore, CREB is involved in both activation and repression of steroidogenesis in Leydig cells depending on its target genes.

C/EBP-Related Factors
Members of the CCAAT/enhancer binding protein (C/EBP) subfamily contain a bZIP DNA-binding domain and regulate gene expression by binding to the sequence (A/G)TTGCG(C/T) AA(C/T) as homo-or heterodimers (62). C/EBPb is the predominant member in Leydig cells (17,63) where it activates Star transcription alone and in cooperation with SF1 and GATA4 (17)(18)(19). C/EBPb also cooperates with NF-kb p50 to stimulate Nur77 promoter activity in Leydig cells (20). The Nur77 gene encodes the orphan nuclear receptor NUR77, which regulates several genes involved in steroidogenesis in Leydig cells (see Section 3.1.2, NGFI-B/NR4A Receptors, below).

Class of Nuclear Receptors With C4 Zinc Fingers
TFs belonging to the nuclear receptor class respond to extracellular and intracellular signals to regulate gene expression. They also regulate cellular functions within the cytoplasm (64). In this section we present the nuclear receptors for which the roles and mechanisms of action are, or have begun to be, characterized in Leydig cells. Detailed information can be found in a review article dedicated to nuclear receptors in Leydig cells (65).
COUP-TFII (NR2F2, ARP1) and COUP-TFIII (NR2F6, EAR2). NR2Fs have been implicated in various physiological and developmental processes by regulating the expression of numerous genes [reviewed in (66,67)]. Via their double zinc finger DNA-binding domain, NR2F factors bind as monomers to the nuclear receptor element AGGTCA and its variants. They also bind as dimers to direct (DR), inverted (IR), and everted (ER) repeats separated by 1-12 nucleotides (68). Of the NR2F subfamily members, COUP-TFII is by far the most abundant in Leydig cells. Although COUP-TFII is present in mice interstitial cells from early fetal life throughout adulthood, it is only associated with steroidogenically active ALC in postnatal life (24). COUP-TFII is a marker of stem cells giving rise to the ALC population (24,69). In vivo studies using mouse models have shown that COUP-TFII is crucial for Leydig cell development and male reproductive function (70,71). In Leydig cells, COUP-TFII regulates the expression of several genes involved in lipid metabolism, male gonad development, and steroidogenesis (28). COUP-TFII activates Star, Insl3, and Amhr2 expression by binding to their respective promoter sequences (24)(25)(26). It cooperates with SF1 on the Star and Insl3 promoters (24,25) and with SP1 on the Amhr2 promoter (26). The Akr1c14 gene, which codes for the 3a-HSD enzyme that catalyzes the interconversion of dihydrotestosterone (DHT) into 5a-androstane-3a,17b-diol (3a-diol), is activated by COUP-TFII in cooperation with MEF2 (27). COUP-TFII also *The letter preceding the name of the gene refers to the species: m, mouse; r, rat; h, human.
activates the expression of Gsta3 and Inha, genes involved in the inactivation of reactive oxygen species and in the homeostasis of the hypothalamic-pituitary-gonadal axis, respectively (28). Expression of several other Leydig cell genes including Cyp17a1, Hsd3b1 and Cyp11a1 is reduced in Coup-tfii null mice (71) and in COUP-TFII-depleted MA-10 Leydig cells (28), implying a role for COUP-TFII in their expression.

FTZ-F1-Related/NR5A Receptors
The nuclear receptor 5A (NR5A) family comprises two members: steroidogenic factor 1 (NR5A1, Ad4BP, SF1) and liver receptor homolog 1 (NR5A2, LRH1, FTF). Both factors share high sequence similarity, bind to the same DNA motif, regulate common target steroidogenic genes, and exhibit overlapping expression in several tissues [reviewed in (78,79)]. Despite this, they have nonredundant roles and cannot fully compensate for each other [reviewed in (78,79)]. NR5A members regulate gene expression by binding as monomers to the sequence (T/C)CAAGGTCA located in the promoter region of target genes. SF1 was initially identified as a tissue-specific activator of several cytochrome P450 steroid hydroxylase genes (38,80). SF1 is essential for steroidogenesis, reproduction, and male sex differentiation, as revealed by mutations in the SF1 gene in humans and in mouse models where adrenal and gonadal development and function are impaired (37,(81)(82)(83)(84) [reviewed in (85,86)]. Interestingly, Sf1 knockdown in MLTC-1 Leydig cells leads to downregulation of Star and Cyp11a1 and accumulation of neutral lipids and cholesterol (37). Moreover, SF1 is one of only a handful of TFs that can convert fibroblasts into functional Leydig-like cells, revealing the pivotal role of this nuclear receptor in Leydig cells (87,88).

