Multiple Sry genes in the Okinawa spiny rat encode proteins with an A-to-S substitution in the HMG domain that retain DNA-binding ability

ABSTRACT

The mammalian sex-determining gene SRY is highly conserved across species, with only a few exceptions. The Japanese rodent genus Tokudaia is known for its unique sex chromosome evolution. The Okinawa spiny rat T. muenninki (TMU) acquired neo-sex chromosomes with multiple Sry copies by sex chromosome–autosome fusions. All SRY copies in TMU have a substitution from alanine to serine at position 21 in the high-mobility group (HMG) box, a critical DNA-binding domain, suggesting that they are nonfunctional. However, the sex determination system in TMU remains unclear, in part because the species is endangered and it is therefore extremely difficult to obtain experimental samples. In this study, we performed in silico and in vitro analyses to investigate the molecular properties and function of SRY using recently obtained whole-genome sequence and RNA-seq data. A comparison of SRY sequences from 225 species showed that TMU is the only species with a substitution at the 21st position. This result highlights the rarity and specificity of this substitution. Structural predictions, DNA docking simulations, electrophoretic mobility shift assays and fluorescence anisotropy showed that although the affinity was slightly lower than that of the mouse homolog, DNA-binding ability was retained. However, Sry expression was not detected in the testis, liver or brain in adult TMU. The complete absence of Sry expression in the adult tissues, despite an intact sequence, strongly indicates a loss of regulatory function. These findings provide insight into the unique evolution of the Sry gene in this species.

INTRODUCTION

Therian mammals generally have heteromorphic sex chromosomes called X and Y chromosomes. Sex determination is primarily contingent on the differential development of bipotential embryonic gonads into the testis or ovary. In most placental mammals, testis differentiation is triggered by the activation of a sex-specific gene called sex-determining region Y (SRY) located on the Y chromosome (Berta et al., 1990). SRY binds to its specific target upstream of SOX9 via the high-mobility group (HMG) domain and promotes testis differentiation (Kashimada and Koopman, 2010). Mutations of SRY can have dramatic effects, with the potential to cause disorders of sex development (DSD); for example, 20% of patients with Swyer syndrome (pure gonadal dysgenesis) harbor an SRY mutation and/or deletion (Culha et al., 2012). SRY mutations in patients with DSD are generally found in the HMG domain (Graves et al., 1999). Several recent reports have revealed that a single amino acid substitution in the HMG domain, such as N65H (Assumpção et al., 2002), R76S (Ambulkar et al., 2021) and A66T (Wang et al., 2018), results in a female phenotype in genetically male individuals (2n = 46, XY), with some studies demonstrating the disruption of HMG–DNA binding activity (Werner et al., 1995). These findings indicate that HMG is a critical domain for sex determination.

The SRY-dependent sex determination mechanism is conserved in most mammalian species, with very rare exceptions. One exception is the genus Tokudaia, which includes three species of rodents native to Japan, all of which inhabit a small area of the Ryukyu Islands. As a consequence of deforestation, logging, hunting by domestic feral cats and introduced mongoose, and competition with the common black rat, all Tokudaia species are now considered endangered and are protected by the Japanese government as natural monuments (Yamada et al., 2011). All Tokudaia species have unique sex chromosomes and sex determination mechanisms. Two of the three species, Amami spiny rat (T. osimensis) and Tokunoshima spiny rat (T. tokunoshimensis), have completely lost their Y chromosome along with the Sry gene (Honda et al., 1977; Murata et al., 2010). Both species show an odd number of chromosomes (T. osimensis is 2n = 25 and T. tokunoshimensis is 2n = 45) (Honda et al., 1977; Kobayashi et al., 2007) with only a single X chromosome present in both sexes (XO/XO), indicating that a new Sry-independent sex-determining mechanism has evolved (Kuroiwa et al., 2010; Kimura et al., 2014; Otake and Kuroiwa, 2016). Recently, we reported a new sex-determining mechanism in which a heterologous duplication that includes cis-regulatory elements upstream of Sox9 is essential for testicular differentiation in the XO/XO spiny rat (Terao et al., 2022).

