Human Sex Determination at the Edge of Ambiguity

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent (“default”) cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity.

Regulation of alternative genetic programs is central to the logic of metazoan development (1). Broad mechanistic insight has been obtained from studies of prokaryotic toggle switches (2) constructed within synthetic operons (3), based in part on the bistable transcriptional circuitry of lysogenic phages (4,5).
Analogous principles are thought to govern developmental decisions in metazoans (6) wherein organogenesis may be directed by alternative states of gene-regulatory networks (7,8). Such control systems have been delineated in model organisms, in particular through molecular-genetic analysis of sex determination and sex-specific gene regulation (9,10). In this and previous studies (11)(12)(13)(14)(15), we have sought to characterize biochemical determinants of a binary switch in human gonadogenesis (16).
Male development of therian mammals is (with rare exception (17,18)) initiated by the stage-and lineage-specific expression of Sry (19), a gene contained within the sex-determining region of the Y chromosome (20). Sry encodes an architectural transcription factor (TF) 3 whose expression in the embryonic gonadal ridge activates a developmental program leading to a wave of Sertoli cell differentiation and ultimately to testis formation (Fig. 1A) (21). Assignment of Sry as the testis-determining factor was demonstrated in transgenic mice (20) and extended to human embryogenesis through identification of diverse mutations in SRY associated with a distinct disorder of sex development (DSD) (22), designated Swyer syndrome (Fig.  1B). Such mutations cluster in SRY's high mobility group (HMG) box, a sequence-specific DNA-bending domain shared by a conserved family of TFs (designated SRY-related HMG box, Sox (23)). Most Swyer mutations arise de novo as meiotic errors in paternal spermatogenesis (green arrowheads in Fig.  1B) and are commonly associated with marked defects in specific DNA binding relative to wild-type (WT) SRY (15,22,24). The three-dimensional structure of a specific SRY HMG box-DNA complex (Fig. 1C) has provided a foundation for interpretation of many such mutations (25)(26)(27).
This study has focused on a Swyer mutation of complementary structural and biological interest, F109S (consensus box position 54; Fig. 1D) (28). This mutation lies at the back surface of the HMG box; substitution of a large side chain by a smaller one would be predicted to create a destabilizing crevice (Fig. 1, E and F) (25,26). First identified in a 46-chromosome XY adolescent female presenting with primary amenorrhea and gonadoblastoma in situ (28), the same mutation was found in her father, two brothers, and a paternal uncle. Such differences * This work, a contribution from the Cleveland Center for Membrane and in phenotype (also known as "variable genetic penetrance") are likely to reflect either autosomal background (29) and/or stochastic variation in gene expression (30). Analogous backgrounddependent XY sex reversal has been observed among strains of laboratory mice (31)(32)(33), highlighting the tenuous function of murine Sry at the threshold of developmental ambiguity (34).
The multiplicity of inherited Swyer mutations in human SRY (filled red arrowheads in Fig. 1B) presumably reflects the diversity of molecular mechanisms (beyond specific DNA binding and bending) by which a developmental switch may be perturbed in vivo. Patient-directed studies of such representative mutations (e.g., V60L and I90M at respective box positions 5 and 35 (22,24)) have illuminated, for example, the contribution of SRY's nucleocytoplasmic shuttling (NCS) to the robust specification of human testicular development (11,14,35,36). NCS may be a general requirement of SOX TFs coupled to their post-translational modification (37). In this context the properties of F109S SRY are particularly intriguing. Consensus position 54 is broadly conserved as an aromatic residue (Phe or Tyr (15)) throughout the metazoan superfamily of specific HMG boxes, and yet Phe-109 is not part of any of the following five signature features of human SRY: (i) its angular DNA-binding surface (26); (ii) its basic tail as accessory DNA-binding element (27) and kinetic clamp C-terminal to the HMG box (12); (iii) N-terminal bipartite nuclear localization signals (NLS) (38); (iv) C-terminal monopartite NLS (39); and (v) central nuclear export signal (NES) (40).
What does Phe-109 contribute to the structure and function of SRY, and why does Swyer mutation F109S SRY lead to variable male or female somatic phenotypes? To address these questions, our investigation had two parts. We first undertook biochemical and biophysical studies of the variant HMG box. These experiments demonstrated that the mutation is profoundly destabilizing, and yet its specific DNA-binding and DNA-bending properties are similar to those of the WT SRY domain. We next pursued cell biological studies of the WT and variant SRY (as epitope-tagged intact proteins introduced by transient transfection) in diverse rat and human cell lines. The latter studies employed two rodent and two human cell lines differing in tissue of origin and state of differentiation. Whereas all four lines share the universal ubiquitin-dependent (26S) and default (20S) proteasomal pathway of protein degradation (41)(42)(43), only one cell line, an XY lineage derived from the rat embryonic gonadal ridge just prior to onset of Sry expression and morphological differentiation (44), supports SRY-directed transcriptional activation of the male program ( Fig. 1A) (21,45). This ras-immortalized pre-Sertoli cell line (designated CH34 (44)) thus provides a model of the site and stage of endogenous Sry expression (13,46). A control rat XY cell line (CH15) lacking pre-Sertoli markers (and unresponsive to transfected SRY) was also obtained from the embryonic bipotential ridge and presumably represents a non-Sertoli-related lineage (46). The two SRY-unresponsive human cell lines (HEK 293T and Hs1.TES) were respectively derived from the embryonic kidney (47) and fetal testis (Leydig cell lineage (48)).
Remarkably, F109S SRY underwent, irrespective of particular cellular context, enhanced polyubiquitination and accelerated proteasomal degradation, reducing the mean intracellular ). F, expanded view of Phe-109 side chain (red stick) in crevice boxed in C. Asterisks indicate main-chain borders, whereas neighboring side chains are without asterisks. Coordinates for structural models were obtained from Protein Data Bank (PDB) code 1J46 (27). SRY concentration at a given level of transfected mRNA expression. In contrast, no significant changes were obtained in the in vitro susceptibility of the variant domain to degradation by the 20S core proteasome. In SRY-responsive CH34 cells, accelerated ubiquitin-associated degradation led to decreased occupancy of a target enhancer element (the testis-specific enhancer of Sox9 (TES) (49)) and in turn with attenuated transcriptional activation of this, the principal endogenous target gene in the program of testicular differentiation (red box in Fig. 1A) (14,49). Treatment of the cells with chemical proteasome inhibitor MG132 equalized expression of WT and F109S SRY, leading in turn to a striking restoration of native TES enhancer occupancy and rescue of native Sox9 expression. Such subtle effects of the F109S substitution on the structure and function of SRY are in general accordance with past studies of inherited Swyer mutations unrelated in their respective molecular mechanisms of perturbation (13,14).
Our results demonstrate that a conserved aromatic residue at the back surface of a specific HMG box functions to seal the hydrophobic core and thereby enhances thermodynamic stability and cellular lifetime. F109S and WT SRY differ in effi-ciency as substrates for polyubiquitination and therefore in proteasomal turnover but exhibit, on a per molecule basis, similar gene-regulatory properties. Together, these findings and their clinical correlation highlight enhanced ubiquitination and accelerated degradation of a master transcription factor as a molecular mechanism of impaired human sex determination. To our knowledge, this is the first observation in a metazoan that such a proteasome-based mechanism may impair the robustness of organogenesis leading to phenotypic variation within a family tree.

Results
Clinical Mutation Destabilizes the HMG Box-Thermal stabilities of the free WT and variant domains were assessed by circular dichroism (CD) (Fig. 2, A and B). The unfolding transition of the variant domain (red in Fig. 2A Fig. 3E). The spectrum of each domain likewise exhibits a blue shift in emission maximum at the lower temperature (relative to its spectrum at the higher temperature). The human domain contains an additional non-conserved Trp exposed on its surface (Trp-107; box position 52) that is absent in the murine domain. D-F, far-UV CD spectra of the three domains are similar at 4°C, but their respective ␣-helical signatures exhibit distinct patterns of attenuation at 25 and 37°C. Relative to the WT human domain (black), loss of structure is more marked in the variant domain (red) than in the murine domain (blue) in accordance with their respective thermal unfolding transitions (A and B). G-I, guanidine (Gu⅐HCl)-induced unfolding at 4, 25, and 37°C as monitored by CD at 222 nm. The variant and murine domains each exhibit greater sensitivity to chemical denaturation than does WT SRY (color code as in D-F). Estimates of C mid and ⌬G u were obtained at 4°C by application of a two-state model ( Table 1). Use of this model at the higher temperatures was limited by possible non-two-state behavior.
at both 15 and 37°C (respective filled and open circles in Fig.  2C), suggesting that the core of the variant domain is less stably packed and so more exposed to solvent water. These emission spectra contain contributions from two conserved Trp residues in the core of the major wing (Trp-70 and Trp-98; box positions 15 and 43 as shown in green in Fig. 3E) and an additional nonconserved Trp on the protein surface (Trp-107; box position 53). The similar emission intensities at the two temperatures is likely to reflect offsetting effects of thermal unfolding (enhancing the fluorescence of core Trp side chains) and solvent quenching (more effective at the higher temperature).
