Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.     

Floppy SOX: mutual induced fit in hmg (high-mobility group) box-DNA recognition

The high-mobility group (HMG) box defines a DNA-bending motif of broad interest in relation to human development and disease. Major and minor wings of an L-shaped structure provide a template for DNA bending. As in the TATA-binding protein and a diverse family of factors, insertion of one or more side chains between base pairs induces a DNA kink. The HMG box binds in the DNA minor groove and may be specific for DNA sequence or distorted DNA architecture. Whereas the angular structures of non-sequence-specific domains are well ordered, free SRY and related autosomal SOX domains are in part disordered. Observations suggesting that the minor wing lacks a fixed tertiary structure motivate the hypothesis that DNA bending and stabilization of protein structure define a coupled process. We further propose that mutual induced fit in SOX-DNA recognition underlies the sequence dependence of DNA bending and enables the induction of promoter-specific architectures.     

Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal

The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.     

A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.     

A novel missense mutation (S18N) in the 5′ non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives

Mutations in the sex-determining region of the Y chromosome (the SRY gene) have been reported in low frequency in patients with 46,XY gonadal dysgenesis. We investigated 21 Brazilian 46,XY sex-reversed patients, who presented either complete or partial gonadal dysgenesis or embryonic testicular regression syndrome. Using Southern blotting, polymerase chain reaction, denaturing gradient gel electrophoresis and direct sequencing, we analyzed deletions and point mutations in the SRY gene. We found a missense mutation at codon 18 upstream of the 5′ border of the HMG box of the SRY gene in one patient with partial gonadal dysgenesis. This variant sequence was also found in DNA obtained from blood and sperm cells of his father and in blood cells of his normal brother. The S18N mutation was not found in 50 normal males, ruling out the possibility of a common polymorphism. We identified a novel familial missense mutation (S18N) in the 5’ non-HMG box of the SRY gene in 1 of 21 patients with 46,XY sex reversal. Received: 6 May 1997 / Accepted: 2 October 1997     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday     

The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.

To establish the basis of sequence-specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence-specific HMG box of TCF1 alpha into their equivalent position in the non-sequence-specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N-terminal and 25 C-terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four-way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence-specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N-terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.     

Rapid binding of T7 RNA polymerase is followed by simultaneous bending and opening of the promoter DNA

To form a functional open complex, bacteriophage T7 RNA polymerase (RNAP) binds to its promoter DNA and induces DNA bending and opening. The objective of this study was to elucidate the temporal coupling in DNA binding, bending, and opening processes that occur during initiation. For this purpose, we conducted a combined measurement of stopped-flow fluorescence anisotropy, fluorescence resonance energy transfer (FRET), and 2-aminopurine fluorescence. Stopped-flow anisotropy measurements provided direct evidence of an intermediate resulting from rapid binding of the promoter to T7 RNA polymerase. Stopped-flow FRET measurements showed that promoter bending occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly bent. Similarly, stopped-flow 2-aminopurine fluorescence changes showed that promoter opening occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly melted. The indistinguishable observed rate constants of FRET and 2-aminopurine fluorescence changes indicate that DNA bending and opening processes are temporally coupled and these DNA conformational changes take place after the DNA binding step. The results in this paper are consistent with the mechanism in which the initial binding of T7 RNAP to the promoter results in a closed complex, which is then converted into an open complex in which the promoter is both sharply bent and melted.     

Evidence for increased prevalence of SRY mutations in XY females with complete rather than partial gonadal dysgenesis.

The Y chromosome gene SRY (sex-determining region, Y gene) has been equated with the mammalian testis-determining factor. The SRY gene of five subjects with 46,XY complete gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Müllerian ducts, and streak gonads) was evaluated for possible mutations in the coding region by using both single-strand conformation polymorphism (SSCP) assay and DNA sequencing. Mutations were identified in three subjects, of which two gave altered SSCP patterns. Two of them were point mutations causing amino acid substitutions, and the third was a single-base deletion causing a frameshift. All three mutations caused alterations in the putative DNA-binding region of the SRY protein. Genomic DNA was obtained from the fathers of two of the three mutant patients: one mutation was demonstrated to be de novo, and the other was inherited. The presence of SRY mutations in three of five patients suggests that the frequency of SRY mutations in XY females is higher than current estimates.     

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997     

Equilibrium binding assays reveal the elevated stoichiometry and salt dependence of the interaction between full-length human sex-determining region on the Y chromosome (SRY) and DNA

In an effort to better define the molecular mechanism of the functional specificity of human sex-determining region on the Y chromosome (SRY), we have carried out equilibrium binding assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3epsilon gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the protein to the labeled target DNA. Our results indicate that the full-length human wild-type SRY (SRY(WT)) forms a complex of high stoichiometry with its target DNA. Moreover, we have demonstrated a strong salt dependence of both the affinity and specificity of the interaction. We have also addressed the DNA bending properties of full-length human SRY(WT) in solution by fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher than the classical 1:1. Oligomerization thus appears, at least in vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the mutant protein SRY(Y129N), identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well.     

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.