Stimulated by retinoic acid gene 8 (STRA8) interacts with the germ cell specific bHLH factor SOHLH1 and represses c‐KIT expression in vitro

Abstract STRA8 (Stimulated by Retinoic Acid Gene 8) controls the crucial decision of germ cells to engage meiotic division up and down‐regulating genes involved in the meiotic programme. It has been proven as an amplifier of genes involved in cell cycle control and chromosome events, however, how STRA8 functions as negative regulator are not well understood. In this study, we demonstrate that STRA8 can interact with itself and with other basic Helix‐Loop‐Helix (bHLH) transcription factors through its HLH domain and that this domain is important for its ability to negatively interfere with the Ebox‐mediated transcriptional activity of bHLH transcription factors. Significantly, we show that STRA8 interacts with TCF3/E47, a class I bHLH transcription factors, and with SOHLH1, a gonadal‐specific bHLH, in male germ cells obtained from prepuberal mouse testis. We demonstrated that STRA8, indirectly, is able to exert a negative control on the SOHLH1‐dependent stimulation of c‐KIT expression in late differentiating spermatogonia and preleptotene spermatocytes. Although part of this results were obtained only ‘in vitro’, they support the notion that STRA8 interacting with different transcription factors, besides its established role as ‘amplifier’ of meiotic programme, is able to finely modulate the balance between spermatogonia proliferation, differentiation and acquisition of meiotic competence.

ATRA treatment 4,5 is expressed at relatively high levels in male and female pre-meiotic germ cells. [6][7][8] Moreover, Stra8 expression appears indispensable for the mitotic/meiotic switch in female PGCs and in male germ cells. 6,[9][10][11] Besides, STRA8 promotes spermatogonial differentiation. 12 Despite the well-demonstrated importance of STRA8 in both sexes, its molecular function/s. has only recently been highlighted. 13,14 First described as a cytoplasmic protein, 5 it has been successively demonstrated that it actually shuttles between the nucleus and cytoplasm. 15 It has also been shown that STRA8 can bind DNA and possess a transcriptional activation domain in the C-terminal region of its molecule. [13][14][15][16] Very recently, it has been demonstrated that STRA8 directly up-regulates a large set of genes by binding to their promoter in the male germ cells at the preleptotene stage. 12 Ishiguro et al, (2020) 14 identified a STRA8-interacting protein, MEIOSIN that is also required for the mitosis-meiosis switching. Both MEIOSIN and STRA8 possesses a conserved region of the protein containing a Helix-Loop-Helix (HLH) domain. This is a homo-or hetero-dimerization domain that characterizes the large family of HLH transcription factors and consists of highly conserved amphipathic helices separated by a loop of variable length and sequence. 17 HLH proteins, through the regulation of gene expression, orchestrate cell cycle, cell lineage commitment and cell differentiation. 18 Different groups of HLH proteins can be distinguished based on the presence or absence of additional functional domains. 17,18 Almost all HLH proteins possess a region of basic residues adjacent to the HLH domain that facilitates binding to DNA at a specific sequence motif known as EBox (CANNTG) or at the related NBox (CACNAG). 17 X-ray crystallographic analyses of bHLH proteins have defined the invariant basic sequence ERXR as the determinant for EBox recognition. 19 Class I bHLH factors, also known as E proteins, are encoded by Beside, other HLH proteins function as transcriptional repressors, for example, HAIRY, HES and STRA13/DEC2 and have an unusual DNA-binding domain in which a proline is present in the basic region which gives specificity for NBox binding. 20 A distinct subfamily of HLH proteins, the ID proteins (Inhibitor of DNA binding), lacks the basic region adjacent to the HLH domain which is essential for DNA binding. In mammalian cells, the ID family contains four proteins (ID1-4) 21 that affect the balance between cell growth and differentiation by negatively regulating the function of bHLH transcription factors. 22 ID proteins bind to both class I and class II bHLH proteins and inhibit their ability to bind DNA through the formation of inactive heterodimers. 22 Consequently, the expression of genes that possess the EBox sequence in their regulatory elements is repressed.
In the first helical region of the STRA8-HLH domain, there is a basic Nuclear Localization Sequence that might mediate DNA binding to Ebox sequence. However, STRA8 lacks the first glutamate and last arginine residues of the ERXR motif for Ebox recognition.
In the present paper, we aimed to characterize the action of STRA8 as a transcriptional regulator and to investigate whether its HLH domain, by mediating the interaction with others HLH protein/s including the germ cell specific bHLH factor SOHLH1 could modulate their transcriptional function.