Comparison of far-UV CD spectra suggested that the variant domain achieves a native-like fold at 4°C (Fig. 2D) with progressive loss of ␣-helical content at 25 and 37°C (Fig. 2, E and F). Respective thermodynamic stabilities at these three temperatures were probed by chemical denaturation (Fig. 2, G-I). At each temperature, the variant domain exhibited greater sensitivity to guanidine-induced loss of structure than did the WT domain. Application of a two-state model (12,50) at 4°C enabled the guanidine concentration at the midpoint of unfolding (C mid ) to be estimated as 1.1(Ϯ0.1) M (variant) and 2.2(Ϯ0.1) M (WT; Table 1). Inferred thermodynamic stabilities (⌬G u ) were 1.0(Ϯ0.1) (variant) and 3.1(Ϯ0.1) kcal/mol (WT); the variant's loss of stability (⌬⌬G u ) was thus 2.1(Ϯ0.2) kcal/ mol. Respective m values (denaturant dependence of free energies of unfolding) were 0.9(Ϯ0.03) and 1.4(Ϯ0.03) kcal/mol/M; the reduced m value of the variant domain suggested that in the absence of guanidine-HCl its nonpolar surfaces are less efficiently desolvated than those of the WT domain (and so undergo a smaller further change in solvation on denaturation) in accordance with their contrasting Trp fluorescence emission spectra (above).
The reduced stability of the F109S HMG box of human SRY resembled (in exaggerated form) the WT HMG box of murine Sry (shown in blue in Fig. 2). The midpoint unfolding temperature of the murine domain (33(Ϯ0.5)°C; Table 1) was thus intermediate between the WT and variant human domains (41 and 28°C, respectively). Similar intermediate trends were observed in temperature-dependent ␣-helical CD signatures (Fig. 2, D-F) and in sensitivity to chemical denaturation (Fig. 2, G-I). At 4°C, the thermodynamic stability of the murine domain (⌬G u ) and C mid guanidine concentration was likewise intermediate between the WT and variant human domains (Table 1). These findings are in general accordance with an analogy proposed between ISS alleles of human SRY and WT murine Sry by Eicher and co-workers (51) (see "Discussion").
Whereas comparative two-state modeling of protein denaturation was feasible at 4°C, unfolding of the variant domain at 25 and 37°C lacked significant cooperativity and so could not be fitted to this model (ND in Table 1). In accordance with these CD studies, 2D 15 N-1 H "fingerprint" NMR spectra of the variant domain at 15, 25, and 35°C exhibited less marked chemical shift dispersion than the corresponding spectra of the WT domain (Fig. 3, A-C). Furthermore, the variant's fingerprint NMR spectrum at 35°C resembles the 15 N-1 H fingerprint of the WT domain on denaturation in 5 M urea (Fig. 3D). Evidence that the variant domain exhibits two or more conformations at 25°C was provided by observation of at least five indole 15 N-1 H cross-peaks for the variant domain ( Fig. 3G) rather than the expected three as in the WT spectrum (Fig. 3F). Upon specific DNA binding, the expected three cross-peaks were observed for both WT and variant (Fig. 3, H and I), providing evidence of a single bound conformation.
Clinical Mutation Preserves Native-like Specific DNA-binding and DNA-bending Properties-Despite its marked instability, the variant domain retains specific DNA binding and bending. WT and variant dissociation constants (K d ), determined using a fluorescence resonance energy transfer (FRET)-based equilibrium assay (13), were indistinguishable at 15 and 37°C (Table 1 and Fig. 4A). Stopped-flow FRET studies of protein-DNA dissociation nonetheless demonstrated that the mutation caused less than a 2-fold increase in rate constant (k off ) at each of four temperatures tested (6,15,25, and 37°C) ( Table 2 and Fig. 4B). That the WT and variant domains exhibited similar specific protein-DNA affinities in turn implied that the variant domain exhibits a compensating increase in association rate (k on ). Conversely, these findings suggest that pre-organized structure within the WT domain imposes a kinetic barrier to specific DNA binding and release.
The WT and variant domains exhibited indistinguishable specific DNA-bending properties. Preservation of native specific DNA bending was demonstrated by a FRET probe of endto-end distances in a 15-bp DNA duplex at 15°C (inset in Fig.  5A) and corroborated by permutation gel electrophoresis at 4°C (Fig. 5, B and C). The decrease in donor emission at 520 nm (fluorescein) corresponded to an enhancement in FRET efficiency (tetramethylrhodamine). Previous studies of the WT SRY domain have shown that this enhancement corresponds to a reduction in mean end-to-end distance from 61 Å (free DNA) The m-value (slope d(⌬G)/d(M)) correlates with extent of hydrophobic surfaces exposed on denaturation. c T m is the apparent midpoint of thermal unfolding of the free domains or equimolar protein-DNA complex (bound) at 25 M as monitored by CD (15). d Dissociation constants (K d ) pertain to the equilibrium between the specific SRY domain and a 15-bp DNA site containing a consensus SRY target site (5Ј-ATTGTT-3Ј and complement): units are in nM as determined by FRET-based titrations (14). e ND indicates not determined due to apparent non-two state unfolding behavior. f Values are as reported previously (14).
Variant Domain-DNA Complex Exhibits Native-like Structure-Far-UV CD spectra of the variant domain-DNA complex (containing a 15-bp DNA duplex with central 5Ј-ATT-GTT target site) at low temperatures closely resembled spectra of the WT complex. At 37°C, restoration of DNA-dependent ␣-helix content of the variant complex was substantial but incomplete (red and black circles in Fig. 5E). Calculated difference spectra indicated that the extent of DNA-induced ␣-helical structure in the variant complex was greater than in the WT . For wild-type SRY, three cross-peaks corresponding each Trp side-chain indole NH were observed at 15 and 25°C, respectively, whereas multiple sets of Trp side-chain cross-peaks were observed at 35°C, indicating that wild-type Trp residues exist in multiple local conformation at 35°C. C and D, for F109S mutant, multiple sets of Trp side-chain cross-peaks were observed even at 15°C, also the main chain amide cross-peaks crowded in the center region ( 1 HN chemical shift range in 7.5-8.5 ppm) at 25°C (C); and the HSQC spectral pattern is similar as that of WT in 5 M urea (D), suggesting that F109S mutant exists in multiple conformation at lower temperature (15°C) and partially random-coiled conformation at 35°C. The results are consistent with the temperature dependence of 1D 1 H NMR spectra of wild-type SRY and F109S mutant. E, ribbon model of major wing of the WT human SRY HMG box; helices ␣1-␣3 are as labeled. Invariant Trp residues of the core (Trp-70 Table 1. The variant domains are in red (trace and axis label). B, stopped-flow FRET traces to determine dissociation rate constant (k off ) values reported in Table 2; WT traces are in black and Phe-109 in red.
complex (red asterisk in Fig. 5F), reflecting the more complete thermal unfolding of the unbound variant domain at 37°C (see above) and its partial stabilization on specific DNA binding. The greater extent of ␣-helical induction at 37°C in the variant complex is highlighted by a double-difference spectrum (purple triangles in Fig. 5F).
In the near-UV region of the CD spectra (wherein the DNA contribution predominates) binding of the WT or variant domain was associated with a blue-shift in spectral maximum (from 282 (Ϯ0.5) nm (free DNA) to 269 (Ϯ0.5) nm (bound DNA)). This 13-nm shift (bracket in Fig. 5E) is reminiscent of a classical B3 A double-helical transition (52) in accordance with the A-like DNA conformation observed in the solution structure of the complex (26,27). A bp-specific view of the bound DNA conformation was provided by 1 H NMR spectroscopy (Fig. 6). Relative to the spectrum of the free DNA site (15 bp; Fig. 6A), the chemical shifts of the DNA imino protons (guanosine N 1 -H and thymidine N 3 -H; mediating Watson-Crick base pairing) exhibited marked changes on specific DNA binding (both upfield and downfield; Fig. 6, B-E). The WT and variant domain-DNA complexes are each in slow exchange on the NMR time scale (arrows in Fig. 6B) in accordance with the stopped-flow FRET studies above. The pattern of complexation shifts was in each case similar, indicating a general correspondence of bound DNA structures. Subtle differences between the complexes were observed at bp 6 -8 (green box at top in Fig. 6), which adjoin the major wing of the WT HMG box-DNA complex (25,27) and hence presumed site of mutational perturbation in the core. By contrast, the large complexation shifts at positions 10 and 11 (adjoining the minor wing of the HMG box) were essentially identical in the two complexes (violet in Fig. 6).
