Sry Length Regulates Its Cellular Stability and Hence The Robustness of Testis Determination

A seeming paradox is posed by metazoan gene-regulatory networks (GRNs) that are robust yet evolvable. Insight is obtained through studies of bistable genetic circuits mediating developmental decisions and in particular transcriptional thresholds operated by a key transcription factor (TF). A model is provided by human SRY, a Y-encoded TF that initiates testicular differentiation, in turn enabling male development.

Function domains in SRY (204 residues) cluster in its high mobility group (HMG) box (residues 56-141). The functions of N- and C-terminal non-box segments are not well characterized. In addition, the paucity of experimental systems connecting SRY structure to function (as tested in cell models and in vivo) has limited progress. Here, we have sought to measure the transcriptional threshold of SRY based on cell-based and transgenic studies of a genetic boundary between organization and dysgenesis. Our findings demonstrate a threshold length in the C-terminal domain of human SRY that determines the protein’s proteasome-enforced half-life in the cell. In a rodent pre-Sertoli cell line, the consequent reduction in the intracellular concentration of truncated SRY constructs is associated with a twofold attenuation of the male-specific downstream GRN. In vivo CRISPR-Cas9-edited XX mice expressing the 1-164 fragment of human SRY cannot redirect to male differentiation whereas that expressing 1-200 of SRY exhibit male GRN development.

This study provides insight into the robustness of human SRY and illustrates a powerful strategy to link biochemical properties in cell culture to developmental outcomes in vivo. Our study unmasks a checkpoint in a mammalian TF serving as a key node in a sex-specific GRN. An experimental “control knob” in cell-based studies is provided by proteasome inhibition. Our approach probes molecular determinants of cell fate and so promises to extend structure-function studies of SRY from its HMG box to flanking non-box domains.