Tokudaia muenninki (TMU) is the only species in the genus with XX/XY sex chromosomes (Tsuchiya et al., 1989). Due to sex chromosome–autosome fusions, the sex chromosome of TMU is extremely large, and more than 70 copies of Sry are distributed along the entire long arm of the Y chromosome (Murata et al., 2012). Among these, at least three copies retain an open reading frame (Murata et al., 2010). The function of the Sry gene is thought to be lost in all copies sharing the same amino acid substitution from alanine (A) to serine (S) at position 21 of the HMG domain, critical for SRY–DNA binding ability (Murata et al., 2010). Duplication of the Sry gene has also been observed in common rats, Rattus norvegicus; however, the amino acid substitution from A to S has not been observed (Prokop et al., 2013). Analyses of Sry expression in embryonic gonads at the sex-determining stage are necessary for understanding sex determination in TMU. However, it is nearly impossible to obtain experimental materials, such as embryonic gonads, from this endangered species. To study mammalian SRY evolution, cytogenetic and molecular biological analyses of TMU have been conducted using fibroblast cells collected from a small tissue sample from adult animals captured and released during ecological research in September 2008 (Yamada et al., 2011).

Several previous studies have evaluated the effects of the A-to-S substitution in the HMG domain on sex determination. For example, we previously evaluated the function of TMU Sry using transgenic (TG) mice; however, the inserted SRY proteins were not stably expressed and became fragile in TG mouse cells, and the function in sex determination could not be examined (Ogata et al., 2019). TMU SRY does not contain a long polyglutamine tract (Q-rich domain), which is a mouse-specific structure (Bowles et al., 1999). The Q-rich domain might play an essential role in protein stabilization in mice; similarly, human SRY does not promote testis differentiation in mice during the sex determination stage (Tsuji-Hosokawa et al., 2022).

In this study, we performed in silico and in vitro analyses using information from assembled whole-genome data for TMU as well as recombinant SRY proteins. We compared the structure of the HMG domain, HMG–DNA binding activity and affinity in TMU SRY with these features of the HMG domain in mouse SRY to improve the accuracy of functional prediction, aiming to reveal the unique sex determination system in this rodent species.

RESULTS

A-to-S substitution is unique in TMU SRY

We evaluated the A-to-S substitution at the 21st position of the HMG domain in the TMU Sry gene through a comparative sequence analysis. We obtained 537 SRY sequences from various mammalian species available in National Center for Biotechnology Information (NCBI) databases. We trimmed these sequences to retain only the first 76 amino acids of the HMG domain, which is highly conserved across mammalian species. To screen for intra-specific variation, 228 sequences from 225 species were finally retained (Supplementary Fig. S1). This dataset included three previously identified TMU SRY sequences: SRY1, SRY2 and SRY3 (Murata et al., 2010). A multiple sequence alignment of the HMG domain was generated using the ClustalW algorithm, revealing strict conservation at position 21 of the HMG domain across mammalian species, with the exception of TMU (showing an A-to-S change) (Fig. 1, Supplementary Fig. S1). We also employed the Maximum Likelihood Estimate of Substitution Matrix to further elucidate the nature of amino acid substitutions in the HMG domain. A matrix was generated with 1,000 bootstrap iterations. Substitution patterns and rates were estimated under the Jones–Taylor–Thornton (Jones et al., 1992) model (+G). A discrete gamma distribution was used to model evolutionary rate differences among sites (five categories, (+G), parameter = 1.1882), with relative values of instantaneous rates (r values) simplified by setting the sum of r values to 100. The amino acid frequencies were 7.69% (A), 5.11% (R), 4.25% (N), 5.13% (D), 2.03% (C), 4.11% (Q), 6.18% (E), 7.47% (G), 2.30% (H), 5.26% (I), 9.11% (L), 5.95% (K), 2.34% (M), 4.05% (F), 5.05% (P), 6.82% (S), 5.85% (T), 1.43% (W), 3.23% (Y) and 6.64% (V), and A was most likely to be replaced with threonine (T) (r = 1.82), followed by serine (r = 1.55) and valine (r = 1.16) (Supplementary Fig. S2). These results suggest that although A can be easily replaced with S, the residue is highly conserved across mammals other than TMU. These results strongly indicate that the 21st amino acid in the HMG domain is functionally important.

Fig. 1. Sequence alignment of the SRY HMG domain in representative mammalian species. The Okinawa spiny rat T. muenninki is the only species with the A-to-S substitution at position 21 of the HMG domain (red box).