| Male and female germ cells isolation
Postnatal male germ cells were obtained from 10 days post-partum (dpp) CD1 albino mice as reported. 23 When indicated, separation of KIT-positive from KIT-negative male germ cells was performed by magnetic-activated cell sorting (mini-MACS) with CD117 conjugated microbeads (Miltenyi Biotech) as previously described. 23 PGCs from 12.5 dpc ovaries were purified using the Mini-MACS immunomagnetic sorting method. 24 Female germ cells were isolated from 14.5 and 16.5 days post-coitum (dpc) ovaries following digestion for 10 minutes in Trypsin/EDTA solution (Lonza) and mechanical disaggregation in a monodispersed cell suspension. The cells were cultured for 30 minutes at 37°C and 5% CO 2 in air in DMEM and samples of cells remaining in suspension, roughly consisting of 70%-80% of oocytes, were collected for RNA preparation.

| DNA constructs
Plasmids expressing the fusion protein GFP-STRA8 and Myc-STRA8 were constructed as reported. 15

| Cell transfection
HEK293T cells were transiently transfected with the different plasmids by using the jetPei TM Polyplus transfection reagent (Polyplus-Transfection SA, SIC, Italy), according to the manufacturer's protocol.

| Immunofluorence analysis
For immunohistochemistry, serial 6 μm thick sections were obtained from testes of 10 dpp mice, fixed in buffered formalin and paraffin embedded. Slides were dewaxed, rehydrated and microwaved in 10 mmol/L sodium citrate buffer, pH 6 for 20 minutes. After blocking with 10% goat serum (GS), sections were incubated with rabbit

| Purification of GST fusion proteins
The

| Pull-down assay
Transfected HEK293T cells and male germ cells were lysed in a pulldown buffer (PB: 25 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 0.5% Triton-X100, 1 mmol/L EDTA, 5% glycerol and PIC) and protein concentration was determined using a BCA protein assay kit (Pierce). Cross-link reaction was blocked with 100 mmol/L ethanolamine pH 8.9, and the beads were washed in PBS and used for pull-down experiments. Cell extracts (500 µg-1 mg) were pre-cleared on glutathioneagarose beads for 1 hour at 4°C, then incubated under constant shaking with GST or GST fusion proteins, that were cross-linked or not to the beads, for 2 hours at 4°C. After three washes with the PB buffer, adsorbed proteins were eluted in SDS sample buffer and analysed by Western blotting.

| Western blot analysis
Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer and boiled for 5 minutes. Protein was separated on either 10% or 8% SDS-Page gels and transferred to PVDF Transfer Membrane Hybond ™ (Amersham Bioscience). Membranes were saturated with 5% non-fat dry milk in PBS containing 0.1% Tween20

| Statistical analyses
All experiments were replicated at least three times. Data were expressed as mean ± Standard Deviation (SD). Student's t-test and one-way ANOVA analysis were performed using Graphpad Prism software.

| STRA8 interacts with itself in vitro and in vivo through the HLH domain
Since STRA8 possesses a well conserved HLH domain (aa 17-84) that could mediate protein-protein interaction, we tested the ability of the protein to interacts with itself. GST and GST-STRA8 fusion proteins were used in pull-down assays with lysates obtained from HEK293T transfected with pcDNA3-Myc-STRA8 or from 10 dpp male germ cells. As shown in Figure 1, both recombinant (1A) and endogenous (1B) STRA8 were able to specifically bind to GST-STRA8 and not to GST alone, thus indicating that the protein efficiently interacts in the in vitro assay. We further investigated whether STRA8 was able to form complexes in intact cells by a co-immunoprecipitation (CO-IP) assay. Efficient co-precipitation of transfected Myc-STRA8 with GFP-STRA8 ( Figure 1C) and endogenous STRA8 ( Figure 1D) was observed.
To verify if HLH domain was involved in the interaction, we generated  processes. 26 Single cell RNA-sequencing analysis from mouse testis in the perinatal period 27 or from sorted KIT-negative and KIT-positive cells, 28 indicated that E47 is expressed in both spermatogonia populations. Moreover, this gene is also expressed in the preleptotene cells. 13 To verify the expression of E47 in male germ cells, we performed RT-PCR with total RNA obtained from immunomagnetic-purified undifferentiated (KIT-negative) and differentiating (KIT-positive) P10 dpp germ cells. As shown in Figure 2A, we confirmed that E47 was expressed in both cell populations. As a control for the enrichment of the cell separation, Stra8 expression was evaluated, confirming its presence prevalently in the KIT-positive germ cell population 23,29 (Figure 2A).