DAX-Related/NR0B Receptors
The DAX-related receptor (NR0B) family comprises two members: critical region on the X chromosome gene 1 (NR0B1, DAX1) and small heterodimer partner (NR0B2, SHP). They lack the typical zinc finger DNA-binding domain and therefore act mainly as transcriptional repressors by inhibiting the activity of other TFs (90,91). Both members are present in Leydig cells and act as homodimers or heterodimers (92).
In Dax1-deficient mice, testis cord organization is compromised and FLC development is arrested (93). In vitro studies in Leydig cell lines revealed that DAX1 represses steroidogenesis by inhibiting Star expression, while silencing Dax1 expression increases Star transcription leading to enhanced steroidogenesis (45). DAX1 interacts with and represses the activity of NUR77 and SF1, inhibiting Star expression (36,45). Interestingly, Dax1 knockdown in MA-10 Leydig cells decreases Cyp11a1 and Star expression suggesting that DAX1 could also act as a coactivator in addition to its repressor role (94).
SHP is a repressor of steroidogenesis. In mouse Leydig cells, Shp expression is reduced by hCG treatment (95). In Shpdeficient mice, testosterone levels as well as Star, Cyp11a1, and Hsd3b1 mRNA levels are increased leading to premature sexual maturation (96). SHP inhibits steroidogenesis by interacting and repressing the activity of LHR1 (96). Shp mRNA levels are significantly reduced in COUP-TFII-and MEF2-depleted Leydig cells, indicating that Shp expression requires these two TFs (28, 97).

Two Zinc-Finger GATA Factors
The six GATA members (GATA1 to 6) are crucial for the development and function of several tissues, including the male gonad [reviewed in (98,99)]. GATA factors regulate gene expression by binding via their two zinc fingers to the DNA sequence (A/T)GATA(A/G) in the promoter region of target genes. Of the six GATA factors, GATA4 is the most abundant in Leydig cells in vivo (100)(101)(102). Its expression is also the broadest being present from the onset of testis morphogenesis and into adult life (103). Considered one of the first gonadal markers in both sexes, GATA4 is required for urogenital ridge development in mice and later for mammalian gonadal differentiation (103,104). A Sf1-Cre mouse line, which expresses the Cre recombinase in several tissues including Leydig, Sertoli and adrenal cells, was used to conditionally inactivate Gata4. The resulting males were undervirilized and had small testes lacking mature sperm (105), thereby supporting a role for this factor in male reproductive function. Transcriptomic analysis of GATA4-depleted MA-10 Leydig cells revealed several deregulated pathways, including cholesterol metabolism and steroidogenesis (46). Consistent with this, GATA4 stimulates the transcription of several genes expressed in Leydig cells such as HSD3B2 (32), Cyp19a1 (106), Star (46, 106), Inha (106), Sf1 (106), Amhr2 (46), and Srd5a1 (46). GATA4 also cooperates with cJUN, C/EBPb, and MEF2 to upregulate Star expression (7,18,47). These results emphasize the indispensable role of GATA4 in the differentiation and function of FLC and ALC (46,107). The critical nature of GATA4 in the Leydig cell differentiation is further supported by the demonstration that GATA4, along with SF1 and DMRT1 or NUR77, are sufficient to reprogram fibroblasts toward the Leydig-like cell fate (87,88).

Forkhead Box (FOX) Factors
The forkhead box A3 (FOXA3) is the only member of the FOXA subfamily present in the testes, mainly in ALC (51,108,109). So far, the only direct target identified for FOXA3 in Leydig cells is the gene coding for the platelet-derived growth factor receptor alpha (Pdgfra) (51), that in response to PDGF signaling, acts in Leydig cell differentiation and testis organogenesis (110). In cAMP-induced steroidogenesis, FOXA3 is proposed to repress Nur77 expression, which in turn reduces steroidogenic gene expression and testosterone production (111). These findings indicate that FOXA3 participates actively in the control of Leydig cell function and male fertility.

