Supplementary figures
Figure 1. Synchronization of clock gene expression in NIH3T3 cells by haem. Synchronization of mPer2 and Bmal1 expression by haem was transient, lasting less than 52 h with decreasing signal amplitude. Note the inverse temporal profile of mPer2 and Bmal1 mRNA expression. Northern blot analysis was carried out by simultaneously probing with mPer2 and Bmal1 cDNAs.
Figure 2. a). Haem inhibits wheel-running activity during subjective night. Representative double-plotted wheel running activity from mice injected at CT6 with solvent or haem. Note the transient wheel-running activity after injection. b) Single-plotted wheel-running activity from mice injected at CT22. Note the decreased in activity on the following DD cycle and the loss of wheel-running activity in all mice that received haem injection. The timing of solvent or haem injections is indicated by the red arrow. Arrows on the right side indicate the days when animals were injected and the shaded area indicates the period in darkness.
Figure 3. NPAS2 and mPER2 mediate haem effects on mPer1 and mPer2 expression. a) Expression of mPer2 in mPer1m/m, Npas2m/m and wild type mice over a 24 h period. b) Expression of mPer1 in mPer2 m/m, Npas2m/m and wild type mice over a 24 h period. The levels of mPer1 and mPer2 mRNA were normalized to the Gapdh mRNA level as an internal control. Error bars indicate SD (n=2). Note that overall mPer2 expression was dampened in Npas2m/m mice. Haem repression of mPer2 expression was moderately diminished in mPer1m/m mice, suggesting that mPER1 is a lesser regulator of this response.
Figure 4. mPER1 is a minor regulator of NPAS2. a) Northern analysis of RNA isolated from livers of wild type and mPer1 mutant mice injected with solvent or haem. The blot was probed with 32P labeled Gapdh and Npas2 cDNAs. b) Reporter assay studies with the mPer1 promoter. COS-1 cells were transfected with the reporter construct mPer1-Luc and various combinations of expression constructs as described in Methods. Luciferase activity in samples transfected with the reporter plasmid alone was arbitrarily set at 1.0. Error bars indicate SEM (n=6).
Figure 5. A model of reciprocal regulation between the circadian clock and haem biosynthesis. Mammalian Period protein 2 (mPER2), Neuronal PAS domain protein 2 (NPAS2), Brain Muscle ARNT Like protein 1 (BMAL1), Haem-Oxygenase (HO), Aminolevulinate Synthase 1 (ALAS1), Carbon Monoxide (CO), and Haem (4 red squares with Fe).
Figure 6. c-Myc is regulated by NPAS2 and is modulated by haem. a) Total RNA was isolated from livers of wild type and Npas2m/m mice injected with solvent or haem as described in Methods. Each RNA sample was analyzed sequentially by Northern blot hybridization with 32P labeled c-Myc and Gapdh cDNAs. The level of c-Myc mRNA was normalized to the internal Gapdh mRNA level and the lowest relative value is arbitrarily set at 1.0. Error bars indicate SD (n=2). Expression of c-Myc mRNA in untreated animals was similar to those injected with solvent21.