CURRENT PROJECTS, continued

I. The front door – Genetic studies of the circadian clock and its influence upon human behavior.

One major obstacle in studying the human circadian oscillator is the difficulty of measuring properties such as period length. So far, this task has been achieved in only a few heroic studies employing extensive subject observation under controlled conditions. We have developed a technique that allows the real-time recording of the period length of circadian gene expression in human fibroblasts derived from skin biopsies. The method employs a lentivirally-delivered circadian reporter shielded by enhancer-blocking activities to infect cells cultivated directly from these biopsies. Circadian rhythms in infected cells can then be analyzed by real-time bioluminescence after prior synchronization of the cell population by serum or dexamethasone (Brown et al., 2005a).

Figure 1. Circadian reporter lentiviruses use the promoter of the Bmal1 gene to drive circadian expression of a luciferase cassette that contains the Bmal1 3’untranslated region. They are shielded from insertion site effects by either the chick Bglobin FII insulator element (Recillas-Targa et al., 2002) or by another upstream promoter. In a typical experiment, 50,000 primary human fibroblasts are infected with reporter virus. Subsequently, circadian rhythms within the cell population are synchronized by brief dexamethasone treatment, and bioluminescence is recorded by means of a homemade photomultiplier device.

In a pilot study of nineteen individuals, biopsies from the same subject had periods that differed by only thirty minutes, but average periods among different subjects differed by up to four hours. The average value determined in this fashion for human period length closely matches what has been determined from the human behavior experiments. Similarly, in mice with defined genetic mutations affecting clock function, circadian period measured in this way correlated with the period of wheel-running behavior. Moreover, mutations at circadian loci that affected periodicity invariably had more extreme phenotypes upon fibroblast period than upon wheel-running period, so this method provided increased sensitivity to genetic differences in clock function. Thus, we think that our procedure could be an ideal tool for quantitative trait analysis of human circadian rhythms, and we are actively searching individuals suitable for such studies.

Figure 2. A) Biopsies were taken from the abdomen, buttocks, or foreskin of 19 individuals. Fibroblasts from these biopsies were cultivated as described in Figure 1. Individual subjects are designated by the letters A-S. B) Graph of the period lengths in constant conditions of wheel-running activity (dark grey) and of fibroblast circadian gene expression (light grey) of different mouse strains. These period lengths are expressed as differences from the 24-hour solar day. Strain genotypes, from left to right, are Per2brdm/brdm, Per1brdm/brdm, wild-type, Cry2-/-, Cry2-/-;Per1brdm/brdm, Cry2-/-;Per2brdm/brdm.

 

Since its initial publication, this technology has been improved to allow its application to blood and even to outer root sheath stem cells from single human hairs. We are applying it to study circadian differences between old and young individuals (in collaboration with Anne Eckert, Christian Cajochen, and Lucia Pagani, University of Basel), between depressed and normal individuals (in collaboration with Klaus Martigny, University of Copenhagen), and between individuals of early and late chronotype (in collaboration with Dieter Kunz and Achim Kramer, Humboldt University Berlin)

Figure 3. Phase contrast microscopy of hair outer root stem cells cultivated on a feeder layer of mitotically inactivated primary human fibroblasts. Left panel, 200x magnification, photo of a single focus after 5 days of cultivation of cells issued from a single hair. Right panel, 400x magnification, same focus after two weeks of cultivation.

 

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Brown, S. A., Fleury-Olela, F., Nagoshi, E., Hauser, C., Juge, C., Meier, C. A., Chicheportiche, R., Dayer, J. M., Albrecht, U., andSchibler, U. (2005a). The period length of fibroblast circadian gene expression varies widely among human individuals. PLoS Biol 3:e338. 

Recillas-Targa, F., Pikaart, M. J., Burgess-Beusse, B., Bell, A. C., Litt, M. D., West, A. G., Gaszner, M., and Felsenfeld, G. (2002). Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities. Proc Natl Acad Sci U S A 99:6883-6888. 

Wiznerowicz, M., and Trono, D. (2003). Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 77:8957-8961.