| STRA8 interacts in vitro and in vivo with class I bHLH E47 through its HLH domain
Immunolocalization of STRA8 in the testis from P10 mice showed an uneven distribution of the protein in different tubules as expected 8 with spermatogonia and preleptotene positive cells ( Figure 2B). E47, on the other hand, is expressed in the nuclei of the cells in each tubule thus indicating that STRA8-positive cells also express the bHLH factors at this age. RT-PCR performed in pre-meiotic (12.5 dpc) and meiotic (13.5-14.5 dpc) female germ cells obtained by mouse embryos, showed that E47 transcript was detected in all female germ cell samples analysed ( Figure 2C).
By using the co-immunoprecipitation assay, we next investigated whether STRA8 and E47 interacted. A Myc-tagged Stra8 expression vector was transfected into HEK293T cells together with pcDNA3-E47-Flag using a Flag-empty vector as a control and total extracts were subjected to immunoprecipitation with an anti-FLAG antibody. The results in Figure 3A showed that STRA8 was co-immunoprecipitated with E47 and the specificity of the immunoprecipitation was confirmed with an isotype-matched nonspecific mouse IgG. Co-immunoprecipitation performed using total lysates from P10-isolated male germ cells, that include both STRA8 and E47 expressing cells ( Figure 2B) confirmed that the anti-E47 antibody co-immunoprecipitated STRA8 also in these cells ( Figure 3B). The same result was obtained when anti-STRA8 antibody was used to immunoprecipitate E47 from nuclear extracts of the germ cells ( Figure 3C).
To analyse which domain of STRA8 was important for the interaction with E47, we performed a GST pull-down assay. Cross-linked GST-STRA8 and GST-MYOD fusion proteins were used for interaction with Flag-E47 that was transiently transfected in HEK293T cells.
GST protein was used as a negative control. As shown in Figure 3D,

GST-STRA8 as GST-MYOD specifically interacted in vitro with E47
whereas the control GST protein exhibited no interaction. When this assay was repeated with the deletion mutants GST-HLH-only-STRA8 and GST-ΔHLH-STRA8 fusion proteins, only the mutant with the HLH domain formed a specific complex with E47 ( Figure 3E), thus indicating that this region mediates also the interaction of STRA8 with the bHLH factor.

| STRA8 inhibits bHLH-dependent transcriptional activity through its HLH domain in transfected cells
Although STRA8 is described as a HLH-transcriptional regulator, 9,13,15 in the literature it is not clear if the HLH domain is involved in its transcriptional activity, also because when the HLH region was deleted or muted, STRA8 did not localize to the nucleus. 13,15 Moreover, STRA8 did not bind the regulated gene promoters to the canonical EBox motif recognized by the tissue specific bHLH transcription factors. 13 Figure S1). Therefore, we hypothesize that STRA8, binding to bHLH factors might negatively modulate their action as the basic region-lacking ID protein do. 21 To test this, we investigated if STRA8  As showed in Figure 5C, recombinant STRA8 was able to interact with over-expressed SOHLH1 also in intact cells. The interaction between STRA8 and SOHLH1 was evident also when the co-immunoprecipitation experiments were repeated immunoprecipitating SOHLH1 from male germ cells nuclear extracts incubated with STRA8 antibody ( Figure 5D). It has been demonstrated that SOHLH1 can positively regulate c-Kit expression by binding its promoter at EBox containing sequence. 29,32 To analyse the effect of  Figure 5E c-Kit promoter activity was induced by SOHLH1 construct, while recombinant STRA8 had no effect. Moreover, the co-transfection of STRA8 with SOHLH1 vector reduced the transcriptional activation in a dose-dependent manner. The inhibitory activity of STRA8 was related to the HLH domain because when the Myc-HLH-only-STRA8 deletion mutant was used in the transcription assay, it was able to reduce the stimulation of luciferase activity by SOHLH1 in a dose-dependent manner ( Figure 5F).

