DNA insulator elements are thought to affect gene regulation at least in part by influencing chromatin structure and nuclear organization. Insulator function is determined by sequence-specific DNA binding proteins that bind to them. The insulator protein BEAF-32 usually binds near transcription start sites, suggesting it might play a direct role in promoter function. One example is the scs’ insulator, which contains divergently transcribed promoters with a BEAF-32 binding site by each. Identifying protein partners of BEAF-32 through a detailed analysis of the scs’ insulator would provide insight into the role of BEAF-32 in the insulator and promoter activity of scs’. Half of scs’ retains insulator activity, and this is lost if the BEAF-32 binding site is mutated. Through a linker scanning analysis of this half of scs’, we found that mutation of a 20 bp sequence decreases the insulator activity of the BEAF-32 binding site. Additional mutations are being tested to determine if other sequences also play a role. These variations are also being tested for effects on promoter activity to determine whether or not insulator and promoter activity can be separated. For instance, promoter activity is impaired if the BEAF-32 binding site or 20 bp sequence is mutated. In addition, we are using DNA affinity chromatography to purify proteins that bind to the identified sequence with the goal of identifying proteins by mass spectrometry. Characterization of minimal scs’ sequences required for insulator and promoter activity, and identification of proteins that bind these sequences, will provide insight into BEAF-32 function.
Progress on this project to identify proteins that work together with BEAF-32 for proper insulator and promoter function will be presented..