Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
A Kinetic Code for SRY-Dependent Transcriptional Activation Based on DNA Intercalation
The HMG (High-Mobility-Group) box defines a superfamily of DNA-bending proteins remarkable for partial DNA intercalation by “cantilever” side chains. Here, we describe the role of the cantilever residue of SRY, the human male-determining factor encoded by the Y chromosome. Mutations at this and related sites abutting the sharp DNA bend cause gonadal dysgenesis leading to chromosome sex reversal. A yeast-one-hybrid model was designed to enable rapid and efficient screening of mutational libraries in relation to the database of clinical substitutions and evolutionary relationships among HMG boxes. Our results demonstrate (a) that the only allowed cantilevers are those observed among mammalian SRY and related SOX sequences (Ile, Met, Leu, and Phe); (b) that mutations associated with de novo sex reversal in vivo markedly impair DNA binding; and (c) that variant cantilevers are associated with a range of kinetic on- and off-rates. To correlate the biophysical properties of the bent protein-DNA complex with gene-regulatory activity, a transfection assay was developed in an immortalized rodent embryogenic cell line derived from the differentiating gonadal ridge at the time of onset of SRY expression. A biological read-out was provided by real-time quantitative PCR analysis of the endogenous Sox9 gene, a key downstream target of SRY in the male transcriptional program. Remarkably, transcriptional potency was observed to correlate with the kinetic life time of the bent DNA complex and not its thermodynamic stability. Whereas a variant Val cantilever (too short to fully insert within the DNA double helix) suffices in vitro to direct formation of a well-organized but short-lived SRY-DNA complex of near-native DNA bend angle, no gene activation was observed in the rodent cell culture model. We propose that the active cantilever side chains act as a kinetic anchor to provide a long-lived DNA bend, in turn enabling formation of a functional male-specific transcriptional pre-initiation complex.
Department of Biochemistry