A-to-S substitution does not have a major impact on HMG domain structure

To understand the potential functional implications of the A-to-S substitution, we analyzed the three-dimensional (3D) structure of the HMG domain. Four models with the highest prediction scores, corresponding to wild-type mouse HMG, mouse HMG with an A-to-S substitution, wild-type TMU HMG and TMU HMG with an S-to-A substitution, were generated using the Robetta computational platform. An alignment of these 3D models revealed structural similarities across all samples, regardless of whether A or S was present at the 21st position (Fig. 2A and 2B). Remarkably, angstrom error estimates were low for all of the predicted models (Supplementary Fig. S3). For residues just after A or S at position 21 of HMG, specifically beginning from glutamine (Q) at position 23, the error exceeded 1.0 Å, peaking at 3.4 Å around S at position 26 and decreasing to less than 1.0 Å by Q 31. Subsequent significant angstrom error estimates were observed after tyrosine (Y) at position 71, reaching over 18 Å at position 80 of the HMG domain, indicating that the lack of the Q-rich domain in the C-terminus region of the SRY protein contributes to the difficulties in model prediction. However, the Q-rich domain varies considerably among species and is not highly conserved, even within the rodent family; its inclusion in the structural model could interfere with the interpretation of the A-to-S substitution in the HMG–DNA complex. Therefore, we focused exclusively on the HMG-box domain, which is the critical DNA-binding domain of the SRY protein.

Fig. 2. 3D docking results showing the similarity in HMG–DNA binding between TMU (A) and mouse (B). The positions magnified in (C) and (D), respectively, are indicated by red boxes. Molecular properties of the S variant (green) indicate more hydrogen bonds within the folded protein structure compared with those for the A variant (pink) in both TMU (C) and mouse (D).

The structure of the DNA–HMG domain interaction remains intact in A and S variants

To assess the impact of the A-to-S substitution on the DNA–HMG domain interaction, we performed in silico DNA-binding predictions using the pyDockDNA platform. The selected HMG models in PDB format were used as receptors, while the conserved rodent Enh13 nucleotide sequence (5′-TCCACTTCTAAACAAACAGCTGAGGGGAGT-3′) was used as the ligand (Gonen et al., 2018). The docking analyses revealed that the DNA-binding patterns of the TMU HMG were similar to those of mouse HMG, regardless of whether A or S was present at the 21st position (Fig. 2, Supplementary Fig. S4). The A-to-S substitution was positioned near the protein surface; however, it did not exhibit direct contact with the Enh13 DNA model in the docking results when the interaction bond length was set at 4 Å. This suggests that the substitution does not impact the DNA–HMG domain interaction substantially. Of note, the hydroxyl group in S resulted in the formation of an additional hydrogen bond within the folded protein structure between S at position 21 and arginine (R) at position 17. The length of this hydrogen bond was 2.767 Å in TMU HMG and 2.773 Å in the mouse homolog (Fig. 2C and 2D). These subtle structural differences suggest that the A-to-S substitution influences intramolecular stability, although the effect is weak and unlikely to alter or disrupt the DNA–HMG domain interaction, as evidenced by the lack of significant change between the A/S and S/A transitions. The protein contact map results retrieved from Mapiya revealed that rather than A/S at position 21 of the HMG domain, in TMU the nearby area R17 exhibited obviously strong interactions with the nucleotide molecules (Supplementary Fig. S5). Notably, the bonds were weaker and some contact was lost in TMU HMG compared with that of mouse, suggesting that the additional hydrogen bond between S at position 21 and R at position 17 detaches R17 or widens the area of contact with DNA.

HMG with both S and A exhibits DNA-binding ability

To validate the presence and expression of recombinant SRY proteins of mouse and TMU, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of recombinant proteins corresponding to the HMG proteins with the A-to-S or S-to-A substitution in a bacterial system was confirmed by Coomassie Blue staining after SDS-PAGE. The electrophoresis results showed clear bands between the 10-kDa and 15-kDa molecular weight markers at the predicted size of the HMG protein, which is approximately 12.9 kDa (Fig. 3A). Western blotting using an anti-SRY antibody showed single bands at positions consistent with those observed in the Coomassie Blue-stained gels (Fig. 3B). Together, these findings confirmed the validity of the recombinant HMG proteins for in vitro HMG–DNA binding analyses.

Fig. 3. SDS-PAGE (A) and western blotting (B) results for recombinant HMG proteins after removal of the chaperone protein TF.

