The proposed research will characterize two genomic regions of the mouse: one on Chromosome 1 and the other on 12. The region on 1 is not only important to many behaviors, but contains regulatory elements for hundreds of genes on distant chromosomes as well. The region on 12 has genes known to interact with components of the biochemical pathway called the circadian clock. This program has focused on differences in daily (circadian) timekeeping in strains of mice. Some strains wake up earlier each day while the others wake up later each day. The first is a model for humans who are "early birds" and the other a model for "night owls". The goal is identifying genes that underlie this difference. With former funding, intercrossed mouse strains produced new strains whose genomes carried elements influencing some heritable parts of daily timekeeping. The new elements came from another strain of "early birds." Importantly, the new elements imparted some of the characteristic to the new strains. The goal is pinpointing genes within the elements (small genomic segments) that impact timekeeping. The way forward is to associate changing behaviors with changing gene function. Measuring function requires three technically challenging assays: the quantitative real-time polymerase chain reaction (qRT-PCR);the microarray system;and a specialized cell culture system called gene dosage network analysis (GDNA). Hypothesis: Aspects of circadian timekeeping and sleep come from certain genes on mouse chromosomes 1 and 12. A comparison throughout a day of the use of a gene from one strain with its use in a strain whose behavior varies, tests the hypothesis. For chromosome 1 the comparison requires a microarray assay. This powerful assay simultaneously compares all functioning genes in one experiment. The material assayed is extracts of suprachismatic nucleus (SCN) of the anterior hypothanamus dissected from brain slices and harvested around the clock. A gene's use should reflect the behavior variations: activity or rest. If as a gene's usage spikes behavior also changes, then some of the activated genes must be behavior modulators. A comparison of an immediate gene-product from chromosomes 12 between the special lines of mice with qRT-PCR tests this hypothesis also. Again it is important to compare gene-product levels around the clock. If the function of the target gene (Zfp277) changes with the behavior, it is a clue that Zfp277 influences behavior. GDNA is for the most important candidate genes. The assay comprises special cultured cells engineered to light up as their circadian clocks are active. Furthermore, when the culture is challenged with a dose of steroid, then in synchrony once a day for six days, the entire culture lights up. Add a small interfering RNA (siRNA) corresponding to the complimentary strand of the candidate-gene's transcript to the culture's media, and the cells'ability to make the coded protein slows and stops. Should the missing protein interact with the cells' clockwork, not only flashing frequency but also levels of key clock genes (qRT-PCR assay) change. A candidate gene's part in a well-regulated sleep and arousal system follows from a positive assay. Fulfilling the proposal's goal also means continuing to breed new strains. Introducing and fixing the outcome of recombination generates not only informative but analysis-friendly genomic substrates. Since new strains developed go out to other laboratories, they serve the research objectives of others interested in this important segment of chromosome 1 as well as ours. This work capitalizes not only on the unique and irreplaceable mouse lines developed at the Indianapolis VA Medical Center but also the rich resources available in the Laboratory of Dr. Hogenesch at University of Pennsylvania and in the Medical Genomics and Biotechnology Core Facilities at Indiana University.
Impact on Veterans The research proposed in Quantitative Trait Genes for Circadian Phenotypes addresses genes that regulate sleep and waking. Sleep problems contribute to poor quality of life for many veterans. Sleep problems are especially notable in schizophrenia, bipolar disorder, depression and Post-traumatic Stress Disorder (PTSD). Our goal is to identify genes that regulate sleep/wake patterns. We have found two groups of mouse genes that make some mice early to rise and others night-owls. We propose studies to identify which genes are involved. We think these might allow some people to easily adapt to certain shifts, rotating shift work, or jet lag. Our work might eventually help develop drugs to help people adjust. One of the regions we are studying controls hundreds of other genes, affecting many other functions in the body. Our research may show which gene is the master gene controlling all the others.