| STRA8 interacts with the germ cell specific bHLH factor SOHLH1 and represses its induced c-KIT expression in vitro
To verify if STRA8 was also able to negatively modulate the expression of c-Kit, we over-expressed SOHLH1 in P19EC cells (that expresses low level of c-Kit) with or without Myc-STRA8. After 24 hours from transfection, we observed that SOHLH1 induced a significant increase of the c-Kit transcript and when STRA8 was co-transfected with the bHLH factor, it was able to almost completely abolish the c-Kit stimulation by SOHLH1 ( Figure 5G). This result was also observed when c-KIT protein expression was analysed by Western blot analysis ( Figure 5H).

F I G U R E 3 STRA8 interaction with E47 in vivo and in vitro. A, Western blot analysis with anti-Myc, anti-FLAG and anti-GAPDH
antibodies of the immunoprecipitation assay performed with control mouse IgG or anti-FLAG antibody of total lysates (TL) of HEK293T over-expressing FLAG-E47 and pcDNA3-Myc-STRA8. B-C, Co-immunoprecipitation assay for endogenous E47 and STRA8. B, TLs obtained from P10 dpp germ cell suspension were incubated with anti-E47 or rabbit IgG as a control. Immunoprecipitated proteins were analysed by Western blot using anti-STRA8, anti-E47 and anti-GAPDH antibodies. C, Proteins from nuclear extracts obtained from the same cell suspension were immunoprecipitated with anti-STRA8 antibody and E47, STRA8 and HISTONE H3 were evaluated by Western blotting. D, Western blot analysis for Flag-E47 in pull-down assays of TL of HEK293T cells expressing Flag-E47 with fusion proteins GST-STRA8 and GST-MYOD (positive control). GST was used as negative control. Coomassie staining shows the purified GST and GST fusion proteins (lower panels). E, Pull-down assay was repeated using the same TLs probed with GST-HLH-only-STRA8 or GST-ΔHLH-STRA8 and analysed by Western blot with anti-Flag antibody. The amount of GST fusion proteins used in the assay was showed in a Coomassie staining of the gel The results were expressed as relative luciferase activity setting that of the 4RE-Luc vector as 1 (mean ± SD, *P < 0.05, **P < 0.01 compared to mock: § §P < 0.01compared to MYOD; #P < 0.05 compared to E47). B, pHA-MyoD and pcDNA3-E47-Flag were co-transfected in HEK293T cells with increasing amounts of pcDNA3-Myc-Stra8 and luciferase assays were performed as above (mean ± SD, *P < 0.05, **P < 0.01). C, Different concentrations of Myc-HLH-only-Stra8 deletion mutant were analysed in the same EBox-mediated luciferase assay system (mean ± SD, **P < 0.01). D, Specificity of the STRA8 inhibitory action. HEK293T cells were transiently transfected with the indicated reporters (200 ng) in which luciferase transcription was driven by AP1, CRE and RARE elements. A pcDNA3-Myc-Stra8 vector was added as indicated. For AP1 and CRE-mediated transcription, an expression vector for CREB was used as a positive control. For RARE activity stimulation, the cells were transfected with a RARγ expression plasmid and treated for 24 h with 1 µmol/L ATRA. (mean ± SD, **P < 0.01 compared to Ctrl; ns: not significant) and TCF3/E47 interacting with SCLERAXIS, regulate the hormonal-induced gene expression. 42 It is interesting that the TCF3 EBox binding motif is enriched in the differentiating spermatogonia 27 and that Tcf3/E47 is among the genes whose transcript is significantly down-regulated at meiotic initiation in the preleptotene population. 13 Our data indicate that at P10, E47 is expressed in the nucleus   36 and in KIT-negative-undifferentiated spermatogonia. 