Identification of Novel Transcriptional Regulators in the Nervous System

Increasingly, it is becoming recognized that transcriptional modulation of genes by sequence variations, DNA methylation, and alterations in transcription factor expression can play a major role in the behavioral phenotype throughout its development. Hence, the understanding of the transcriptional mechanisms that regulate key candidate genes for mental illness has taken on new significance. In a step-by-step approach we describe the crucial experiments to identify and characterize mechanisms of transcription regulation of the 5-HT1A receptor gene, which has been implicated in major depression, anxiety disorders, and suicide. These steps include: identification of transcription start sites by RT-PCR, primer extension, 5′-RACE, and RNase protection assay; characterization of upstream promoter, enhancer, and repressor regions using 5′-deletion luciferase reporter constructs and transfection assays in cell lines and primary cultures; identification of protein-DNA interactions using EMSA, super-shift and CHIP assays; cloning of novel transcription factors using yeast one-hybrid screening and functional characterization using Gal4-DBD system and by siRNA approaches. Using these approaches in combination with bioinformatics searches it is possible to identify and characterize the basic transcriptional regulatory mechanisms of virtually any candidate gene of interest and to identify specific proximal DNA elements and transcriptional factors that mediate its regulation.