The 1 H NMR signature of DNA-dependent minor wing ( Fig.  7A) stabilization was essentially identical in the 2D NOESY spectra of the WT and variant complexes (Fig. 7B). This signature is provided by the upfield ring-current shift of the ␥ 1 -methyl resonance of the valine (near 0.0 ppm) due to its inter-residue NOE-associated packing within an "aromatic box" comprising His-120, Tyr-124, and Tyr-127 and consensus positions 65, 69, and 72 (arrow and horizontal brackets in Fig.  7B). Although Val-60 does not contact the DNA (violet side chain in Fig. 7A), Tyr-127 projects near a solvated DNA interface.
Despite their subtle differences in DNA imino 1 H NMR chemical shifts adjoining the major wing of the HMG box, the WT and variant domain-DNA complexes exhibited similar NMR signatures of partial side chain intercalation by "cantilever" residue Ile-68 (consensus box position 13). This aliphatic  ). E and F, CD studies of mutual induced fit in SRY-DNA complex at 37°C. E, CD spectra of the free DNA (green), WT complex (black), and variant complex (red). Bracket indicates blue-shift of DNA band reminiscent of classical B 3 A transition. F, CD difference spectra relative to spectrum of free DNA (green). Black and red spectra represent respective difference WT and variant spectra, obtained by subtracting the spectra of the free domain and free DNA from the spectrum of the complex (a buffer control was also added). Purple spectrum represents the difference between the spectra of the WT complex and variant complex (or equivalently, between the red and black curves). Asterisk highlights marked stabilization of ␣-helical structure in variant on specific DNA binding. side chain inserts between successive AT base pairs at positions 8 and 9 ( Fig. 7, C and D) (26,44). Such insertion leads to a large upfield ring-current shift in the ␦-methyl resonance of Ile-68 (53), which is identical in the 1 H NMR spectra of the WT and variant complexes (Fig. 7, D and E). This upfield methyl resonance exhibits prominent intermolecular nuclear Overhauser effects (NOEs) with the flanking imino resonances of thymidines 8 and 9 (Fig. 7D) and to the C 2 -H resonances of adenines 8 and 9 in the expanded DNA minor groove (box in Fig. 7B). Evidence for the restoration of a native-like major-wing core adjoining the site of mutation was provided by the upfield 1 H NMR chemical shift of the ␦ 1 -methyl resonance of Leu-101 (box position 46) near 0.1 ppm (Fig. 7E, right) and its preserved NOE to the C 2 -H proton of Trp-98 (box position 43; greenlabeled cross-peaks in Fig. 7B and green side chains in Fig. 7A).
In contrast to the above similarities in the 1 H NMR NOESY spectra of the DNA-bound domains, subtle but illuminating differences were observed in the respective 1 H NMR spectra of the free domains. Whereas the respective ␦ 1 -methyl resonances of Leu-101 in the WT and variant free domains exhibit an attenuated upfield shift (Fig. 7E, left), reflecting greater conformational flexibility than in the DNA-bound domains, this methyl resonance is broader and closer to its random-coil value in the spectrum of the variant (upper panel of Fig. 7E, left) than in the spectrum of the WT domain (lower panel of Fig. 7E, left), presumably due to intermediate exchange between folded and unfolded states at 25°C in accordance with the above CD studies.
Variant SRY Undergoes Accelerated Proteasomal Degradation in Diverse Cell Lines-To test whether the instability of the F109S HMG box (as an isolated domain) and its marked loss of structure at 37°C (as probed by CD) affected the mean intracellular protein concentration of full-length F109S SRY relative to WT SRY, respective steady-state levels of expression 24 h following transient transfection were probed by SDS-PAGE followed by anti-HA WB (Fig. 8A). In each of the four cell lines employed (CH15, CH34, HEK 293T, and Hs1.TES), expression of the variant was reduced. The extent of reduction (relative to WT) was 3-fold in the rat cell lines and 6-fold in the human cell lines. In these studies 1ϫ transient transfection was employed (i.e. 1 g of SRY-encoded plasmid per 10 6 cells without addition of the parent plasmid; see under "Experimental Procedures").
To test whether the decreased level of F109S SRY at the protein level was due to proteasomal degradation, chemical pro- teasome inhibitor MG132 was added 12 h after transient transfection, and the cells harvested 12 h later. In each cell line addition of MG132 resulted in enhancement of the F109S SRY band (Fig. 8B) to a level similar to that of WT SRY in the absence of MG132 (at left in Fig. 8B); addition of MG132 did not enhance WT protein expression. Control studies of subcellular localization (see below) indicated that the differential rates of proteasomal degradation were not due to mislocalization of the variant SRY in the cytoplasm as had been observed in unrelated proteins (54). Control studies of the mRNAs encoding HAtagged F109S or WT SRY indicated in each cell line that the mutation did not lead to reduced levels of mRNA (which could in principle have resulted from decreased efficiency of transcription or accelerated mRNA decay in the presence of the mutant codon (55)).
That similar decrements in cellular accumulation of F109S SRY (relative to WT) were observed in the four unrelated cellular contexts provided evidence that its mechanism was not dependent on species (rat or human), cell lineage (pre-Sertoli, embryonic gonadal stroma, fetal kidney, or fetal testis of Leydig origin), or stage of development (rat embryonic day E14.5 (46), embryo or human mid-trimester fetus). This conclusion was corroborated through analysis of respective cellular lifetimes following general translational arrest by addition of cycloheximide (56). In each cell line, the apparent lifetime of F109S SRY was shorter than that of WT SRY (Fig. 8C). Quantitation of these bands (relative to ␣-tubulin loading controls) provided a time course of degradation (semilog plot in Fig. 8D) yielding estimates of apparent cellular lifetimes (half-lives in Fig. 8E). The relative half-life of F109S SRY in CH34 cells observed here (2.8 Ϯ 0.1 h) was reduced relative to WT SRY (5.6 Ϯ 0.4 h) but greater than that implied by a previous study of a clinical SRY variant (de novo mutation W70L; half-life of 2.1 Ϯ 0.1 h) whose HMG box was found to be almost completely unfolded at 37°C (15).
F109S SRY Exhibited Enhanced Polyubiquitination-To probe the cellular mechanism of accelerated proteasomal degradation of F109S SRY (Fig. 9A), we investigated the extent of its ubiquitination (relative to WT SRY) in each of the above four cell lines. This post-translational modification provides a general signal targeting protein to the 26S proteasome in eukaryotic cells (57). Our protocol exploited the HA tag to immuno-precipitate the transfected proteins. I68A SRY was included to test whether loss of specific DNA binding might influence the extent of ubiquitination. The immunoprecipitated proteins were resolved by SDS-PAGE and probed by an anti-Ub monoclonal antiserum (Fig. 9, B and C). Whereas only trace ubiquitination could be observed in the absence of MG132 (lanes 1-4 in Fig. 9, B and C), as expected treatment with MG132 led to accumulation of multiple ubiquitinated species (lanes 5-8). In each cell line, F109S SRY exhibited enhanced mono-and polyubiquitination (respective lanes 7 in Fig. 9, B and C). Extent of ubiquitination of I68A SRY was similar to that of WT SRY. Quantitation of these results (using the bands contained within the dashed red boxes in the shortest film exposures; Fig. 9, B and C) is shown by the histogram in Fig. 9D (see also Table 3). The fold-increase of ubiquitination of F109S SRY depended on the cell line within the approximate range 2-3 (Table 3). In these assays input-and general loading controls were, respectively, provided by total accumulation of SRY variants (as detected with a polyclonal anti-HA antiserum) and monoclonal blotting of ␣-tubulin (lower two panels in Fig. 9, B and C). These findings  suggest that the decreased cellular half-lives of epitope-tagged F109S SRY and W70L SRY (15) (relative to WT SRY) reflect enhanced modification of the variant proteins by one or more E3 ligases (58) in association with thermodynamic destabilization and segmental unfolding.
Destabilization of the SRY HMG Box by F109S Did Not Affect 20S Proteasomal Degradation-Short-lived proteins in eukaryotic cells may be degraded by a ubiquitin-independent default pathway through the 20S core proteasome (Fig. 9E) (59). Because F109S perturbs the structure and stability of the free SRY HMG box, we sought to test whether the variant domain was a more efficient substrate for ATP-independent 20S proteasomal degradation in vitro. Assays were conducted at 37°C (to capture the differential thermal unfolding of the SRY domains under physiological conditions; see Fig. 2 above) at a SRY/core proteasome molar stoichiometry of 3000:1. Under these conditions no significant differences were observed in the rate of 20S degradation between the F109S and WT domains (Fig. 9, F and G). It is possible that their similar susceptibilities to default 20S degradation in vitro reflect the baseline partial unfolding of even the WT domain at 37°C as demonstrated by the above CD studies (Fig. 2).
