The period length of fibroblast circadian gene expression varies widely among human individuals

Abstract

Mammalian circadian behavior is governed by a central clock in the suprachiasmatic nucleus of the brain hypothalamus, and its intrinsic period length is believed to affect the phase of daily activities. Measurement of this period length, normally accomplished by prolonged subject observation, is difficult and costly in humans. Because a circadian clock similar to that of the suprachiasmatic nucleus is present in most cell types, we were able to engineer a lentiviral circadian reporter that permits characterization of circadian rhythms in single skin biopsies. Using it, we have determined the period lengths of 19 human individuals. The average value from all subjects, 24.5 h, closely matches average values for human circadian physiology obtained in studies in which circadian period was assessed in the absence of the confounding effects of light input and sleep-wake cycle feedback. Nevertheless, the distribution of period lengths measured from biopsies from different individuals was wider than those reported for circadian physiology. A similar trend was observed when comparing wheel-running behavior with fibroblast period length in mouse strains containing circadian gene disruptions. In mice, inter-individual differences in fibroblast period length correlated with the period of running-wheel activity; in humans, fibroblasts from different individuals showed widely variant circadian periods. Given its robustness, the presented procedure should permit quantitative trait mapping of human period length.

Figure 1. Circadian Bioluminescence Can Be Recorded in Fibroblasts Infected with a Lentiviral Luciferase Expression Vector

(A) Circadian reporter constructs used in these studies. Each contains the mouse Bmal1 promoter, the firefly luciferase coding region, and the Bmal1 3′UTR, flanked by the long terminal repeats (LTRs) of a lentiviral packaging vector. In (i), a dimerized chick β-globin FII element is inserted between each LTR and adjacent Bmal1 sequences. In (ii), a DNA segment composed of the EF1α promoter and a SV40 terminator is inserted between the upstream LTR and Bmal1 promoter, and the gfp coding region between the Bmal1 UTR and the downstream viral LTR. (B) 3T3 cells were infected with the lentiviral vectors shown above, and equivalent infection levels were verified by real-time PCR to detect integrated viruses. Four days after infection, cells were shocked with dexamethasone to synchronize circadian rhythms, and luciferase output was measured by real-time luminometry. (C) 3T3 cells were infected with different concentrations of the lentiviral reporter vector ii (see [A]), and circadian rhythms were measured as in (B). 10× represents unconcentrated filtered viral supernatant, 1× represents a 10× dilution of this, and 100× was a 10× concentration by ultracentrifugation. The number of viral infection units/plate were approximately 10,000 (1×), 30,000 (3×), 100,000 (10×), 300,000 (30×), and 1,000,000 (100×).

Figure 2. Circadian Bioluminescene Recordings from Primary Human Blood Cells, Fibroblasts, and Keratinocytes

(A) For two different individuals, 50,000 non-immortalized adult primary human skin fibroblasts were infected with lentiviral reporter vectors. Four days after infection, cells were shocked with dexamethasone, and circadian rhythms were measured by real-time luminometry. (B) For two different individuals, 50,000 human blood monocytes were treated as in (A). (C) A skin biopsy was taken from one individual, and 50,000 fibroblasts were treated as in (A) and (B). In parallel, hairs were plucked until a hair was withdrawn that contained hair root keratinocytes clinging to the proximal end. These were cultivated and amplified to 50,000 cells, then infected and measured identically to fibroblasts.

Figure 3. Circadian Bioluminescence Cycles in Fibroblasts from Different Human Individuals

Biopsies were obtained from buttocks, foreskin, or abdomen of 19 individuals (see Materials and Methods for details). Fibroblasts were isolated from each biopsy, infected with lentiviral circadian reporter vectors as in Figure 2, and analyzed by real-time luminometry. Individuals are designated with the letters A–S. (A) Representative BMAL1-luciferase oscillations measured from biopsies of four different individuals. Individuals N, L, A, and P are shown. (B) Summary of the period lengths of BMAL-luciferase oscillations from all 19 individuals. Each value shows the average plus or minus the standard deviation from two different trials of two different infections of fibroblasts from two to five biopsies per subject. The probability by Student's t-test that the most different individuals (A and S) have the same period length is ≤0.00001; the probability that the second most different (B and R) are equal is ≤0.004. (C) For four subjects from whom two to five biopsies were taken, the average plus or minus the standard deviation of period length from two infections and four measurements of each skin biopsy is shown. The probability that the individuals that differ the most (C and R) do not differ in period length is ≤0.000002.

Figure 4. Comparison of the Period of Wheel-Running Behavior and of Fibroblast Bioluminescence among Mouse Strains Containing Different Circadian Gene Disruptions

Mice of nine nearly isogenic genotypes were analyzed to obtain the period of running-wheel behavior (τrw) and the period of fibroblast luminescence (τf). For each genotype, a single running-wheel profile of the behavior of a single mouse kept first in normal light/dark conditions and then in constant darkness is shown. Periods of darkness are shaded on the graphs. Standard double-plotted actogram format is used, with consecutive rows representing consecutive days of activity, and x-axis showing time. Period lengths are presented below the graphs as average plus or minus the standard deviation for two mice, each measured twice. For each genotype, a single 100-h representative fibroblast recording is also shown. The x-axis shows time in hours; the y-axis shows arbitrary light units. Period lengths are presented above the graphs as averageplus or minus the standard deviation for two measurements of two biopsies of each mouse.

Figure 5. Genetic Differences in Circadian Period Length Measured from Fibroblasts Are Larger than Those Measured from Animal Behavior

Data from Figure 4 depicted in stacked bar graph format. In each rhythmic strain measured, the period of wheel-running activity is shown in light grey, expressed in the difference in hours from the 24-h solar day. On top of this is shown the change in period of fibroblasts from the same animals, also measured in the difference in hours from the solar day. Genotypes depicted, from left to right, are Per2^brdm/brdm, Per1^brdm/brdm, wild-type, Cry2^+/−, Cry2^−/−;Per1^brdm/brdm, Cry2^−/−, Per2^brdm/brdm;Cry2^−/−. Because Per2^brdm/brdm;Cry2^−/− mice had unstable periods that ranged widely from individual to individual, this genotype is shown twice at the extreme right, with representative mice with periods both less than and greater than 24 h separated into two groups. Arrhythmic fibroblasts are designated “arr.”