The DNA-binding domain of Lef-1 places the protein into the HMG family of DNA-binding proteins. The laboratory chose to study the closely related HMG family member SRY because of the role of SRY protein in determining male sex in humans. The principle discovery of the study of the HMG-domain of SRY bound to DNA was the presence of a novel mode of protein-induced distortion of DNA structure via protein sidechain intercalation. The insertion of a tetrad of amino acids unstacks a single basestep and drives the helical unwinding of the DNA. By unwinding the DNA, the DNA can be bent by approximately 75o, thereby bringing distant regions of DNA into contact with one another. Because of the close similarity in the SRY and Lef-1 HMG domains, it is clear that the same process occurs for the Lef-1/DNA interaction. Thus, SRY and Lef-1 act as architectural elements in the enhancer, creating the structural environment in which other proteins may interact with one another; for example, the ETS1 and ATF/CREB proteins bind at sites located 70 basepairs apart, yet interact with one another in the complex in the presence of Lef-1. This occurs as a consequence of Lef-1 bending the DNA.

To date, three structurally distinct protein families have been shown to utilize protein sidechain intercalation as a means of driving the distortion of DNA necessary to assemble a higher order complex. Each protein family utilizes a similar wedge of hydrophobic amino acids to unstack consecutive DNA basepairs and bend the DNA.

SRY

The DNA-binding domain of Lef-1 places the protein into the HMG family of DNA-binding proteins. The laboratory chose to study the closely related HMG family member SRY because of the role of SRY protein in determining male sex in humans. The principle discovery of the study of the HMG-domain of SRY bound to DNA was the presence of a novel mode of protein-induced distortion of DNA structure via protein sidechain intercalation. The insertion of a tetrad of amino acids unstacks a single basestep and drives the helical unwinding of the DNA. By unwinding the DNA, the DNA can be bent by approximately 75o, thereby bringing distant regions of DNA into contact with one another. Because of the close similarity in the SRY and Lef-1 HMG domains, it is clear that the same process occurs for the Lef-1/DNA interaction. Thus, SRY and Lef-1 act as architectural elements in the enhancer, creating the structural environment in which other proteins may interact with one another; for example, the ETS1 and ATF/CREB proteins bind at sites located 70 basepairs apart, yet interact with one another in the complex in the presence of Lef-1. This occurs as a consequence of Lef-1 bending the DNA.

To date, three structurally distinct protein families have been shown to utilize protein sidechain intercalation as a means of driving the distortion of DNA necessary to assemble a higher order complex. Each protein family utilizes a similar wedge of hydrophobic amino acids to unstack consecutive DNA basepairs and bend the DNA.

Determination of Sex in Mammals (TIBS 21:302 (1996))

Sex determination in mammals is a complex process in which critical developmental decisions are made early in embryonic development. The study of sex determination in mammals is hampered by the lethality of most mutations that arise during the developmental process. In the mouse, the determination of male sex occurs early, around day 10, and requires the active stimulation of the male pathway by switching on differentiation of testis. This developmental switch is the testis-determining factor SRY. Establishment of maleness is also dependent on inhibiting further development of female structures with the protein Müllerian Inhibitory Substance (MIS). The expression of SRY and MIS appears to be linked, although further studies are required to establish this firmly.

Failure to express functional SRY leads to the phenomenon known as 46X,Y sex reversal in which an individual with an X,Y (i.e. male) karyotype can appear to be phenotypically female, but, sterile. The molecular basis of SRY-based sex reversal was fully explained from the three-dimensional structure of the protein/DNA complex. Generally, the failure in SRY function that leads to sex reversal is principally a failure to bend DNA stably. Thus, the loss in the architectural function of the protein leads directly to the inability to form a higher order complex required for normal gene expression.

The explanation of SRY-related sex reversal has led to the hypothesis that SRY-like proteins, also called SOX proteins, may play a larger role in controlling development by promoting the assembly of enhancer complexes at the appropriate time in a developing embryo. It is hoped this hypothesis could be used to identify other proteins which might directly interact with SRY or SOX proteins and thereby play a role in the sex-determination pathway. Projects are currently being designed to search for such proteins.

Hypothetical Model for an Architectural Hierarchy in Development (TIBS 21:302 (1996))

The hierarchy of regulatory events in embryogenesis is suggested to be coupled to the assembly of higher-order nucleoproteins complexes whose assembly is promoted by SOX proteins (triangles). Early events, which may involve one or more additional DNA-binding proteins, separate kidney, gonad, adrenal and skeletal developmental pathways. Arrows merely represent different pathways, but do not imply exact timing or extent of development along that path. The monolith represents a point of committment in embryonic development after which certain group(s) of cells are committed to specific organ system developmental pathways. Failures in the actions of several proteins implicated in the mammalian sex determination pathway which lead to clinical disorders are highlighted. The events which disrupt the function of these proteins affect the indicated organ system and gonadal differentiation, thereby reinforcing the importance of sex determination as an early developmental decision.Hypothetical Model for an Architectural Hierarchy in Development (TIBS 21:302 (1996))