From Sleeplessness to Stroke: Functional Characterization of a Pleiotropic Transcription Factor.

Researchers at the MRC recently discovered a mouse that had no trouble waking up in the morning. In fact, due to an aberrant internal body clock, the mouse would wake up earlier and earlier each day. This was found to be caused by a mutation in a gene named ATBF1. This gene has been previously found to play roles in breast cancer, gastric cancer, and stroke development. ATBF1 is known as a pleiotropic gene, a single gene that influences many independent traits, and we are proposing research to help understand what roles ATBF1 performs in the body. Instead of studying
potential effects of ATBF1 on a case-by-case basis, we propose using an innovative new technique called ChIP-Seq. In this way, we can analyze how ATBF1 regulates each gene in the human genome. Our research will help us understand the functions of this fascinating gene and may one day lead to treatments for medical conditions ranging from sleeplessness to stroke.

Recent analysis of circadian systems has expanded on the classical 'core clock protein' concept to accommodate a complex of interacting transcription factors which orchestrate the rhythmic transcription of hundreds of genes. One such factor has been identified recently in a forward-genetics screen at Harwell. The mutation, named short circuit (Sci), lies in the AT-motif-binding-factor 1 (ATBF1) gene sequence and shortens the period of a mouse's internal body clock by 1 hour. ATBF1 encodes a 404-kDa transcription factor (TF), exhibits rhythmic oscillating expression patterns, and interacts with 'core clock' proteins.
In addition to its circadian role, ATBF1 has been independently shown to be pro-differentiative in neural and muscle tissue and to function as a tumor-suppressor in breast and gastric cancers. The gene is also associated with a number of human cardiovascular conditions. ATBF1's pleiotropic effects make it a prime candidate for high-throughput functional genomic analyses. The aim of our research is to observe ATBF1 DNA binding on a genome-wide scale in order to identify the downstream targets, developmental processes and disease pathways that it regulates. As part of this analysis, we also aim to identify the mechanism of action for the Sci mutation.
To accomplish this, we will perform ChIP-Seq experiments at two circadian time points in both wild-type and Sci mutant mouse tissue. Analyzing the resulting global map of binding sites will allow us to identify downstream genes regulated by ATBF1. Comparing the results of the four ChIP-Seq analyses will provide valuable insight into the role of ATBF1 binding. Of particular interest are enhancers where binding varies between wild-type and mutant mice as these regions may hold the key to explaining the Sci phenotype. We will validate the regulation of biologically interesting enhancer regions via luciferase reporter assays.
To predict co-factors which interact and bind with ATBF1, we will analyze bound regions in silico. Using our newly developed software tool, MotiFSA, we will search the sequence around bound regions for overrepresented motifs which correspond to known TF recognition patterns. Comparing analyses across genotypes may reveal intriguing differences between ATBF1-cofactor interactions in wild-type and mutant mice, which can be validated through Co-IP experiments.
ATBF1 is one of the largest annotated transcription factors and has been implicated in processes ranging from tumor suppression to circadian regulation. Our research promises to lead to the functional characterization of this complex pleiotropic gene and shed light on its many functions in physiology and disease.