Determination of Specific RAI1 Binding Sites

Smith‐Magenis Syndrome (SMS) is a neurobehavioral disorder characterized by mental retardation, sleep disturbances, obesity and self injurious/attention seeking behavior. The major symptoms of SMS are caused by haploinsufficiency of the retinoic acid‐inducible 1 (RAI1) gene. This gene is thought to be a transcription factor. Another member of our group performed a Chromatin Immunoprecipitation‐microarray chip (ChIP‐chip) experiment to identify genes regulated by RAI1. One gene identified was Circadian Locomotor Output Cycles Kaput ( CLOCK ). The region of CLOCK located on the chip was PCR amplified and cloned into the pGL3 luciferase reporter vector and preliminary experiments showed that CLOCK was regulated by RAI1. The purpose of this project is to confirm RAI1 as a regulator of CLOCK and identify specific RAI1 binding sites within the cloned region. To confirm that RAI1 regulates CLOCK, additional luciferase assays were conducted. A statistically significant increase in luciferase activity was seen when RAI1 was cotransfected with the pGL3‐ CLOCK construct. To identify specific RAI1 binding sites, we are currently creating deletion constructs for future luciferase assays. Identification of a specific RAI1 binding site would facilitate the identification of other genes regulated by RAI1. Knowing this cohort of genes may provide a better understanding of the specific function of RAI1 and the cause of SMS.