Variant SRY Exhibits Reduced Transcriptional Activity in Proportion to the Reduced Mean Intracellular Concentration of the TF-Gene regulatory activity of the WT or variant SRY proteins were evaluated by quantitative reverse-transcriptase polymerase chain reaction (qPCR)-based measurement of endogenous Sox9 mRNA accumulation following their transient transfection (14). The studies were performed at a series of expression-plasmid dilutions, designed to explore a broad range of intracellular SRY concentrations. Whereas standard transient transfection with the undiluted WT plasmid ("1ϫ") gave rise to a mean protein expression level of 7 ϫ 10 5 molecules per cell, transient transfection of 50ϫ dilution with the empty parent plasmid (yielding the same total DNA dose per cell) gave rise to 2 ϫ 10 3 mean molecules per cell. By contrast, 1ϫ transfection of the F109S SRY construct led to mean expres-sion of only 2 ϫ 10 3 molecules per cell. Systematic characterization of the relationship between plasmid dilution and SRY expression level is provided in Table 4. This experimental maneuver thus enabled adjustment of the transfected TF concentration either within the physiological range (10 2 -10 4 molecules per cell; Ref 60) or under conditions of marked overexpression (10 5 -10 6 molecules per cell).
Transcriptional analysis of transfected SRY in the four cell lines required prior characterization of the chromatin state pertaining to its principal endogenous autosomal target gene, Sox9 in rat (chromosome 10) and ortholog SOX9 in human (chromosome 17). To this end, representative histone marks were analyzed in the TESCO element based on side-chain methylation of conserved lysine residues in the N-terminal arm of histone H3 (61). H3 modifications associated with active chromatin (or chromatin accessible to transcriptional activation) were provided by mono-, di-, and tri-methylation of Lys-4 (left-hand panel of Fig. 10A; designated me1 (red), me2 (blue), and me3 (green)), mono-methylation of Lys-9 (middle panel of Fig. 10A), and mono-methylation of Lys-27 (right-hand panel of Fig.  10A). H3 modifications associated with inactive chromatin or chromatin inaccessible to transcriptional activation were provided by di-and tri-methylation of Lys-9 (middle panel of Fig.  10A), and tri-methylation of Lys-27 (right-hand panel of Fig.  10A). Such characterization of orthologous TESCO elements indicated that CH34 cells exhibited activating H3 marks whereas the other three cell lines exhibited repressive H3 marks.
The above H3 epigenetic codes in the untransfected cell lines were associated with TESCO occupancy by WT SRY on its transient transfection (under 1ϫ conditions). Although such transfection led to similar overexpression of WT SRY in each cell line (Fig. 8A, above), no SRY-specific chromatin immunoprecipitation (ChIP)-based signal was observed in the CH15, HEK 293T, or Hs1.TES cells (Fig. 10B). The ChIP pattern in CH34 cells with primer sets a, b ,and c was as described previously (14). Primer sets a and c were designed based on the results of Sekido and Lovell-Badge (49) to enable amplification of a DNA segment containing multiple SRY target sites, whereas primer set b provided a negative control within TESCO. In light of these results, comparative studies of the transcriptional activity of F109S SRY and WT SRY were restricted to CH34 cells.
Comparative ChIP studies of F109S SRY and WT SRY in CH34 cells demonstrated 2-fold reduction in TESCO occupancy in the absence of MG132 and equal occupancy in the presence of MG132 (Fig. 10C). Treatment of the cells with MG132 did not affect TESCO occupancy by WT SRY. Such  7 ϫ 10 5 3 ϫ 10 5 ϳ1900 ϳ2200 ϳ1100 ϳ170 6 ϫ 10 5 3 ϫ 10 5 ϳ2000 a Chemical proteasome inhibitor MG132 enhanced accumulation of F109S SRY in the 12-h post-treatment but does not have significant effect on accumulation of WT as described (13, 14). b A mammalian cell typically contains 300 pg of total protein (103); the ␣-tubulin content is about 3% of total protein (104).  61): left, mono-, di-, and tri-methylation of Lys-4 (modifications me1, me2, or me3 in H3K4; shown in red, blue, and green, respectively). Fold-enrichment in TESCO (ChIP primer set a; see "Experimental Procedures") was evaluated in untransfected cell lines CH34, CH15, Hs1.TES, and HEK 293T; middle, corresponding ChIP analysis of mono-, di-, and tri-methylation of Lys-9; right, ChIP analysis of mono-and tri-methylation of Lys-27. CH34 cells exhibited activating marks at H3K and H3K9 and reduction in repressive mark me3 at H3K9; the other three cell lines exhibited attenuated activating marks and repressive marks at H3K27. B, TESCO occupancy by WT HA-tagged SRY was selectively observed in CH34 cells (left-hand lane of gel with relative quantitation in histogram at right). Transfection conditions were "1ϫ." Primer sets a, b, and c were as defined by Sekido and Lovell-Badge (49) as homologous sets 4, 6, and 8 in mouse TES. Set b provided a negative control due to absence of specific SRY-binding sites. C, comparison of the relative TESCO occupancies of WT SRY and F109S SRY (variant, v) in CH34 cells. The variant exhibited 2-fold reduction in enhancer occupancy in the absence of MG132 but native occupancy on rescue of protein accumulation by MG132. Transfection conditions were 1ϫ. Extent of attenuation in TESCO binding by F109S SRY is less marked under these conditions than its fold reduction in protein accumulation, reflecting baseline overexpression WT SRY under 1ϫ transfection conditions. A negative control was provided by I68A SRY, which contains a substitution that blocks specific DNA binding (62). enhancer binding required DNA recognition by the HMG box as indicated by negative control I68A SRY, a variant that is well folded but without detectable specific DNA binding activity in vitro (62). The 2-fold reduction in TESCO occupancy by F109S SRY was associated with a 2-fold reduction in SRY-directed transcriptional activation of Sox9 under conditions of overexpression (1ϫ transient transfection; left-hand side of Fig. 10D) or reduced expression (50ϫ transient transfection; right-hand side of Fig. 10D). In each case treatment of the cells with MG132 rescued transcriptional activation of Sox9 (blue labels in Fig.  10D). MG132 treatment did not enhance the apparent potency of WT SRY as shown previously (14).
To evaluate the relative transcriptional potencies of F109S SRY and WT SRY on a per molecule basis, plasmid dilution ratios were carefully adjusted to give similar intracellular protein concentrations (Fig. 10E, left). Anti-HA WB band intensities of WT and F109S SRY were similar (top panel in Fig. 10E, left) relative to the ␣-tubulin loading controls (middle panel) following respective 1ϫ and 25ϫ plasmid dilutions as monitored by GFP co-transfection (bottom panel). Negative controls were provided in this experiment by SRY variants with negligible specific DNA binding activity (I68A and G95R (22,62)). Results of qPCR-based studies of Sox9 mRNA abundance are shown at right in Fig. 10E. Whereas the negative controls lead, as expected, to no change in the baseline abundance of the Sox9 mRNA, the per molecule potencies of WT and F109S SRY were similar. Although not statistically significant, the small attenuation of the F109S-associated signal was in accordance with the small attenuation of the F109S WB signal (Fig. 10E) due to technical limitations of this protocol.
The above functional analysis was supported by two additional sets of control experiments. The first employed genespecific qPCR to assess the specificity of SRY-directed transcriptional activation of Sox9 and in particular to exclude induction of a general state of enhanced transcriptional activity. To this end, other members of the Sox family uninvolved in testis determination were probed (Sox2, -3, -4 and -17); none of these genes exhibited SRY-dependent transcriptional activation (left-hand side of Fig. 11A). Similarly, transient transfection of WT SRY did not alter the expression of housekeeping genes (right-hand side of Fig. 11A). Finally, to demonstrate that the relative transcriptional activities of WT and F109S SRY were unaffected by a differences in subcellular localization, an immunocytochemical assay was employed to evaluate the extent of nuclear localization (Fig. 11B). Quantitative analysis (ϳ500 cells per SRY variant; see Table 5) revealed indistinguishable patterns of nuclear or pancellular protein distributions for WT and F109S SRY (left-hand side of Fig. 11C), whereas control mutations I90M (impairing nuclear export and hence NCS) and R62G (impairing nuclear import) gave rise to the expected perturbations in accordance with past studies (right-hand side of Fig. 11C) (14,35).