To explore the binding ability of the DNA–HMG domain with the A-to-S substitution, we employed electrophoretic mobility shift assays (EMSAs) using digoxigenin-labeled oligonucleotide probes. We designed DNA probes to simulate the HMG-binding region on Enh13, a testis-specific Sox9 enhancer sequence in the mouse genome (Gonen et al., 2018); this region contains the essential SRY/SOX9 binding motif (5′-A/T AACAA A/T-3′) (Ferrari et al., 1992; King and Weiss, 1993; Giese et al., 1994; Murphy et al., 2001; Phillips et al., 2004). We confirmed that the binding motif sequence was conserved in the upstream region of Sox9 in TMU (Supplementary Fig. S6).

As shown in Figure 4, the EMSA results revealed shifted bands corresponding to the formation of DNA–protein complexes in all HMG samples derived from both mouse and TMU. The presence of these shifted bands in all Enh13 BS(+) binding complexes having a complete SRY core binding motif sequence indicated that both A- and S-containing HMG domains were capable of binding to the DNA probes. Importantly, no bands were observed in the negative control lanes Enh13 BS(-), which lacked the SRY core binding motif (5’-AAACAAA-3’), confirming the specificity of the interactions. The HMG–DNA binding intensity was comparable for the A or S variant within both mouse and TMU, suggesting that this specific amino acid substitution did not critically impair the HMG–DNA interaction. This finding aligned with computational predictions, which indicated that the A-to-S substitution does not play a critical role in modulating the DNA-binding activity of TMU SRY, at least when considering the HMG domain in isolation. However, the HMG–DNA complexes formed by TMU proteins, for both the A and S variants, exhibited weaker shifted-band intensity than those of the corresponding mouse HMG–DNA complexes by observation. This suggested that while TMU SRY retained the ability to bind DNA through its HMG domain, its binding affinity was lower than that of the mouse SRY protein.

Fig. 4. EMSA results for mouse WT (A), mouse A to S, TMU WT (S) and TMU S to A. The negative control, Enh13 BS (-), showed no band, unlike the HMG–Enh13 binding complex Enh13 BS (+).

A-to-S substitution causes a slight reduction in DNA-binding affinity

To complement the qualitative EMSA results, we performed fluorescence anisotropy assays to quantify the effects of the A-to-S substitution on DNA-binding affinity. Fluorescence anisotropy, also known as fluorescence polarization, is a sensitive technique used to assess molecular complex formation by measuring the degree of polarization of emitted light after excitation (Lacowicz, 2006). When a fluorophore-labeled DNA molecule binds to a protein, the rotational motion of the complex slows, decreasing the polarization of emitted light. This method thus provides a more precise understanding of the binding affinity between HMG proteins and DNA. By diluting the HMG proteins over a range of concentrations, we traced the extent of complex formation from the anisotropy values. The highest anisotropy values observed at 10 µM HMG were 0.178 for the S-substituted mouse variant, 0.179 for both the S and A variants of TMU, and 0.181 for the wild-type mouse SRY, which indicate that all HMGs form a similar DNA complex at a certain DNA concentration.

As the dilution progressed and protein concentrations decreased, the anisotropy values declined gradually, until they reached approximately 0.067 at 0.0019 µM protein. An anisotropy plot revealed notable differences in binding affinity patterns between species and variants. For concentrations ranging between 0.01 µM and 1 µM, the reduction in fluorescence anisotropy initiates at higher concentration levels in the S mutants of mice and TMU, compared to the wild-type mouse SRY, indicating a decreased affinity for the target DNA. This trend was more pronounced in the S variant of TMU, suggesting a weaker interaction with DNA than that for the A variant (Fig. 5).

Fig. 5. Fluorescence anisotropy results on a log scale for comparisons of binding affinity among mouse WT (A), mouse A to S, TMU WT (S) and TMU S to A in a reaction solution containing 0–10 µM HMG protein. The fluorescence anisotropy was measured after exactly 15 min of incubation with excitation and emission at 495 and 515 nm, respectively. Error bars represent the standard deviation of three independent experiments. The solid lines represent fit to the Hill equation.

To quantify the binding affinities further, we calculated the dissociation constant (K_d) for each HMG–DNA complex. The calculated K_d values revealed a gradient in binding affinity across the different variants. The lowest K_d value, indicating the highest affinity, was observed for the wild-type mouse HMG–DNA complex (K_d = 0.12 ± 0.02 µM) followed by the serine-substituted mouse HMG protein with a slightly higher K_d of 0.14 ± 0.03 µM. For TMU HMG, both variants displayed higher K_d values, with the A variant showing a K_d of 0.16 ± 0.02 µM and the S variant exhibiting the weakest binding affinity with a K_d of 0.22 ± 0.03 µM (Table 1). These findings supported the conclusion that in TMU the A-to-S substitution resulted in a slight reduction in DNA-binding affinity, particularly in the TMU HMG protein. However, the A-to-S-substituted mouse HMG showed slightly different affinity within the margin of error compared to wild type, although both still exhibited a higher binding affinity than the wild type of TMU (S-variant). This observation suggests that the differences in binding complexes and affinities are species-specific, influenced by other residues within the HMG-box domain and their interactions with neighboring amino acids.

