[1881] "Calling Cards" Is a Novel Next-Gen Sequencing Approach That Identifies SRY Targets

Gabriel A Bien-Willner, David Mayhew, Robi Mitra. Washington University School of Medicine, St. Louis, MO

Background: SRY is a tissue-specific transcription factor that is necessary and sufficient to drive male sexual determination. While its function as a male-determining factor has been well studied, no mechanism of action for SRY has been proven. It has been proposed that SRY drives male sexual determination by up-regulating SOX9, as well as inhibiting female-specific factors activated by WNT signaling. Interestingly, SRY is also expressed in the adult Sertoli cell, implying it has other functions besides sexual development. SRY has been postulated to be involved in the formation of gonadoblastomas and sex-cord stromal tumors. Further elucidation of the function of SRY is severely hampered by the fact that no functional antibodies specific to the SRY protein have been produced, precluding chromatin immunoprecipitation (ChIP) or western-blot analysis. Here we present a novel approach to identify protein-DNA interactions and use it to reveal novel SRY binding sites. This technique dubbed "Calling Cards" functions independent of antibodies. Furthermore, it does not rely on freezing DNA-protein interactions and thus can record such interactions over time in cell culture.
Design: The novel “Calling Cards” technique employs a transposase tethered to a transcription factor of interest to insert a marked transposon into the genome at the site of DNA-protein interaction. A SRY-transposase construct was created and transfected into mouse ES cells and primary human ovarian cultures enriched for granulosa cells (the female counterpart to the Sertoli cell), marking sites of interaction. The locations of genomic insertions were mapped using massively-parallel sequencing and clusters of SRY-directed insertions were identified.
Results: Using the “Calling Cards” technique, significant SRY peaks were found in the 5' and 3' UTR regions of several spermatogenesis-related genes including Rnf168, Hormad2, Sox11, and Bnc2. Over 5,000 unique insertions were identified throughout the genome per experiment.
Conclusions: The “Calling Cards” technique is a novel way of identifying protein-DNA interactions independent of antibodies. Using this method, we uncovered several previously unknown targets of SRY, suggesting a role in spermatogenesis in the adult testis. Finding genes in this pathway may serve as targets for therapy in mixed germ cell and/or sex-cord stromal tumors.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer

Monday, March 19, 2012 9:30 AM

Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 1, Monday Morning

 

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