Discussion
This study has focused on an inherited mutation in the HMG box of SRY associated in the proband with somatic XY sex reversal, primary amenorrhea, and gonadoblastoma in situ (63). The same mutation was found in the Y chromosomes of the proband's father, fertile paternal uncle, and two brothers (Fig.  12A). Such a DSD pedigree (unusual among Swyer families) suggests that the variant SRY allele is compatible with either male or female somatic phenotypes depending on genetic background or stochastic gene expression (64). Despite the marked biophysical perturbation of the variant HMG box, four members of this pedigree bearing this mutation are male, and only the proband is female. Although such family trees are rare in human populations (65), similar variable DSD phenotypes have commonly been observed among laboratory strains of mice due to Y chromosome-autosome incompatibility (31)(32)(33)51).
Clinical Mutation F109S Provides Biophysical Insight into General Architectural Rules-Our goal was to decipher possible molecular mechanisms underlying the incomplete genetic penetrance of this mutation. Why was the proband female when the same variant Sry allele was found in male members of her family, in two individuals with evidence of fertility? The Ser substitution at residue 109 alters a core Phe residue (Fig. 12, B  and C). This position in the HMG box is (a) invariant among Sry and Sox domains (consensus position 54; lower two panels of Fig. 12D) and (b) conserved as an aromatic residue (Phe or Tyr) among other families of HMG boxes (upper two panels of Fig.  12D) (66 -68). This substitution results in loss of aromaticity, hydrophobicity, and side-chain volume, and this was predicted by Clore and co-workers (26) (based on the three-dimensional structure of the SRY HMG box) to be destabilizing. The present biochemical and biophysical studies of the WT and variant box have verified this prediction. These findings are in accordance with mutational studies of diverse globular proteins whose respective hydrophobic cores seldom tolerate such non-conservative substitutions (69,70).
Although the marked instability of the F109S SRY domain readily rationalizes the phenotype of the proband, what may account for the divergent phenotype of her father and uncle (as fertile males)? The F109S SRY domain was found to retain native-like specific DNA-binding affinity with unperturbed DNA bend angle in a consensus DNA-domain complex. In striking contrast to the temperature-dependent unfolding of the free domain, CD and 1 H NMR studies indicated that the native-like structure was largely intact in the specific DNA complex. Such structure was either regained on specific DNA binding (induced fit) or captured by the specific DNA site to predominate in the equilibrium complex (conformational selection) (71). These biophysical mechanisms are of evolutionary interest as the near-native functional properties of unstable or unfolded polypeptides may rationalize how the exquisite structural organization of modern proteins emerged through stepwise stabilization of nascent partial folds (72).
Despite the native-like structure of the variant protein-DNA complex, a subtle increase in rate of protein-DNA dissociation (and hence a corresponding increase in rate of association) was observed in stopped-flow FRET studies. This finding suggests that pre-organized structure in the major wing of the WT domain imposes, at least to this extent, a kinetic barrier to specific DNA binding and release. Analogous compensation between on-and off-rates (likewise associated with specific DNA-dependent protein folding with native-like DNA bending) was observed in studies of the adjoining aromatic substitution W70F in the SRY HMG box (consensus box position 15; Fig. 12C) (15). Although the latter substitution has not to date been observed in a patient, a cavity-associated mutation at the same site (W70L) was identified as a de novo Swyer mutation (63). This mutation (occurring at a site less exposed than is Phe-109) caused a more severe decrement in stability with marked reduction in specific DNA affinity (15). Whereas the present proband had gonadoblastoma in situ, the W70L patient presented with gonadoblastoma as a discrete tumor (63).
Comparative studies of clinical variants promise to uncover general architecture rules governing the structure and function of the SOX HMG box. The failure of kinetic compensation in the case of W70L, for example, was ascribed in unresolvable structural frustration in the bound state due to steric clash imposed by the tetrahedral configuration of the Leu ␥-carbon and hence non-planarity of the mutant side chain (15). It would be of future interest to investigate F109L and other non-planar substitutions to compare and contrast these two sites of conserved aromatic residues (box positions 15 and 54; Fig. 12, C and D). We speculate that box position 54, due to its partial exposure to solvent and less constrained packing environment, may tolerate diverse substitutions with respect to maintenance of high affinity specific DNA binding, although we have shown that that Trp-70 (box position 15) can only be substituted by other aromatic amino acids (15).
Clinical mutations have also been found at box position 43 (W98R (46)), at which an invariant Trp adjoins box position 54 a Swyer mutation I90M occurs within the Leu-rich nuclear export signal of SRY (40), impairing NCS and hence an activating phosphorylation N-terminal to the HMG box (14). b Swyer mutation R62G occurs within the N-terminal NLS, impairing nuclear entry and hence TESCO occupancy (14,35). (Fig. 12C). The indole ring contributes both to core packing and to specific DNA binding (as part of the cantilever-associated "hydrophobic wedge" in the minor groove and via a hydrogen bond from the indole NH to the DNA backbone (26,27)). Because this Trp (unlike that at box position 15) is invariant even among distant structure-specific HMG boxes (top panel of Fig. 12D), representing an evolutionary depth of more than 500 million years (66), we imagine that no other natural side chain can fulfill both roles. It is noteworthy that substitution of the homologous Trp by Arg has also been observed in the homologous HMG boxes of SOX9 (in a patient with campomelic dysplasia with XY sex reversal (73)) and SOX10 (in a patient with Kallmann syndrome (74)). Such corresponding mutations highlight how general architectural rules shared by a family of lineage-specific TFs may underlie diverse clinical presentations. Similarities and Differences between the F109S Human HMG Box and the HMG Box of Mouse Sry-The HMG box of mouse Sry (like the orthologous boxes of muroid rodents in general) is divergent relative to other mammalian taxa (75). Such divergence has been ascribed to the evolutionary dynamics of a muroid-specific C-terminal Gln-rich domain (76,77), which functions as a transcriptional activation domain (78). This domain is the result of an ancestral invasion of the muroid Y chromosome by a DNA microsatellite and its fortuitous location in-frame within the Sry gene (76). The encoded Gln-rich domain provides intragenic complementation for deleterious mutations in the HMG (76) and also protects the protein from proteasomal degradation (77). Such complementation at the protein level has been proposed to enable the DNA microsatellite to function as a genetic capacitor, permitting the accumulation of cryptic genetic variation in the HMG box and its discharge as a source of evolutionary novelty (76). We speculate that the Gln-rich domains of rodent Sry orthologs either (i) protect these divergent and unstable HMG boxes from polyubiquitination or (ii) hinder degradation of these TFs (once ubiquitinated) from 26S proteasomal degradation.
The instability of the murine Sry HMG box is broadly consistent with an analogy proposed by Eicher and co-workers (51) between Swyer syndrome and the widely observed phenomenon of murine Y/autosome strain incompatibility leading to intersexual phenotypes (33,79). Our CD studies indicate, however, that the inherited Swyer mutation renders the human SRY HMG box even less stable than the murine HMG box with respect to both thermal unfolding and chemical denaturation. Furthermore, studies of intrinsic Trp fluorescence suggest that the hydrophobic core of the F109S domain is less well protected from solvation than is the core of the murine domain. That the majority of XY humans bearing the F109S mutation develop as males (and in at least two cases as fertile males (28)) suggests that human testis determination is more robust that murine testis determination. It would be of future interest to develop quantitative methods to assess input-output relationships among orthologous developmental switches. Such studies would in principle be akin to those pioneered by Ptashne and co-workers (81) in studies of the phage cI and Cro repressors. Quantitative differences in the molecular robustness of a developmental switch may in turn correlate with macro-evolutionary differences the pace of evolutionary change in a taxon, an overarching goal in the emerging "evo-devo" synthesis (10,18,29).
Clinical Mutation F109S Provides Biological Insight into Transcriptional Regulation-The biophysical instability of the F109S HMG box and its near-complete unfolding at 37°C are associated with accelerated proteasomal turnover (relative to WT) of epitope-tagged full-length F109S SRY following transient transfection in four cell lines. These cell lines share the universal eukaryotic proteasomal pathway (82) but otherwise differ in species of origin (rat or human), developmental stage, lineage, and degree of transformation. Despite these marked differences, cellular studies of protein degradation following translational arrest by cycloheximide are remarkable for the uniformity of results.