Table 1. K_d values for HMG-DNA complexes

Protein	Kd(µM)
Mouse WT (A)	0.12 ± 0.02
Mouse A to S	0.14 ± 0.03
TMU WT (S)	0.22 ± 0.03
TMU S to A	0.16 ± 0.02

No expression of Sry in brain, testis or liver tissues of adult male TMU

Sry is typically expressed in various adult tissues in addition to the embryonic gonads at sex determination. Human SRY is expressed not only in adult gonadal tissues but also in the adrenal glands, kidneys, lungs, heart and brain (Clépet et al., 1993; Salas-Cortés et al., 1999; Dewing et al., 2006). In mouse, Sry is expressed in the brain (Lahr et al., 1995). In rats, there are tissue-specific differences in the expression patterns of Sry copies in adult testis and adrenal glands (Turner et al., 2007). We therefore examined the mRNA expression levels of Sry in adult brain, testis and liver tissues of male TMU to further explore its functions in TMU. Although high-quality genome assemblies and gene annotations for TMU have been reported (Okuno et al., 2023), these annotations do not include the Sry copies identified in earlier studies (Murata et al., 2010). Therefore, we predicted localization within the genome to assess whether Sry mRNA was actively expressed in adult tissues. The expression analysis revealed that none of the Sry copies (Sry1, Sry2, Sry3) were expressed in any of the adult tissues of TMU (Fig. 6).

Fig. 6. Expression analysis in brain (A), testis (B) and liver (C) in adult male TMU. Sry copies (Sry1, Sry2 and Sry3) were not expressed. The TATA box binding protein (Tbp) gene was used as a control.

DISCUSSION

SRY typically functions via two mechanisms, DNA binding and DNA bending, both of which are critical for Sox9 activation and lead to the promotion of testis development from bipotential gonads. As a transcription factor, SRY binds to the target DNA via the HMG domain and recognizes the minor groove containing AACAA. In human SRY, 10 hydrogen bonds between the DNA and amino acids have been confirmed in R7, N10 and Y74 on one side with I13 as an axle, and the other side in R20, N32 and S36, corresponding to R4, N7, Y71, I10, R17, N29 and S33, respectively, in the HMG domain alignment shown in Figure 1 (Racca et al., 2024). All of these positions are highly conserved among mammalian species (Supplementary Fig. S1). The HMG domain induces the sharp bending of DNA (at an angle of approximately 70°–85°), facilitating chromatin remodeling for gene regulation through the hydrophobic intercalation of amino acid residues, such as I13 (I10 in Fig. 1), into the DNA (Racca et al., 2024). This sharp bend may provide kinetic stability for SRY–DNA transcriptional activation, and a change in the angle caused by an amino acid substitution is expected to alter overall transcriptional activity dramatically (by several fold) (Ukiyama et al., 2001; Li et al., 2006), potentially leading to de novo sex reversal (Pontiggia et al., 1994).

Our results confirmed the A-to-S substitution at the 21st position of the HMG domain in TMU. The A at this position is highly conserved across mammals and was detected in 228 SRY sequences from 225 species evaluated in this study; this high conservation suggests that the site is critical in sex determination. The discovery of the substitution in TMU raises important questions about the evolutionary forces driving this unique substitution in the species. Given that the A-to-S substitution was not observed in any other mammalian species, it likely represents an isolated evolutionary event, specific to TMU, that occurred before SRY duplication events. However, except for I10, which is M in rodent species, and R4, which is C in TMU SRY1 (BAJ08419) and H in TMU SRY3 (BAJ08421), other amino acids involved in binding to the minor groove of DNA are strictly conserved. Although the additional hydrogen bonds between S21 and R17 have been confirmed and likely explain the relatively weak bond between DNA and R17, in silico and in vitro results indicated that the SRY of TMU retains its DNA-binding ability, at least via the HMG domain. There was a slight reduction but not complete loss of the DNA-binding activity of SRY. Our findings resolve, in part, a critical question regarding the effects of the A-to-S substitution on protein–DNA interactions and suggest that it influences HMG–DNA bending rather than DNA-binding activity. Further studies of HMG–DNA bending of TMU SRY may shed light on the effects of the A-to-S substitution and its consequences for sex determination in this species.