29,32,35 In the adult mouse testis, SOHLH1 is prevalently F I G U R E 5 STRA8 interacts with SOHLH1 and down-regulates SOHLH1-induced c-KIT expression in vitro. A-B, Western blot analysis with anti-Myc (A) and anti-SOHLH1 (B) antibodies of the pull-down assays performed with total lysates (TL) from HEK293T expressing pcDNA3-Myc-SOHLH1 (A) or from P10 dpp-isolated male germ cells and fusion proteins GST-STRA8 and GST (as negative control). The amount of purified GST fusion proteins used in the assay was determined by Coomassie staining (A,B lower panels). C, Western blot analysis with anti-STRA8, anti-Myc and anti-GAPDH antibodies of the immunoprecipitation performed with control mouse IgG or anti-Myc antibody of total lysates (TL) of HEK293T expressing pcDNA3-Myc-SOHLH1 and/or pCMV-STRA8. D, The co-immunoprecipitation assay was repeated with nuclear extracts obtained from P10 dpp-isolated male germ cells. SOHLH1 immunoprecipitated by anti STRA8 antibody was revealed in Western blot with anti SOHLH1 antibody (upper panel) and as a control of immunoprecipitation, the membranes was incubated with STRA8 and HISTONE H3 antibodies (lower panel). E, The bar graph represents the relative luciferase activity in HEK293T cells transfected with c-Kit promoter reporter in combination or not with pcDNA3-Myc-SOHLH1 and/or different amount of pcDNA3-Myc-STRA8. A schematic representation of the Eboxes in the c-Kit promoter is showed. Data represent mean ± SD of three replicates. *P < 0.05; ***P < 0.001; ns, not significant related to control (ordinary one-way ANOVA, Tukey's multiple comparisons test). F, Different concentrations (300 and 600 ng) of Myc-HLH-only Stra8 mutant were analysed in the same EBox-mediated luciferase assay (mean ± SD, ***P < 0.001; ****P < 0.0001). G-H, qPCR (G) and Western blot (H) analyses of c-KIT expression in P19EC cells transfected with Myc-STRA8 and/or Myc-SOHLH1 vectors or control plasmid (mock). G, Fold change of c-Kit expression relative to Gapdh expression was calculated by the ΔΔCq method. Data represent mean ± SD of three replicates. ****P < 0.0001; ns, not significant (ordinary one-way ANOVA, Tukey's multiple comparisons test). H, Western blot analysis of c-KIT expression in P19EC cells transfected as above. Over-expression of recombinant proteins was evaluated with anti-Myc antibodies. GAPDH was evaluated as loading control expressed in differentiating KIT-positive spermatogonia. 29,30,31 Besides, it resulted expressed also in preleptotene enriched germ cell population 12 and up-regulated by ATRA treatment 29 50 In line with the notion that the differentiating spermatogonia are therefore heterogeneous cell populations, [51][52][53] we previously observed that in prepuberal testis there are cells SOHLH1+/c-KIT+/STRA8+ (differentiating spermatogonia), but also cells SOHLH1+/c-KIT-/STRA8+; this latter population could represent early differentiating spermatogonia or the competent-late pre-meiotic spermatogonia. 12,51 Since c-KIT is important for spermatogonia differentiation and in fact is expressed at high levels in such population, we hypothesize that STRA8 takes part to its negative regulation at the beginning of meiosis interfering with SOHLH1 in late differentiating spermatogonia and preleptotene spermatocytes.
In conclusion, it is possible to postulate that STRA8 is crucial to regulate the spermatogonia cell cycle and differentiation and at the same time for triggering the meiotic programme in different ways. First, with a positive feedback mediated by its direct binding to its own promoter (a consensus STRA8-binding motif CNCCTCAG is located −782 bp from TSS) 51 and to the promoter of the positively regulated genes. 12,13 Moreover, STRA8 is able to modulate negatively genes regulated by regulation is not only related to spermatogenesis, but extend also to testicular germ cell tumours. Alteration as mutations, copy number and expression of c-KIT and c-KIT-PI3K pathway are significantly associated to seminoma. 54,55 STRA8 and the premature germ cell differentiation are associated to susceptibility to testicular teratomas. [56][57][58] Therefore, other than for fertility preservation, it is important to understand how STRA8 works to design future therapeutic strategies.

ACK N OWLED G M ENTS
We thank Prof. C. Sette, Catholic University of the Sacred Heart, Rome, Italy, for kindly providing the pGL3-CMV-LUC and pGL3-

CO N FLI C T O F I NTE R E S T
The authors confirm that are not conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated during the study are available from the corresponding author on request.