Mammalian Testis-determining Factor SRY and the Enigma of Inherited Human Sex Reversal

Phillips, Nelson B.; Racca, Joseph; Chen, Yen Shan; Singh, Rupinder; Jancso-Radek, Agnes; Radek, James T.; Wickramasinghe, Nalinda P.; Haas, Elisha; Weiss, Michael A.

doi:10.1074/jbc.m111.260091

Cited by 20 publications

(82 citation statements)

References 104 publications

(165 reference statements)

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“…This protocol employed a 15-bp DNA duplex of sequence 5Ј-TCGGTGATTGTTCAG-3Ј ("upper strand") and complement; consensus target site is underlined. Use of a 15-bp DNA site restricted protein binding to the 1:1 high affinity complex (41). To provide a fluorescent donor, the upper strand was extended at its 5Ј terminus by 6-carboxyfluorescein (6-FAM); the dye was flexibly linked to the DNA through a hexanyl linker.…”

Section: Methodsmentioning

confidence: 99%

“…Transcriptional Activation Assay-Following transient transfection (see above), SRY-mediated transcriptional activation of Sox9 was measured in triplicate by quantitative real time Q-rtPCR (qPCR) as described (41). Cellular RNA was extracted using RNeasy (Qiagen, N.V., Hilden, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Mammalian Plasmids-Plasmids expressing full-length human SRY or variants were constructed by polymerase chain reaction (PCR) (41). Following the initiator Met, the cloning site encoded a hemagglutinin (HA) tag in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Y1H Screening-Reporter strains of Saccharomyces cerevisiae (derived from strain YM4271) were as described previously (41). Expression of an SRY HMG box fusion protein (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Studies of representative variants demonstrated that the core tryptophan both contributes to the folding of the HMG box and enables critical cellular processes pertinent to biological function. Whereas significant residual function was observed among aromatic variants (W70F and W70Y) in accordance with sequence patterns in the HMG box superfamily (36), the clinical variant W70L and related aliphatic variant W70A were associated with pleiotropic molecular and cellular 4 Structural and biophysical studies of SRY and homologous SOX TFs by this laboratory (31,41,90) and other laboratories (28,91,92) have been restricted to their respective HMG box domains. In our hands, the intact human SRY readily aggregates in vitro leading to progressive loss of specific DNA binding activity (40).…”

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confidence: 99%

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Structure-Function Relationships in Human Testis-determining Factor SRY

Racca

Chen

Maloy

et al. 2014

Journal of Biological Chemistry

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Background: Testis-determining factor SRY provides a model of bent protein⅐DNA complex. Results: Mutation of an invariant Trp perturbs multiple biochemical, cellular, and transcriptional activities. Conclusion: Folding and function of a sequence-specific HMG box require a core "aromatic buttress." Significance: Mutations in SRY causing human sex reversal probe the architecture and evolution of a DNA-bending motif.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Y1H Screening-Reporter strains of Saccharomyces cerevisiae (derived from strain YM4271) were as described previously (41). Expression of an SRY HMG box fusion protein (Fig.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Structure-Function Relationships in Human Testis-determining Factor SRY

Racca

Chen

Maloy

et al. 2014

Journal of Biological Chemistry

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Generation and mutational analysis of a transgenic mouse model of humanSRY

et al. 2021

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SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage the pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field.To address this issue, we generated a mouse model carrying a human SRY transgene able to drive testis determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRY's three domains (N-terminal, HMG box, and C-terminal) and performed a detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and present a versatile and powerful system in which to functionally analyze variations of SRY including known and novel pathogenic variants found in DSD.

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SRY protein function in sex determination: thinking outside the box

Zhao

Koopman

2011

Chromosome Res

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Even though the mammalian sex-determining gene Sry has been intensively studied for the two decades since its discovery, the regions outside the conserved HMG box DNA-binding domain have received less attention due to a lack of sequence conservation and of obvious structural/functional motifs. Here, we summarize the available evidence for function beyond the HMG box, identify the known and postulated biochemical functions of the non-HMG-box domains in sex determination, and present possible explanations for the puzzling diversity of these non-HMG-box domains.

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Mammalian Testis-determining Factor SRY and the Enigma of Inherited Human Sex Reversal

Cited by 20 publications

References 104 publications

Structure-Function Relationships in Human Testis-determining Factor SRY

Structure-Function Relationships in Human Testis-determining Factor SRY

Generation and mutational analysis of a transgenic mouse model of humanSRY

SRY protein function in sex determination: thinking outside the box

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