To delineate mechanisms contributing to the decreased halflife of the mutant SRY, we sought to characterize the ubiquitination state of the variant and WT SRY under conditions of chemical proteasome inhibition. Protein turnover is regulated by various mechanisms, including proteasomal degradation (82,83). Proteins that misfold due to mutation, chemical damage, or improper maturation are often targeted and hydrolyzed by this process. Two routes of proteasomal degradation have been characterized (57,58,82) as follows: (i) ubiquitin-independent hydrolysis by the 20S core proteasome, and (ii) ubiquitindependent pathway and hydrolysis by the 26S proteasome complex. Our results have implicated the latter as F109S SRY exhibited increased polyubiquitination (relative to WT SRY and I68A SRY). Although structural analysis of F109S SRY domain suggests that the variant TF resembles, an unfolded protein within the cell, its in vitro ubiquitin-independent degradation by the 20S core proteasome was similar to that of the WT SRY domain. Together, these findings provided evidence that the accelerated degradation of epitope-tagged F109S SRY is due to the ubiquitin-dependent 26S proteasomal pathway.
Of the four mammalian cell lines employed in this study, only CH34 cells exhibit histone marks in the embryonic TESCO (49) associated with an open chromatin structure amenable to transcriptional activation (84). Such a chromatin structure presumably reflects the site and stage of the cell line's origin in the bipotential gonadal ridge just prior to Sry expression (46). Similarly, it was only in CH34 cells that we could observe the following: (a) TESCO occupancy by WT SRY on its transient transfection and (b) SRY-directed transcriptional activation of Sox9. Evidence that such activation was gene-or pathway-specific was provided by control studies of other members of the Sox family not implicated in testis determination and by control studies of housekeeping genes. Together with the above cell line specificity, the absence of SRY-directed transcriptional activation of the latter two sets of endogenous genes implies that SRY-directed activation of Sox9 in CH34 cells provided a mechanistic model of a developmental switch rather than a general state of enhanced transcriptional activity in a transfected cell line.
In our studies enhanced polyubiquitination and accelerated proteasomal degradation of F109S SRY led to a 2-fold decrease in its TESCO enhancer occupancy (relative to WT SRY) and in turn to a 2-fold decrease in extent of SRY-directed Sox9 expression. The relative impairment of target gene regulation was similar in magnitude under standard transfection conditions (1ϫ, corresponding on average to ϳ7 ϫ 10 5 WT SRY molecules per cell and 2 ϫ 10 3 variant SRY molecules per cell) and upon marked plasmid dilution with the parent plasmid (50ϫ, leading on average to expression of ϳ2 ϫ 10 3 WT SRY molecules per cell and 10 2 variant SRY molecules per cell). Two lines of evidence indicated that the functional defect of F109S SRY is solely due to its instability and accelerated turnover as follows: (i) chemical proteasome inhibition by MG132 fully rescued TESCO occupancy leading to native Sox9 activation and (ii) equalization of protein expression levels (at a mean value of 10 3 SRY molecules per cell) by individual adjustment of plasmid dilution ratio (25ϫ for WT SRY; 1ϫ for F109S SRY) likewise led to essentially equal levels of Sox9 mRNA accumulation.
To date, the following three inherited Swyer mutations in SRY have been characterized in biochemical detail (65): V60L (leading to a partial impairment of nuclear import via the N-terminal NLS), I90M (leading to a partial impairment of nuclear export and hence NCS-coupled phosphorylation), and F109S (leading as above to instability). Each of these impairments is associated, independently of molecular mechanism, with a 2-fold decrease in SRY-dependent Sox9 activation under conditions of physiological TF expression (generally 10 2 -10 4 protein molecules per cell (60)). These findings thus reflect a general feature of human male sex determination, the slim margin by which the output of the SRY-Sox9 regulatory axis functions as a developmental switch. The 2-fold threshold of Sox9 expression is consistent with the pathophysiology of campomelic dysplasia as a syndrome of TF haploinsufficiency (85). In none of these pedigrees has the influence of autosomal genetic variation been distinguished from effects of stochastic gene expression as a mechanism of phenotypic variation.
The remaining four inherited Swyer mutations (Table 6) are each likely to perturb fundamental mechanisms underlying the structure or function of SRY. R30I, for example, adjoins potential sites of serine phosphorylation (bold in sequence LRRSSS-FLC; residues 31-33 in human SRY) recognized by PKA (86). Although SRY has not been shown to be phosphorylated in vivo and the adjoining one or more of these three serines are only conserved within primates (76), and not more broadly among therian mammals (76), evidence has been obtained in cell culture that such phosphorylation enhances transcriptional activity (86) and is coupled to NCS (14). We speculate that R30I impairs PKA recognition of this motif and hence recapitulates the functional consequences of impaired nuclear import or export, a 2-fold reduction in SRY-directed transcriptional activation of SOX9 (14) as a general feature of the inherited Swyer syndrome (65). It would be of future interest to test this prediction and more broadly to investigate the relationship (if any) between PKA site selectivity in SRY and transcriptional potency.

F109S SRY as a Quantitative
Tool-The biophysical instability of a variant SRY HMG box coupled with its retention of native-like specific DNA-binding and -bending properties both underlies the sex-reversed phenotype of the proband and enables the male development of her father, uncle, and brothers. We imagine that such developmental variation is purely a "numbers game," and in the proband the number of variant SRY molecules per cell in the gonadal ridge fell below the threshold to trigger implementation of the Sertoli cell program.
Beyond the intrinsic interest of these results, we envisage that the fortuitous molecular properties of F109S SRY may enable this variant allele to function as a tool in future quantitative studies of testis determination as a model developmental switch in therian mammals. Just as in the pioneering decades of prokaryotic molecular biology, wherein a small molecule (isopropyl ␤-D-1-thiogalactopyranoside) was employed to regulate the activity of Lac repressor (87) and in turn the expression of cI repressor in engineered operons (2), we imagine that MG132-regulated expression of F109S SRY may provide an analogous chemical "rheostat" by which to measure transcriptional input-output relationships. If so, the binary switch between male and female programs of mammalian gonadogenesis may permit quantitative modeling akin to that of the celebrated genetic switch between the lytic and lysogenic programs of lambdoid bacteriophages (4). Such future directions would highlight the prescience of these prokaryotic model studies (88).
Concluding Remarks-The present results have demonstrated a novel mechanism of impaired human organogenesis, the heightened susceptibility of a master lineage-specific TF to polyubiquitination and proteasomal degradation. This mechanism has historical resonance in studies of simpler eukaryotes: precise control of the cellular lifetimes of mating type-specific TFs was first investigated in Saccharomyces cerevisiae (MAT␣2 and MATa1 (Ref. 89 and references therein)). Although the latter TFs contain homeodomains as modules of specific DNA binding (90), and so are unrelated to SRY, the corresponding mating-type gene matA in fungus Aspergillus nidulans encodes an HMG-box TF (91). Remarkably, matA may functionally be replaced by human SRY to regulate both early and late stages of  (92). Such complementation suggests that SRY-related HMG boxes have deep evolutionary roots in Eukarya. Furthermore, the fundamental role of ubiquitin-directed proteasomal degradation in cell fate decisions (as exemplified in both the yeast mating-type switch (89) and mammalian gonadogenesis) is independent of molecular motif of DNA recognition.
The present results have further shown that the specific DNA-binding and DNA-bending activities of the SRY HMG box are robust to its structural destabilization. Yet, the cellbiological properties of F109S SRY support the notion that the WT TF functions at the edge of developmental ambiguity (13,14). That a tenuous genetic switch (i.e. a mere 2-fold decrease in SRY-directed transcriptional activation of SOX9) underlies inherited XY sex reversal may seem surprising but is in accordance with the genetics of human DSD as a downstream syndrome of TF haploinsufficiency (93,94). These clinical correlations highlight the narrow margins of decision-making by opposing sex-specific gene-regulatory networks in the bipotential gonadal ridge (34,95). In such decisions ubiquitin-directed protein degradation in principle provides a mechanism to control both TF abundance and its time course in relation to morphogenesis. Indeed, exquisite control of the timing of Sry expression in mice has been shown to be critical to the proper initiation and maintenance of testis formation (96 -98). We envisage that systematic studies of human SRY variants, as identified in Swyer patients (22,60) and as tested in model organisms (72), may decipher molecular mechanisms of an archetypical yet enigmatic genetic switch (99). Like the F109S "molecular rheostat" characterized here, the diverse human mutations associated with inherited XY sex reversal promise to provide tools to probe mechanisms of mammalian organogenesis at the edge of ambiguity.

Experimental Procedures
Protein Purification-WT and variant SRY domains of human SRY were expressed in Escherichia coli strain T LYS (New England Biolabs, Inc., Ipswich, MA) and purified as described (100). Purity was determined in each case to be Ͼ98% by SDS-PAGE. Results of matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were in agreement with expected values.