The lack of Sry expression in adult tissues (testis, brain and liver) of TMU is a significant finding. Sry is expressed in the gonads during embryonic development and plays a key role in initiating testis differentiation across many taxa. However, it is also known to be expressed in adult tissues, although its function is less well understood at this stage. Since Sry was originally identified as a homolog of Sox3 on the X chromosome (Weiss et al., 2003), which is involved in the development of the pituitary and central nervous system, it may function in processes other than sex determination. In fact, Sry is expressed in different kinds of cells and tissues outside the sex determination stage in some mammals. Human SRY is expressed in the adult frontal and temporal cortex and medial rostral hypothalamus (Mayer et al., 1998). Mouse Sry is expressed in the germ cells in adult testis (Hacker et al., 1995). The complete absence of Sry expression in adult brain and testis in TMU, despite an intact sequence, strongly indicates a loss of regulatory function. The regulatory region that controls expression in the adult tissues and embryonic gonads is probably different. The proximal promoter sequence is conserved in the three Sry sequences used in this study (Murata et al., 2010; Ogata et al., 2019). However, even in humans and mice, no regulatory sequences such as enhancers that control tissue-specific expression of SRY/Sry have been reported. Therefore, a comparative analysis of the regulatory regions of each copy will be necessary in the future, because the embryonic gonads of this species are not available and experiments to confirm the expression cannot be performed.

In conclusion, this study provides the first evidence that TMU Sry retains its DNA-binding ability via the HMG domain; the A-to-S substitution at the 21st position results in slightly weaker binding, as measured by in silico and in vitro assays. However, Sry expression was not observed in adult TMU. Further studies, including the crystal structure determined through NMR or HMG–DNA bending analyses, would clarify the changes in HMG–DNA activity caused by the A-to-S substitution. Finally, the lack of Sry expression in adult tissues raises important questions regarding the regulation of the gene in TMU. Our findings provide insight into the unique evolution of Sry gene in this species.

MATERIALS AND METHODS

Gene expression analysis

The coding sequences of TMU Sry from NCBI databases, BAJ08421, BAJ08420 and BAJ08419 (Murata et al., 2010), were used as queries for GMAP v2023.10.10 (Wu and Watanabe, 2005) to annotate the location against the whole genome reported by Okuno et al. (2023). In the annotation results, the predicted locations were replaced with the three curated transcripts. The modified general annotation file and RNA-seq data for adult brain (DRR059293), testis (DRR059295) and liver (DRR059294), which are included in data (DRA004624) in the NCBI Sequence Read Archive (Murata et al., 2016), were mapped to the genome using STAR v.2.7.9 (Dobin et al., 2013). TPM (transcripts per million) values were then calculated using RSEM V.1.3.3 (Li and Dewey, 2011) with default parameters for single-end reads. A bar plot of the TPM per gene was generated using RStudio (V.2012.15.2) (http://www.posit.co/, accessed 10 Nov 2023) and the package ggplot2. The TATA box binding protein (Tbp) gene was used as an internal control.

HMG sequence analysis

All SRY sequences from mammalian species in NCBI databases were downloaded, trimmed to obtain the HMG domain, and filtered to remove duplicates. An alignment was generated using AliView (Larsson, 2014), an alignment viewer released under the Open-Source Software License GNU General Public License, version 3.0 (GPLv3); the source code is available at GitHub (https://github.com/AliView/AliView). MEGA X (Kumar et al., 2018) was used for statistical model prediction and to generate a substitution matrix of the HMG domain via maximum likelihood methods based on the JTT model and gamma-distributed rates with 1,000 bootstrap replicates (Jones et al., 1992).

