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Kinetic Control of a Developmental Switch: Design of a “Kinetic Rheostat” in a Bent Protein‐DNA Complex with Application to Human Sex Reversal
Author(s) -
Racca J.D.,
Chen YS.,
Bhatnagar V.,
Phillips N.B.,
Weiss M.A.
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.880.21
Subject(s) - testis determining factor , biology , enhancer , hmg box , dna , genetics , sox9 , gene , microbiology and biotechnology , y chromosome , dna binding protein , gene expression , transcription factor
Protein‐directed DNA bending is of fundamental importance in gene regulation. Our studies focus on the role of sequence‐specific DNA bending by the high‐mobility‐group (HMG) box in the embryonic specification of human male development by Y‐encoded testis determining factor SRY. This architectural factor binds to an enhancer element in autosomal target gene Sox9 to activate its lineage‐specific expression in the gonadal ridge. We seek to test the hypothesis that SRY's transcriptional activity is regulated by the lifetime of its bent protein‐DNA complex. Our approach employs design of a novel “kinetic rheostat” in the HMG box such that the lifetime of its specific DNA complex may be tuned independently of protein‐DNA affinity and DNA bend angle. This rheostat exploits the “mini‐core” of the L‐shaped box. Function was evaluated in a rodent embryonic gonadal‐ridge cell line. SRY‐directed expression of Sox9 was amplified (or blunted) by substitutions that prolonged (or foreshortened) the lifetime of the specific box‐DNA complex as characterized by a FRET‐based stopped‐flow assay, even in complexes exhibiting similar affinities. Lifetimes correlated with occupancies of the Sox9 enhancer as probed by ChIP. These findings rationalize the phenotypes of transgenic XX mice (male or female) expressing variant human or murine Sry constructs (P. Koopman, R. Lovell‐Badge et al .). Further, a survey of clinical SRY mutations causing somatic sex reversal (Swyer's Syndrome) suggests that kinetic perturbations may explain a subset of such disorders. We envisage that the evolution of SRY has been constrained by a functional kinetic threshold in the assembly of bent transcriptional pre‐initiation complexes.