Circular Dichroism-Far-and near-ultraviolet (UV) CD spectra were obtained at 4, 25, and 37°C in a 1-mm path length quartz cuvette using an Aviv spectropolarimeter equipped with titrating unit (Aviv Biomedical, Inc., Lakewood, NJ). The domains were made 25 M in 140 mM KCl and 10 mM potassium phosphate ((pH 7.4) "standard buffer"). Thermal unfolding was monitored at helix-sensitive wavelength 222 nm. CD difference spectra were calculated as the buffer-corrected difference between the observed spectrum of a protein-DNA complex and the sum of the spectra of the free protein and free DNA site.
Protein Stability-Fractional protein unfolding was monitored as a function of guanidine hydrochloride concentration by CD ellipticity at 222 nm. The domains were made 5 M in standard buffer in a titrating cuvette. The same protein concentration was used in a titrant reservoir containing 7.8 M guani-dine-HCl in the same buffer. Thermal unfolding of the free domains and equimolar protein-DNA complexes (25 m in standard buffer) was monitored using a 12-bp consensus DNA duplex (5Ј-GTGATTGTTCAG-3Ј and complement; core target sequence underlined); CD spectra (200 -320 nm) were acquired from 4 -90°C at 2.5°C increments.
Tryptophan Fluorescence Spectroscopy-Intrinsic Trp fluorescence spectra of the free HMG boxes and their specific DNA complexes were observed in standard buffer at 15 and 37°C at a concentration of 5 M. Emission spectra were acquired from 300 to 500 nm following excitation at 295 nm. Extent of the DNA-dependent inner filter effect under these conditions was estimated through control studies as described (15).
Fluorescence Resonance Energy Transfer-Protein-directed DNA bending was probed by steady-state FRET as described (12). This approach employed a 15-bp DNA duplex (5Ј-TCG-GTGATTGTTCAG-3Ј ("upper strand") and complement ("lower strand"); consensus target site underlined). Use of a 15-bp DNA site restricted protein binding to the 1:1 high affinity complex (13). To provide a fluorescent donor, the upper strand was extended at its 5Ј terminus by 6-carboxyfluorescein; the dye was flexibly linked to the DNA through a hexanyl linker. To provide a compatible acceptor, the lower strand was extended at its 5Ј-end by tertamethylrhodamine, also via a hexanyl linker. The labeled DNA strands were purchased from Oligos, Etc., Inc. (Wilsonville, OR). Photophysical control studies verified the mobilities of the probes and excluded non-FRET-related mechanisms of donor quenching (12). FRET studies employed a DNA concentration of 3 M in 10 mM potassium phosphate, 10 mM Tris-HCl, 140 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol with final pH 8.0 ("FRET buffer").
FRET-based DNA Binding Assays-Steady-state FRET was employed to determine protein-DNA dissociation constants (K d ); the DNA site was as above. Measurements were made in FRET buffer at 15 and 37°C. Varying concentrations of the WT or variant SRY domain were titrated at a constant DNA concentration of 25 nM. Emission spectra were recorded from 500 -650 nm following excitation at 490 nm. Estimates of K d were determined by plotting change in fluorescence intensity at 520 nm against total protein concentration. Data were fit to a single-site ligand-binding model (Equation 1) as described (101) using Origin 8.0 software (OriginLab Corp., Northampton, MA).
In this formalism, ⌬F is the change in donor fluorescence observed on addition of the SRY domain relative to the baseline DNA fluorescence; ⌬F 0 is the maximum fluorescence change obtained in a 1:1 protein-DNA complex; K d is the dissociation constant; D 0 is the concentration of DNA (25 nM); and S is the concentration of SRY domain.
Stopped-flow Kinetic FRET Assay-Rates of protein-DNA dissociation were measured with an Aviv double-mixing stopped-flow apparatus at a fixed temperature (6, 15, 25, or 37°C); the instrument contained a thermo-electric temperature controller. Fluorescence emission was monitored at 520 nm following excitation at 490 nm using an Aviv ATF 105 spectrofluorometer (12). In brief, a 20-fold molar excess (final stoichiometries) of unlabeled DNA in FRET buffer was employed to sequester the WT or variant SRY domain on rapid mixing. Estimates of dissociation rate constants (k off ) were obtained by fitting the traces to a mono-exponential equation; values represent the mean and standard error of four replicates. Control studies of the WT domain indicated that similar time-dependent recovery of donor emission was observed irrespective of the concentration of the unlabeled DNA in the molar excess range 10 -50-fold relative to initial concentration of the specific FRET-labeled DNA-protein complex.
Permutation Gel Electrophoresis-Six DNA fragments (150 bp each) containing an SRY-binding site (5Ј-ATTGTT-3Ј and complement) were PCR-amplified from a plasmid previously described (12) such that the binding site was at varying distances from 5Ј-end (leading to variation in "flexure displacement"). Each fragment (10 nM) was complexed with the WT or variant HMG box (20 nM). Gels were purchased from Bio-Rad, equilibrated, and resolved in 0.5ϫ Tris borate/EDTA buffer (TBE). Protein-DNA complexes were visualized using SYBR Green stain (Life Technologies, Inc.).
NMR Spectroscopy-15 N-Labeled WT and variant Sry domains were prepared by growth of a corresponding overexpression strain of E. coli (12) in minimal medium containing [ 15 N]ammonium sulfate as sole nitrogen source. The proteins were dissolved in a nitrogen-purged buffer containing 10 mM potassium phosphate buffer (10% D 2 O (pH 6.5)) and 50 mM NaCl and placed in a 280-l Shigemi NMR tube. Homonuclear 2D 1 H nuclear Overhauser effect (NOESY), total correlation (TOCSY), and double quantum-filtered correlation spectra were obtained of the free domains and their specific DNA complexes; a 15-bp DNA duplex was employed with sequence 5Ј-TCGGTGATTGTTCAG-3Ј and complement (core target sequence underlined and site of Ile-68-cantilever insertion in boldface). To obtain a spectrum of the unfolded state, the WT 15 N-labeled domain was also dissolved in phosphate-buffered saline (PBS) solution containing 5.4 M urea (10% D 2 O (pH 7.4)). Two-dimensional 1 H-15 N heteronuclear single-quantum coherence (HSQC) spectra were acquired at 25°C using a Bruker Avance 700 MHz spectrometer.
Mammalian Plasmids-Plasmids expressing full-length human SRY or variants were constructed by PCR (13). Following the initiator Met, the cloning site encoded a hemagglutinin (HA) tag in triplicate to enable WB and chromatin immunoprecipitation (ChIP; below). Mutations in SRY were introduced using QuikChange TM (Stratagene). Constructions were verified by DNA sequencing.
Rodent Cell Culture-CH15Ϫ and CH34 cells (kindly provided by Dr. P. K. Donahoe, Massachusetts General Hospital, Boston, MA) (14,46) were cultured in DMEM containing 5% heat-inactivated fetal bovine serum at 37°C under 5% CO 2 . For proteasome-inhibitor studies, transfected cells were maintained for 24 h in serum-free conditions and then treated with proteasome inhibitor MG132 for 6 h followed by 18 h of incubation in 5% serum-containing medium.
Human Cell Lines-Hs1.TES cells (derived from second-trimester human fetal testis; ATCC CRL 7002 TM ) were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium containing 10% FBS in 5% CO 2 atmosphere. HEK 293T cells (derived from the human embryonic kidney (47)) were obtained from ATCC and grown in Dulbecco's modified Eagle's medium with 10% FBS.
Transient Transfections-Transfections were performed using FuGENE 6 as described by the vendor (Hoffmann-La Roche, Nutley, NJ). After 24 h in serum-free medium, cells were recovered using fresh DMEM containing 5% heat-inactivated fetal bovine serum. Transfection efficiencies were determined by ratio of GFP-positive cells to untransfected cells following co-transfection with pCMX-SRY and pCMX-GFP in equal amounts (14). Subcellular localization was visualized by immunostaining for 24 h post-transfection following treatment with 0.01% trypsin (Invitrogen) and plating on 12-mm coverslips. SRY expression was monitored by Western blotting via its triplicate HA tag (above).
Cycloheximide Assay and Western Blotting-24 h following transient transfection, cells were split evenly into 6-well plates and treated with cycloheximide to a final concentration of 20 g/ml in DMEM for the indicated times; cells were then lysed by radioimmunoprecipitation assay buffer (Hoffmann-La Roche). After protein normalization, cell lysates were subjected to 12% SDS-PAGE and WB using anti-HA antiserum (Sigma) at a dilution ratio of 1:5000 with ␣-tubulin antiserum providing a loading control. Quantification was performed by Image J software (rsbweb.nih.gov/ij).
