From the Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia
Received for publication, April 17, 2003
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ABSTRACT |
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INTRODUCTION |
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A number of SOX transcription factors have been shown to play important roles during sex determination and differentiation. For example, the Y-encoded testis-determining factor SRY is believed to be involved in directing the differentiation of the gonadal supporting cell lineage into Sertoli cells and thus acts as the sex determination switch in eutherian mammals (for review see Ref. 6). Complementary results have also implicated SOX9 as a male-specific sex determinant, with deletion or disruption of human SOX9 often resulting in XY sex reversal (7, 8) and overexpression of Sox9 inducing female to male sex reversal in mice (9).
Recently we showed that Sox8, a gene closely related to Sox9, is expressed in the developing brain, branchial arches, limbs, and kidneys and sex-specifically expressed in the testes (10). Structural comparison of SOX8 and SOX9 revealed considerable conservation, particularly within the HMG domain and the C-terminal trans-activation domain. We now report that Sox8 and Sox9 have overlapping expression patterns during testis development in mice and that both proteins bind specifically to the SOX binding site in the Amh promoter and interact with SF1 to synergistically enhance the expression of Amh. Thus we conclude that SOX8 may act redundantly with SOX9 to activate Amh expression during testis development.
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MATERIALS AND METHODS |
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Cell Culture and Transfection AssaysTrans-activation domain analysis of GAL fusion constructs were performed as previously described (10). Co-transfection experiments were performed in triplicate using COS7 cells, in 12-well tissue culture plates containing 500 ng of reporter vector (360-bp Amh promoter in pGL3-basic, see Ref. 16). Transfection controls included 500 ng of pGL3-control. Transfections were performed using Cytofectene (Bio-Rad) according to the manufacturer's instructions. Full-length SF1, SOX9, and SOX2 expression vectors were constructed by sub-cloning the open reading frames from RSV/Ad4BP (16), Sox9-pSG5 (17), and cSox2-pCMV/SV1 (18) into pcDNA3.1+ (Invitrogen). Equal amounts of SOX8 (10), SOX9, and SOX2 expression constructs (20 ng per well) with or without SF1 expression construct (0 or 50 ng per well) were transfected, with the total amount of CMV-containing vector standardized by the addition of different amounts of pcDNA3.1+ to each transfection (total amount of CMV promoter-containing vector was 70 ng per replicate). Luciferase assays were performed essentially as previously described (10) with the following alteration: 80 µl of the 100-µl cell lysate was added to 100-µl Luciferase assay reagent (Roche Applied Science).
The statistical analysis performed on transfection data include a two-way, paired analysis of variance followed by the Student-Newman-Keuls post hoc analysis with a 95% confidence score. These analyses were performed using StatView 5.0.1 (SAS Institute Inc.).
Electrophoretic Mobility Shift AssayEMSA analysis was performed using recombinant, bacterially expressed GST fusion proteins of full-length SF1, SOX2, SOX8, and truncated SOX9 (amino acids 1440). Recombinant plasmids were constructed using the vector pGEX-KG (19) for SOX2, -8, and -9 and pGEX-4T-3 (Amersham Biosciences) for SF1.
Wild-type and mutated Amh promoter fragments were cloned by PCR
into the pGEM-Teasy vector (Promega) using MIS-wt, MIS-R1, MIS-R2, and MIS-R3
(20) as templates and the
following primers: AMHpro-for (AAG CTT GAT ATC GAA TTC CTG) for amplifying R1,
R2, and R3 fragments, AMHpro-for2 (AGT GGA TCC CCC GGG CTG) for amplifying the
wild-type fragment, together with AMHpro-rev (GGA ATT CGC CCC CTA TCA ACA CCA
AA). Fragments were cut out of the respective constructs with EcoRI
and gel-purified. Oligonucleotide probes (S2) harboring two SOX2 binding sites
had the following sequence: GGG ATC CCT TTG TGT CTG GCA ATG CAC AAT ATT G.
