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Browsing by Author "Racca, Joseph"
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Item Inherited Human Sex Reversal due to Loss of a Water-Mediated Hydrogen Bond at a Conserved Protein-DNA Interface(Cold Spring Harbor Laboratory Press, 2021) Chen, Yen-Shan; Racca, Joseph; Amir, Dan; Haas, Elisha; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineMale sex determination in mammals is initiated by SRY, a Y-encoded architectural transcription factor. The protein contains a high-mobility-group (HMG) box that mediates sequence-specific DNA bending. Mutations in SRY causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box. Although such mutations usually arise de novo in spermatogenesis, some are inherited: male development occurs in one genetic background (the father) but not another (the sterile XY daughter). Here, we compare de novo and inherited mutations at an invariant Tyr adjoining the motif’s basic tail (consensus position 72; Y127C and Y127F in intact SRY). Crystal structures of homologous SOX-DNA complexes suggest that the wild-type side chain’s para-OH group anchors a water-mediated hydrogen bond to the DNA backbone. In an embryonic gonadal cell line, Y127C (de novo) led to accelerated proteasomal proteolysis and blocked transcriptional activity; activity remained low on rescue of expression by chemical proteasome inhibition. Y127F (inherited) preserved substantial transcriptional activity: 91(±11)% on SRY overexpression and 65(±17)% at physiological expression. Control studies indicated no change in protein lifetime or nuclear localization. Only subtle biophysical perturbations were observed in vitro. Although though inherited variant’s specific DNA affinity was only twofold lower than wild type, stopped-flow kinetic analysis revealed a sevenfold decrease in lifetime of the complex. Time-resolved fluorescence energy transfer (using a 15-base pair DNA site) demonstrated native mean DNA bending but with a slightly widened distribution of end-to-end DNA distances. Our findings highlight the contribution of a single water-mediated hydrogen bond to robustness of a genetic switch in human development.Item OR15-5 Human Sex Determination at the Edge of Ambiguity: Impaired SRY Phosphorylation Attenuates Expression of the Male Program(Oxford University Press, 2019-04-15) Chen, Yen-Shan; Racca, Joseph; Phillips, Nelson; Weiss, Michael; Medicine, School of MedicineA paradox is posed by metazoan gene-regulatory networks (GRNs) that are robust yet evolvable. Insight may be obtained through studies of bistable genetic circuits mediating developmental decisions. A model in organogenesis is provided by the sex-specific differentiation of the embryonic gonadal ridge to form a testis or ovary. Here, we investigated a Swyer mutation in human testis-determining factor SRY that impairs its phosphorylation in association with variable developmental outcomes: fertile male, intersex, or infertile female (46, XY pure gonadal dysgenesis). The mutation (R30I) abrogates serine phosphorylation within a putative target site for protein kinase A (PKA) N-terminal to the HMG box. Diverse processes can be regulated by protein phosphorylation, including DNA recognition by transcription factors (TFs). Phosphorylation of this site in human SRY (LRRSSSFLCT; italics) in vitro was previously shown to enhance specific DNA affinity. Biological consequences of the mutation were evaluated in SRY-responsive mammalian cell lines following transient transfection. The mutation attenuated in concert occupancy of a target enhancer (TESCO) and SOX9 transcriptional activation. These perturbations were mitigated by acidic substitution (LRIDDDFL) whereas Ala substitutions (RRAAAFL or RIAAAFL) attenuated activity to an extent similar to R30I alone. No differences were observed in nuclear localization. Mutagenesis suggested that the central Ser is most efficiently phosphorylated in accord with PKA targeting rules. Replacement of the native site by an optimized “Kemptide” PKA site (LRRASLGCT) enhanced both SRY phosphorylation and SOX9 transcriptional activation whereas a “swapped” protein-kinase C determinant (LRRSSFRRCT) blocked phosphorylation. Among SRY variants, extent of cellular phosphorylation mirrored relative in vitro efficiencies of synthetic SRY-derived peptides as PKA-specific substrates. Although several kinases are predicted in silico to target this tri-serine motif, cell-based studies implicate PKA as the relevant kinase in vivo. Our results provide evidence that primate Sry requires its phosphorylation for full gene-regulatory activity. A PKA site N-terminal to the SRY HMG box, unique to primates, exemplifies network “tinkering” through recruitment of a new regulatory linkage. Molecular characterization of the R30I inherited Swyer mutation in SRY thus demonstrates that impaired TF phosphorylation can attenuate a human developmental switch at the edge of ambiguity.