Plasmid Dilution Protocol-Dilution of the WT or variant SRY expression plasmid by the parent plasmid (to provide a constant DNA mass per transient transfection) was employed in CH34 cells to modulate the intracellular concentration of SRY as described (13,14). Dilution ratios ranged from 1 to 50ϫ, where 1ϫ represents the undiluted expression plasmid and 50ϫ represents a 1:50 ratio of the expression plasmid concentration (in g/ml) to the parent plasmid concentration.
Estimation of Intracellular SRY Concentration-The intracellular concentration of the WT or variant epitope-tagged SRY was estimated following transient transfection with dilution ratio 1-50ϫ as described (13,14). In brief, ␣-tubulin was employed in each case as an internal standard. In step 1, we assumed that a typical mammalian cell contains by weight the following amounts of tubulin: 300 ϫ 10 Ϫ12 g ϫ 3% ϭ 9 ϫ 10 Ϫ12 g. In step 2, we next estimated the number of tubulin molecules (molecular mass 110 kDa) as (9 ϫ 10 Ϫ12 g/110 ϫ 10 3 g/ mol) ϫ 6.02 ϫ 10 23 molecules/mol ϭ 5 ϫ 10 7 molecules. In step 3, we in turn estimated the number of transfected SRY molecules/cell in the 1ϫ transfection using its Western blotting signal strength relative to ␣-tubulin: 5 ϫ 10 7 /100 (the tubulin-blotting dilution ratio)/2.5 (relative band intensity/tubulin/1ϫ SRY signal)/ 28.9% (transfection efficiency), which yields an estimate of 7 ϫ 10 5 molecules (predominantly in the nucleus in the case of WT SRY and F109S SRY). Finally, in step 4, we estimated the number of transfected SRY molecules/cell following 50-fold plasmid dilution using relative signal strength between 1 and 50ϫ HA-SRY Western blottings: 7 ϫ 10 5 molecules/390, which yields an estimate of near 1900 molecules per cell. This degree of expression is within the middle of the range of cellular abundances expected of a lineage-and stage-specific transcription factor (10 2 -10 4 molecules per nucleus). This four-step procedure assumed that the affinities of the anti-HA and anti-tubulin antisera were similar as indicated by the vendor.
Anti-ubiquitin Western Blottings-To compare relative extents of ubiquitination of HA-tagged SRY variants in the above cell lines on transient transfection, the cells were grown in the absence or presence of MG132 and lysed with radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.05% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride; PMSF) containing protease inhibitors (Hoffmann-La Roche). Lysates were precipitated with monoclonal anti-HA-agarose beads (catalogue no. E6779; Sigma) and resolved by SDS-PAGE (4 -20% gradient gel; Bio-Rad). Ubiquitinated proteins within the immunoprecipitation were detected by immunoblotting using a monoclonal anti-Ub antibody (catalogue no. MAB1510; Millipore). To monitor the total SRY input control, the anti-HA-agarose beads were also subjected to immunoblotting using 10% SDS-PAGE as probed by a polyclonal anti-HA antiserum (catalogue no. H6908; Sigma). WB using an antiserum against ␣-tubulin (catalogue no. T6074; Sigma) provided general loading controls. To facilitate quantification of band intensities, successive dilutions of the immunoprecipitated proteins were tested followed by optimization of film exposure times. Four or five biological replicates were obtained for each cell line. Images were analyzed using ImageJ in triplicate (technical replicates) to estimate relative levels of ubiquitination. Statistical analyses employed the Student's t test.
Default Degradation of SRY HMG Boxes by the 20S Core Proteasome-The purified (Ͼ95%) rabbit 20S core proteasome was obtained from Sigma (catalogue no. P3988) and used without further purification. In vitro digestion of WT or F109S variant HMG boxes was performed at 37°C as described by Yamada et al. (102). The domains were in each case made 100 M in a reaction volume of 100 l in a buffer consisting of 10 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , 1 mM 2-mercaptoethnol, and 0.025% (w/v) SDS. Domains were pre-incubated at 37°C for 15 min, and ϳ2 g (final concentration is 0.03 M) of the 20S proteasome was then added to each reaction. At indicated times, 3-l aliquots of the reaction were quenched at room temperature in Laemmli sample buffer (Bio-Rad). Samples were resolved by SDS-PAGE (4 -20% gradient gels; Bio-Rad). Following Coomassie staining, band intensities were measured using ImageJ software. For each domain, three biological replicates were obtained; for each replicate, three technical replicates of the SDS-polyacrylamide gels were obtained and quantified.
Immunocytochemistry-Transfected CH34 and HEW 293T cells were plated evenly on 12-mm coverslips, fixed with 3% para-formaldehyde in PBS (pH 7.4) on ice for 30 min, treated with cold permeability buffer solution (PBS containing 10% goat serum and 1% Triton X-100; Sigma) for 10 min, blocked with 10% goat serum and 0.1% Tween 20 in cold PBS (Sigma), and incubated overnight at 4°C with FITC-conjugated anti-HA antibody (diluted to 1:400 ratio; Santa Cruz Biotechnology, Santa Cruz, CA). After washing and 4Ј,6-diamidino-2-phenylindole (DAPI) staining, cells were visualized by fluorescence microscopy. Nuclear localization of F109S SRY was evaluated in relation to WT SRY in CH34 cells by the ratio of cells exhibiting nuclear HA-tagged SRY to the total number of GFPpositive cells. To enable robust statistical analysis, ϳ500 SRYpositive cells were counted in each case (exclusive of non-expressing cells with mean transfection efficiency in the range 37-41%) and categorized as predominantly nuclear or pan-cellular in accordance with past studies (14).
TESCO Chromatin Immunoprecipitation-Cells were transfected with plasmids encoding epitope-tagged WT or variant SRY under 1ϫ conditions (undiluted by the parent plasmid) and subjected to ChIP. Recovered cells were cross-linked in wells by formaldehyde, collected, and lysed after quenching the cross-linking reaction. Lysates were sonicated to generate 300 -400-bp fragments and immunoprecipitated with anti-HA antiserum (Sigma) containing a protein A slurry (Santa Cruz Biotechnology) for pre-clearing. A nonspecific antiserum (control IgG; Santa Cruz Biotechnology) served as nonspecific control. After de-cross-linking at 65°C overnight, fragments were treated with proteinase K and RNase (Hoffmann-La Roche), and then extracted by using 1:1 solution of phenol with a 24:1 solution of chloroform and isoamyl alcohol. PCR and qPCR protocols were as described (15). The following pairs of forward (F) and reverse (R) DNA oligonucleotide primers were employed to probe TESCO subsites as defined by Sekido and Lovell-Badge (49). Rat-specific primer sets a-c (pertinent to cell lines CH15 and CH34) are as follows: set a (with conserved SRY-binding sites), F 5Ј-GGAACTCCAACTACGTAC-3Ј and R 5Ј-CCTGTAGTTGGTAGCTGC-3Ј; set b (as negative control), F 5Ј-ATCTCTACAGCTGACTTC-3Ј and R 5Ј-TAG-CTGGGCTCATATCG-3Ј; and set c (with conserved SRYbinding sites), F 5Ј-CTGAGAGCAATCTGAGC-3Ј and R 5Ј-CACACCGTGCAAATGTA-3Ј. Human-specific primer sets a-c (pertinent to cell lines HEK 293T and Hs1.TES) are as follows: set a (with conserved SRY-binding sites), F 5Ј-CTAAACTCCAACTACACACGAA-3Ј and R 5Ј-CCTTGA-CAGCTGCCACCT-3Ј; set b (as negative control), F 5Ј-GTTTTATGCAGCTGATTCT-3Ј and R 5Ј-CTTGTCTGT-GTAGCC-3Ј; and set c (with conserved SRY-binding sites), F 5Ј-GTGGAATGAATGTGCAC-3Ј and R 5Ј-GGTTAGTCT-TTTAATTTTTG-3Ј.
ChIP Studies of Histone Modifications-To probe histone modifications within rat or human TESCO associated with activated (mono-methylation, di-methylation, or tri-methylation of lysine H3K4 (designated me1, me2, and me3), monomethylation of lysine H3K9 or H3K27) or repressed chromatin (di-methylation or tri-methylation of lysine H3K9 or H3K27), ChIP was employed with primer set a in the appropriate species (above) following immunoprecipitation with corresponding anti-me1, anti-me2, or anti-me3 IgG (Millipore Corp., Billerica, MA). Signals were quantified by qPCR as above.
Transcriptional Activation Assay-Following transient transfection (above), SRY-mediated transcriptional activation of Sox9 was measured in triplicate by qPCR as described (13). Cellular RNA was extracted using RNeasy as described by the vendor (Qiagen, N.V., Hilden, Germany). Transfection protocols were performed with the following: (i) SRY expression plasmid only, which contains 1 g of WT or variant SRY-encoded plasmid per million cells ("1ϫ" conditions) and (ii) a