Fragments as well as oligonucleotide probes were labeled using
[-32P]ATP and T4 polynucleotide kinase. Binding reactions
were performed in a final volume of 30 µl in binding buffer (100
mM KCl, 1 mM MgCl2, 10 mM Tris, pH
7.5, 4% glycerol, 0.1% Triton X-100, 1 mg/ml bovine serum albumin, 1 µg of
poly(dI-dC)/poly(dA-dT), 0.5 mM dithiothreitol). Reaction mixtures
containing 100 ng of purified protein were preincubated for 10 min at room
temperature followed by the addition of end-labeled probe. After 20-min
incubation at room temperature, DNA-protein complexes were resolved by
electrophoresis on 4% polyacrylamide gels (containing 2.5% glycerol) at 150 V
for 2 h in 0.5x TBE buffer.
GST Pull-down AssaysFragments of SOX8 were generated using PCR and cloned into the pcDNA3.1+ expression vector. 35S-Labeled proteins were generated in vitro using the T7-TNT system (Promega). GST-SF1 fusion protein was purified from BL21 cells as described previously (21) and left attached to glutathione-agarose beads. Bound SF1 fusion protein was then incubated with different 35S-labeled proteins in NTEN buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) at 4 °C for 1 h. The beads were then washed four times with NTEN, and bound proteins were eluted with 6x Laemmli buffer, boiled, analyzed on a 15% SDS-PAGE gel, and visualized by autoradiography.
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RESULTS |
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We next compared the expression of Sox8 with that of Sox9 and Amh, two key genes involved in testis development, to shed light on the potential regulatory relationships between these genes. At each stage studied, left and right genital ridges or testes from the same fetus were assessed in parallel for Sox8 and Sox9 expression. Sox8 and Amh expression was compared in other samples in a similar fashion (Fig. 1B). As a control, left and right gonads from the same fetus were assessed for differences in expression of each gene, and equal levels were observed in each case (data not shown).
The up-regulation of Sox9 expression was first evident at 11.5 dpc (18 tail somites, Fig. 1B), with expression continuing in the testis cords at 13 dpc through to 16 dpc. Similar to Sox8, expression of Sox9 began in the center of the testis and expanded to both poles by 12 dpc, when the first transcripts of Sox8 were detected in the center of the testis. Thus, Sox9 expression precedes that of Sox8 during sex determination by some 12 h. Sox8 and Amh expression patterns showed considerable overlap, with Amh transcripts first detected at 12 dpc in a patch of cells that was spatially more restricted than that expressing Sox8. The expression of Amh began in the center of the testis and encompassed the poles within 24 h; between 12 and 12.5 dpc the expression domain of Sox8 was more extended than that of Amh (Fig. 1B), suggesting that expression of the former preceded that of the latter. These data, combined with the localization of transcripts of all three genes to the developing testis cords, are consistent with the regulation of Amh expression by Sox9 and Sox8 in vivo.
Previous studies have established that Sox8 expression is associated with developing testis cords in mice (10, 22). To further test the hypothesis that Amh expression may be regulated by SOX8 in addition to SOX9, it was necessary to determine whether Sox8 is expressed in the Sertoli or germ cell component of the testis cords, because both Sox9 and Amh are known to be expressed in developing Sertoli cells (13, 17, 23). We performed in situ hybridization on serial sections of 15 dpc testes using probes for Sox8, Sox9, Amh, or the germ cell marker Oct4. Characteristic Sertoli cell expression was clearly evident for Amh, because it was distinct from the expression pattern of Oct4 (Fig. 2A). This analysis showed that Sox9 and Sox8 are also expressed in Sertoli cells (Fig. 2A). To confirm the expression of Sox8, Sox9, and Amh in the same cells, we performed double-labeling experiments, detecting Sox8 or Amh transcripts by in situ hybridization and SOX9 protein by immunofluorescence (Fig. 2B). All three genes were detected in the same cell type, as evident from the co-localization of signal observed in each case (Fig. 2B).
