Browsing by Author "Racca, Joseph D."

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    Inherited Human XY Sex Reversal and Gonadal Neoplasia Due to Enhanced Formation of Non-Specific Enhanceosomes by an Architectural Transcription Factor
    (Endocrine Society, 2021-05-03) Chen, Yen-Shan; Racca, Joseph D.; Belgorosky, Alicia; Weiss, Michael Aaron; Biochemistry and Molecular Biology, School of Medicine
    The development of organisms is regulated by a fine-tuned gene-regulatory network, which is driven by transcription factors (TFs). In the embryogenesis, these TFs control diverse cell fates and final body plan. This is precisely regulated by a specific DNA-binding process and enhanceosome formation. A model is provided by testis determination in mammals, which is initiated by a Y-encoded architectural transcription factor, SRY. Mutations in SRY cause gonadal dysgenesis leading to various developmental defects. Such mutations cluster in SRY’s high mobility group (HMG) box, a sequence-specific DNA-binding domain shared by a conserved family of TFs. Here, we have characterized several mutations at the same position in HMG box, which are compatible with either male or female phenotypes as observed in an XY father and XY daughter, respectively. These mutations, at a function-unknown motif in the SRY HMG box, markedly disturb the specific DNA affinity. On transient transfection of human and rodent cell lines, the SRY variants exhibit decreased specific DNA-binding activity (relative to wild type) are associated with mis-formed enhanceosomes. The variants’ gene regulatory activities were reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. When engineered mutations that functions to increase the DNA-binding specificity were deployed to SRY variants, the transcriptional activity was in association with restored occupancy of sex-specific enhancer elements in principal downstream gene Sox9. Our findings define a novel mechanism of impaired organogenesis, disturbed specific DNA-binding activity of a master transcription factor, leading to a developmental decision poised at the edge of ambiguity.
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    Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY
    (American Society for Biochemistry and Molecular Biology, 2024) Racca, Joseph D.; Chen, Yen-Shan; Brabender, Adam R.; Battistin, Umberto; Weiss, Michael A.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high-mobility group box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY high-mobility group domain bound to a DNA site containing consensus element 5'-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5'-CATTGT/ACAATG-3', shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever) and Arg20, Asn32, and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles (69-84°) of the distinct box-DNA complexes are similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36, or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10, or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective Ala substitutions exhibited significantly decreased transcriptional activation of SRY's principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.
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    Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity
    (Frontiers Media, 2022-07-26) Chen, Yen-Shan; Racca, Joseph D.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.
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    Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity
    (Frontiers, 2022-12) Racca, Joseph D.; Chatterjee, Deepak; Chen, Yen-Shan; Rai, Ratan K.; Yang, Yanwu; Georgiadis, Millie M.; Haas, Elisha; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain’s minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father’s male development, but at odds with the daughter’s XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably “unclamps” its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.