MEF2 Subfamily
The Myocyte Enhancer Factor 2 (MEF2) factor subfamily comprises four members (MEF2A-2D) that share two highly conserved domains, a MADS box and a MEF2 domain, involved in dimerization and DNA binding [reviewed in (112)]. MEF2 factors form homo-and heterodimers that bind the sequence YTAWWWWTAR (Y=C/T, W=A/T, R=G/A) in the promoter region of their target genes. Because of their conserved DNAbinding domain, MEF2 members share common targets and can compensate for each other. MEF2 members also display unique spatiotemporal patterns in different tissues. Due to their divergent transactivation domain, MEF2 factors respond to different signals and interact with different partners, leading to specific gene expression [reviewed in (112)].
Although widely studied in other organs, the presence of MEF2 in the testes, more specifically in Sertoli and Leydig cells, was only reported in 2014 (52). In Leydig cells, MEF2A and MEF2D and to a lesser extent MEF2C, are expressed from early gonadal development into adulthood (52). MEF2A/2D-depleted MA-10 Leydig cells produce less steroid hormone demonstrating that MEF2 factors have a role in male reproductive function (47). Consistent with this, microarray analysis of MEF2A/2D-depleted MA-10 Leydig cells identified several differently regulated genes known to be involved in fertility, gonad morphology, and steroidogenesis (97). To date, direct gene targets for MEF2 factors in Leydig cells include Nur77 (52), Gsta1-4 (53), Star (involving a MEF2/GATA4 cooperation) (47), and Akr1c14 (through a cooperation with COUP-TFII) (27). The complete network of genes regulated by MEF2 factors in Leydig cells as well as MEF2 interacting partners remain to be fully elucidated.

STAT Factors
The signal transducer and activator of transcription (STAT) family consists of seven proteins [reviewed in (113)]. Cytokines and growth factors activate STAT members through the Janus kinase (JAK) signaling pathway. In the nucleus, STAT factors regulate gene transcription by binding as homo-or heterodimers to the g-interferon-activated sequence (GAS; TTCN 3 GAA) in the promoter region of target genes. So far, STAT5B is the only STAT factor identified in Leydig cells (114). In these cells, STAT5B is activated by growth hormone, an important regulator of steroidogenesis (8). STAT5B activates Star expression directly by binding to a GAS element and in cooperation with cJUN (8). STAT5B also activates the Nur77 promoter (8).

OTHER TRANSCRIPTION FACTORS PRESENT IN LEYDIG CELLS
Other TFs have been described in Leydig cells, but their mechanisms of action remain poorly characterized. This includes the nuclear factor E2-related factor-2 (NRF2, NFE2l2), which is an important modulator of reactive oxygen species levels, especially in aging Leydig cells (115)(116)(117). Furthermore, the brain and muscle arnt-like protein-1 (BMAL1), a component of the circadian clock system, is also directly involved in the control of Leydig cell function in different species, by regulating the expression of Star, Hsb3b, and Cyp11a1 (21,118,119). Finally, members of the nuclear factor kappa-beta (NF-kb) family, involved in immune and inflammatory responses, also contribute to the regulation of steroidogenesis in Leydig cells (20,31,120).

CONCLUDING REMARKS
As described in this mini review, several TFs belonging to different classes and families are pivotal to ensure proper Leydig cell differentiation and function. This underscores the complex regulatory mechanisms involved. Most of the knowledge acquired so far has relied on in vitro analyses of regulatory elements of genes expressed in Leydig cells. Although we are far from fully understanding all the signals, pathways, and TFs involved, technological advances and novel mouse models will certainly lead to significant discoveries in the coming years.

AUTHOR CONTRIBUTIONS
KM wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
Supported by a grant from the Canadian Institutes of Health Research (funding reference number MOP-81387) to JT. KM is the recipient of a studentship from the Fonds de recherche du Quebec-Sante.