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Binding of SOX8 to Amh Promoter SequencesBased on the overlapping expression of Sox8 and Amh in Sertoli cells, we wished to determine whether SOX8 was capable of binding the critical SOX binding site in the Amh promoter (20). We performed an electrophoretic mobility shift assay (EMSA) with wild-type and mutant Amh promoter fragments spanning both the established SOX and SF1 binding sites (Fig. 3A). As expected, SF1 bound to promoter fragments with a wild-type SF1 binding site but not to those with a mutant SF1 site or an oligonucleotide (S2) harboring SOX2 binding sites (Fig. 3B, lanes 15), and SOX9 bound to wild-type but not mutant SOX binding site fragments (Fig. 3B, lanes 1114). In these experiments, SOX8 also bound specifically to probes containing the wild-type but not the mutated SOX site (Fig. 3B, lanes 69), thus demonstrating sequence-specific DNA binding of SOX8 within the Amh minimal promoter. In contrast, the unrelated SOX2 protein was not able to bind to any of the Amh promoter fragments (Fig. 3B, lanes 1619). All three SOX proteins bound to the SOX2 binding site oligonucleotide S2 (Fig. 3B, lanes 10, 15, and 20). SOX8 DNA binding in vitro was consistently weaker than that shown by SOX9.
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Activation of the Amh Promoter by SOX8 and SF1SOX9 has previously been shown to act synergistically with SF1 to activate transcription from the minimal Amh promoter in cultured cells (5). Considering the temporal and spatial expression of Sox8, and the sequence-specific DNA binding to the Amh promoter, we tested directly whether SOX8 is able to stimulate Amh promoter activity. To investigate potential cooperative trans-activation between SF1 and SOX proteins, we performed co-transfection experiments similar to those described by de Santa Barbara and co-workers studying SOX9 and SF1 (5). COS7 cells were transfected with an Amh promoter-reporter construct and co-transfected with expression constructs for SF1 and the SOX proteins SOX8, SOX9, or SOX2.
In agreement with previous studies (5), SOX9 and SF1 synergistically activated transcription from the Amh promoter 4-fold above the levels observed with the reporter vector alone (Fig. 4A). SOX8 was also able to cooperatively activate transcription in the presence of SF1 (Fig. 4A). The synergistic activation of the Amh promoter by SF1 in combination with either SOX8 or SOX9 was found to be statistically significant with a confidence value of 0.05. In over a dozen independent experiments, the activation levels achieved by SOX8 and SF1 were consistently lower than those achieved by SOX9 and SF1, although the difference was not always statistically significant (Fig. 4 (A and B) and data not shown). The unrelated SOX protein SOX2 was found to have no effect on Amh expression in the presence or absence of SF1 (Fig. 4A).
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Finally, we also assessed whether SOX8 and SOX9 are able to act synergistically on the Amh promoter. The level of trans-activation achieved in the presence of SOX8, SOX9, and SF1 (around 5-fold, Fig. 4B) was similar to that observed for SOX9 and SF1 (difference not statistically significant). The small increase in trans-activation in the presence of all three factors shows that SOX8 and SOX9 do not act synergistically.
Potency of the SOX8 Activation DomainActivation of the
Amh promoter in the presence of SOX8 and SF1 was consistently lower
than that observed with SOX9 and SF1. To further investigate this observation,
we compared directly the relative trans-activation domain potency of
SOX8 and SOX9 (Fig.
4C). The entire coding regions C-terminal to the HMG
domains of SOX8 and SOX9 were fused to the GAL4 DNA binding domain and used in
trans-activation domain analyses, similar to those performed in
earlier studies (10,
15). These regions contain a
conserved trans-activation domain at the C terminus and a second
non-conserved trans-activation region
(10,
24). The C terminus of SOX9
consistently showed an 80-fold increase in trans-activation compared
with GAL alone, whereas SOX8 enhanced expression 10-fold
(Fig. 4C). Both fusion
proteins were expressed at similar levels as detected by Western analysis
(data not shown). These data suggest that SOX8 has a weaker C-terminal
trans-activation domain than SOX9.