HMG domain structure and DNA-binding complex prediction

A previously reported TMU Sry sequence, SRY2 (BAJ08420) (Murata et al., 2010), was used for structure prediction based on the highest alignment score for HMG sequences compared with mouse, the model, and species with structural annotations. Along with TMU, mouse SRY (NP_035694) was saved as a FASTA file and the amino acid sequence was trimmed to retain the HMG box. A-to-S substituted sequences were generated and then submitted to Robetta, a protein structure prediction server developed by the Baker lab at the University of Washington (Kim et al., 2004). 3D structures were predicted using a deep learning-based method, RoseTTAFold (Baek et al., 2021). The predicted structure of the HMG-box protein together with the SRY-binding DNA sequences were subsequently analyzed using pyDockDNA, a web server for predicting protein–DNA interactions based on pyDock and FTDOCK algorithms (Rodríguez-Lumbreras et al., 2022). We selected a 30-bp DNA sequence from the testis-specific enhancer of Sox9, Enh13 (706 bp), which is conserved among rodent species, including rat and mouse, as the HMG-binding DNA (Gonen et al., 2018). The PDB files were downloaded and the model with highest prediction score was visualized. The molecular structures were compared between each sample using UCSF Chimera and Chimera X (Pettersen et al., 2004). Protein–DNA contact maps were generated using Mapiya (Badaczewska-Dawid et al., 2022) with default parameters.

Preparation of the plasmid carrying the HMG sequence

For HMG domain recombinant protein constructs, primers were designed to specifically amplify the HMG region. All products were double-digested with restriction enzymes (TaKaRa, Kusatsu, Japan) and ligated into the XhoI/HindIII restriction site of pCold TF, a fusion cold shock expression vector (TaKaRa). Ligation products were electrophoresed using 1% agarose gels stained with MIDORI Green (Nippon Genetics, Tokyo, Japan) and purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL, Düren, Germany). The products were confirmed by DNA sequencing using the Hitachi Applied Biosystems ABI 3100 Avant DNA Sequencer (HITACHI, Tokyo, Japan) after transfection into the JM109 Escherichia coli strain (ECOS, Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. After colony selection using antibiotic (50 µg/ml ampicillin in 1.5% LB agarose medium) and plasmid extraction using the FastGene DNA Extraction Kit (Nippon Genetics), inserted DNA was confirmed again by colony PCR using the SimpliAmp Thermal Cycler (Thermo Fisher Scientific, Tokyo, Japan) and KAPA Taq PCR Kit (Nippon Genetics).

Recombinant HMG domain protein production

To produce the recombinant protein, recombinant plasmid vectors carrying the HMG sequence were transformed into E. coli BL21 (ECOS, Nippon Gene), a bacterial recombinant protein expression system, using the heat shock method. The transfected BL21 E. coli was precultured overnight in 10 ml of 2× Yeast Extract Tryptone liquid medium containing 50 µg/ml ampicillin, incubated at 37 °C, and shaken at 200 rpm in a shaking chamber. On the following day, large-scale recombinant protein production was performed by transferring the precultured E. coli into a flask with 2× YT medium (per liter: Yeast extract 20 g, Tryptone 20 g, NaCl 10 g, 2 M NaOH 3.2 ml). Additional incubation was performed until the cell concentration measured by the optical density at 600 nm reached 0.6, and samples were immediately cooled with ice water for 15 min. After cold shock, the cultures were shaken at 15 °C for 30 min before the addition of 1 mM isopropyl β-d-thiogalactopyranoside, incubation for another 24 h, and collection by centrifugation (10,000 g, 4 °C, 5 min per 500 ml of culture). Cell lysis was performed by mixing the collected suspension with 35 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 0.1% NP-40), 0.05 g of lysozyme, 200 μl of 1 M MgCl₂, 20 μl of 1 M MnCl₂ and 200 μl of 1 mg/ml DNase I, with mixing using a magnetic stirrer at 4 °C for 30 min. The lysed samples then were sonicated using the Branson SONIFIER 250 for 10 min and centrifuged at 18,000 rpm for 30 min to remove the cell debris. The HMG-TF recombinant proteins were purified using the ÄKTA prime plus HPLC system on a HisTrap HP column (Cytiva, Uppsala, Sweden) following the instructions provided. The fusion tag (trigger factor [TF] chaperone) was removed from the HMG-TF recombinant protein by incubating the column-purified HMG-TF proteins overnight at 4 °C with the HRV 3C protease expressed in and purified from E. coli BL21 (DE3). The mixture was purified again using a nickel resin column (TaKaRa) according to the manufacturer’s instructions. The expression of purified recombinant proteins was confirmed through SDS-PAGE and western blotting.

