Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences.

The regulation of gene expression drives many biological processes and alterations in normal regulation are integral in the development of the diseased state. Therefore, the ability to screen genomic DNA for direct targets of DNA binding proteins (DNA-BP) would provide valuable information about the mechanisms underlying these processes. At present chromatin immunoprecipitation (ChIP) and its variants are the primary methods for identifying regulatory elements. However, some DNA-BPs, such as the winged-helix transcription factor FOXO1, are difficult to ChIP thereby detracting from the use of this technique as a nonbiased screen to isolate regulatory sequences. In this report we use an improved in vitro method to Pull Out Regulatory Elements (PORE), which uses purified protein with a stable genomic library to isolate regulatory elements directly bound by a DNA-BP, to identify putative FOXO1 genomic regulatory sequences. We first validate this technique using two known DNA-BP (FOXO1 and Pax3) by demonstrating their ability to bind and amplify identified promoter elements when present in a genomic DNA context or when present in the context of our stable genomic library. Subsequent use of this technique with FOXO1 isolated regulatory elements associated with several genes known to be regulated in a FOXO1-dependent manner and multiple genes whose biological functions are consistent with the known biological functions of FOXO1 proving that the in vitro PORE is a valuable and easy to use alternative to ChIP for the isolation of genomic regulatory elements.