SOX8 and SF1 Interact Directly via the HMG Domain Target specificity of SOX proteins is thought to be mediated by direct interaction with cell type-specific partner proteins and cofactors (25, 26). To support the in vivo significance of the observed synergy between SOX8 and SF1, we next tested the ability of SOX8 and SF1 to interact directly. Radiolabeled full-length SOX8 and fragments representing the N terminus, HMG domain, and C terminus were produced and incubated with GST-SF1 fusion protein attached to glutathione-Sepharose resin. After several washes, only full-length SOX8 and the HMG domain fragment remained bound by GST-SF1 (Fig. 5). These data confirm that SOX8 can bind to SF1 in vitro and that this interaction is mediated by the HMG domain of SOX8. Interestingly, SOX2 was also found to bind SF1 (Fig. 5), as might be expected for an interaction mediated by the highly conserved HMG domain. This latter observation is unlikely to have any in vivo relevance, because SOX2 is not co-expressed with SF1 in Sertoli cells of the developing testis2 and does not activate transcription from the Amh promoter (Fig. 4A). Taken together, our observations suggest that SOX8 and SF1 can bind to adjacent sites on the Amh promoter and form a complex that is able to activate transcription from the Amh promoter in Sertoli cells.
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DISCUSSION |
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Sox8, Sox9, and Amh all show similar spatiotemporal
expression patterns in developing mouse gonads, with expression beginning in
the center of the genital ridge and spreading to the poles within 12 h. This
is the first evidence that the expression of all three genes begins at, and
expands from, the center of the developing testis, and is congruent with
recent evidence that Sry expression also occurs as a wave beginning
in the center of the testis and moving to the anterior and then posterior pole
(28). The similar spatial and
temporal dynamics of expression of Sry, Sox9, Sox8, and Amh
provides support for a link between these genes in the molecular pathway of
testis development. Sox8 expression is up-regulated in Sertoli cells
around 12 dpc, 12 h after Sox9 and just before Amh.
These observations are consistent with the possibility that Sox8,
like Sox9, can contribute to the regulation of Amh
transcription in vivo.
This hypothesis is further supported by our results relating to SOX8 DNA binding and transcriptional activation in vitro. Our EMSA experiments demonstrated that SOX8 and SOX9 were able to bind as a monomer and, surprisingly, also as a dimer to two adjacent SOX binding sites in the Amh promoter. This is the first example of a paired SOX binding site in the promoter of a gene involved in sex differentiation. The binding to these sites is specific to SOX8 and SOX9, because the unrelated SOX2 protein did not bind. Similarly, it has been shown that SOX10 is able to bind to DNA as a monomer and a dimer (29). All three proteins (SOX8, -9, and -10) belong to the group E of SOX proteins, which contain a conserved 40-amino acid domain adjacent and N-terminal to the HMG-box. For SOX10 it has been demonstrated that this domain is essential for cooperative binding to response elements (30). So far, paired SOX binding sites have been only identified in the regulatory regions of a number of target genes involved in chondrogenesis, such as Col2a1 and Col11a2 (31, 32). Mutation of these enhancer sequences has different functional consequences depending on which of the two SOX binding sites is mutated (29). Mutation of the primary SOX binding site (148 to 154 bp) in the Amh promoter results in pseudohermaphroditism in transgenic mice, demonstrating that this site is essential for Amh expression in vivo (20). The functional significance of the newly identified, adjacent SOX binding site, and whether SOX8 and/or SOX9 binds in vivo, is still unknown.
Similar to SOX9, SOX8 acts synergistically with SF1 to activate transcription from the Amh promoter, although the levels are consistently lower than those achieved by SOX9 with SF1. The reduced efficiency of SOX8 to trans-activate transcription in the presence of SF1 is most likely due to a combination of its apparent lower DNA binding affinity to Amh promoter sequences and its less potent trans-activation domains, compared with SOX9. Our observations suggest that adjacent binding as well as physical interaction are important for the synergy between SOX8 and SF1 in transcriptional activation, as it is for SOX9 and SF1.
Sox8 may perform functions additional to regulating Amh during sex determination. For instance, the expression of Sox9 is activated either directly or indirectly by SRY, but Sox9 expression continues long after the cessation of Sry expression (17, 23), suggesting that another element is required to maintain Sox9 expression. Sequence and cell transfection analyses have revealed a SOX binding site in the Sox9 promoter that is essential for maximal activity in gonadal somatic cells (33). Our observations suggest SOX8 as a candidate for a factor required to maintain Sox9 expression during sex differentiation.