Electrophoretic mobility shift assay (EMSA)

Specific double-stranded DNA sequences for SRY-binding activity were designed, annealed stepwise, and labeled with digoxigenin (DIG) according to the DIG Gel Shift Kit, 2nd generation protocol (Roche, Mannheim, Germany). HMG–DNA binding reactions were performed according to the kit instructions. Briefly, the labeled DNA and purified HMG proteins were mixed with EMSA binding buffer, poly [d(I-C)] as a non-specific competitor, and poly-l-lysine and incubated for 15 min at 25 °C. Samples were then immediately cooled on ice to stop the reaction. The DNA–protein complex mixture was separated on an 8% polyacrylamide gel for 80 min at 70 V in 5% TBE buffer. Samples were electro-transferred to a 5% TBE-presoaked Amersham Hybond-N+ nylon membrane (GE Healthcare, Chalfont St. Giles, United Kingdom). The membrane was then baked at 120 °C for 15 min before being treated with filter papers presoaked with 2× SSC solution. DNA fixation was performed following the crosslinking method using a UV transilluminator (Vilber Lourmat, Collégien, France). The membrane was then incubated in blocking solution provided in the EMSA kit for 30 min at room temperature and further incubated with a 1/20,000-diluted DIG-POD (150 U Anti-Digoxigenin-POD Fab fragments) (Roche) antibody for 2 h. Chemiluminescent detection was performed using the LAS-3000 Imager (Fujifilm, Tokyo, Japan) using a chemiluminescent agent, following the instrument’s protocol.

Fluorescence anisotropy spectroscopy

HPLC-grade fluorescent label oligonucleotides were synthesized to resemble those used in EMSAs with an additional fluorescent group, 6-carboxyfluorescein (6-FAM), bound to the 5′ end (Fasmac, Kanagawa, Japan), and dissolved in TEN buffer (10 mM Tris, 0.1 M NaCl and 1 mM EDTA (pH 8.0)). Together with the complementary strand, the oligomers were heated at 95 °C for 5 min and annealed by cooling to room temperature. The DNA–protein complexes were obtained using premixed 50-nM annealed oligomers with HEPES binding buffer (40 mM HEPES, 40 mM KCI, 20% glycerol, 100 mg/ml BSA, pH 7.5) incubated at 37 °C for 15 min, and the HMG protein was titrated from 10 μM with the total volume of the reaction at 1 ml at 25 °C. To avoid non-specific binding, 10 μg/ml sheared salmon sperm DNA (BioDynamics Laboratory, Plainview, USA) was added. Fluorescence anisotropy of serial titrations of HMG–DNA complexes (0.002 to 10 μM HMG) was performed after incubation using an FP-8500 fluorometer (JASCO, Tokyo, Japan). Measurements were taken at excitation and emission wavelengths of 495 and 515 nm, respectively, with spinning at 250 rpm. The average values from three replications were calculated using the anisotropy equation (Equation 1) (Nam et al., 2020). K_d values were compared among samples.

  

$$Anisotropy \left(r\right)=\frac{I_{\mathrm{V}\mathrm{V}}-{GI}_{\mathrm{V}\mathrm{H}}}{I_{\mathrm{V}\mathrm{V}}-2{GI}_{\mathrm{V}\mathrm{H}}}\mathrm{ }\text{(1)}$$

where I is the fluorescence intensity, the first character of the subscript is the polarization direction of the excitation light, and the second character is the direction of the emission. V and H indicate the vertical and horizontal directions, respectively, and G is an instrument correction factor determined by excitation of the protein solution with horizontally polarized light. K_d for the HMG–DNA complex was determined by using the Hill equation and the nonlinear least-squares method (IGOR Pro, Wavemetrics, Lake Oswego, USA):

  

$$Anisotropy\left(r\right)=base+\frac{max-base}{1+{\left(\frac{K_{\mathrm{d}}}{\left[\mathrm{P}\mathrm{t}\right]}\right)}^n}\mathrm{ }\text{(2)}$$

where base is the baseline fluorescence anisotropy in the absence of binding, max is the maximum fluorescence anisotropy, [Pt] is the total protein concentration and n is the Hill coefficient.

DECLARATIONS

Data availability: All data are contained within the manuscript.

Conflict of interest: The authors declare that they have no conflict of interest with the content of this article.

Ethical considerations: The Okinawa spiny rat (Tokudaia muenninki, 2n = 46, XX/XY) is endangered (The IUCN Red List of Threatened Species, DOI: e.T21972A22409515), and has been protected by the Japanese government as a natural monument and national endangered species of wild fauna and flora since 1972 and 2016, respectively. No animal samples were used in this study.

ACKNOWLEDGMENTS

This work was supported by MEXT Scholarship: IGP special Quota, and JSPS KAKENHI Grant Numbers 23K23930, 22H02667 and 19H03267.

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