Shen and Ingraham (34) showed recently that SOX8, like SOX9, can bind to a specific site in the Sf1 promoter. However, SOX8 was found to bind with much less affinity than SOX9, and was not able to specifically induce transcription from this promoter in vitro. These results provide an intriguing counterpoint to our present observations relating to Amh regulation, and it thus appears that the two SOX proteins are not able to act interchangeably in all situations.
SOX8 appears to be capable of performing the same or similar function as SOX9 in regulating Amh expression during sex differentiation, but the overall extent of functional overlap between the two proteins remains to be determined. Functional redundancy has been demonstrated or proposed for a number of SOX proteins (18, 3537) and is considered to reflect evolution in progress. Often the genes involved are closely related and in the process of diverging, where their co-expression and shared functionality are a result of a recent common ancestor (38), as may be the case for Sox8 and Sox9 (39). They may also share common functions in other tissues where expression is overlapping, such as in the developing CNS, spinal cord, and kidneys (10, 12).
Sox8 and Sox9 also show complementary expression patterns in numerous developing tissues, including the developing somites and skeleton. In the somites, Sox9 expression is observed in the sclerotome (12) and Sox8 in the dermomyotome (40). Similarly, Sox9 is expressed in chrondrocytes (12) and Sox8 in osteoblasts (41) during skeletal development. These complementary expression patterns may reflect the adoption of unique functions that were originally performed by a common ancestor. In teleosts, a lineage-specific, genome-wide duplication has resulted in two copies of many genes, and is often used as a model of gene duplication and divergence (38). Two Sox9 genes have been identified in zebrafish, where the expression of the duplicate genes is distinct but overlapping in the brain, head skeleton, and fins (42). Considering these issues, we propose that the conserved function between SOX8 and SOX9 in regulating Amh expression may be an evolutionary remnant. The lower efficiency of SOX8 in regulating Amh expression suggests that this role may no longer be important in vivo, leaving SOX9 as the primary SOX protein required for this role, at least in eutherian mammals. This view is further confirmed with the apparent lack of sex reversal or gonadal dysgenesis observed in Sox8 knockout mice (43).
In view of the biochemical similarity between SOX8 and SOX9, these proteins may share other roles during sexual development. This may explain the inconsistent rate of sex reversal seen in campomelic dysplasia patients (44), who have mutations in one copy of SOX9. We hypothesize that SOX8 at least partially ameliorates the haploinsufficiency of SOX9 in XY campomelic dysplasia patients who do not undergo sex reversal, with the penetrance of this effect likely to be influenced by other genetic modifiers.
It is clear that the SOX binding site in the Amh promoter is functionally important, because mutations in this site severely disrupt Amh expression levels in vivo (20). Previously it has been assumed that this site is important solely due to binding of SOX9. However, the ability of SOX8 to enhance expression of Amh in mammals through interaction with this site may explain the paradox in chicken and alligator sex determination, where Amh is expressed before Sox9 (45, 46). In these species, SOX8 may be important for the regulation of Amh expression. Amh is also expressed in granulosa cells of the adult mammalian ovary in the absence of Sox9 expression (17, 47). This may represent another cellular context where SOX8 could contribute to the control of Amh expression. These possibilities are currently under investigation.
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FOOTNOTES |
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An Australian Professorial Research Fellow of the Australian Research Council.
To whom correspondence should be addressed. Tel.: 61-7-3365-4491; Fax:
61-7-3365-4388; E-mail:
p.koopman{at}imb.uq.edu.au.
1 The abbreviations used are: AMH, anti-Müllerian hormone; dpc, days
post coitum; PBS, phosphate-buffered saline; HISS, heat-inactivated sheep
serum; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; GST,
glutathione S-transferase; HMG, high mobility group.
2 G. Schepers, M. Wilson, D. Wilhelm, and P. Koopman, unpublished
observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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