David R. Weaver

Coordination of circadian timing in mammals

Time in the biological sense is measured by cycles that range from milliseconds to years. Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous and well-studied timing systems. At the core of this timing mechanism is an intricate molecular mechanism that ticks away in many different tissues throughout the body. However, these independent rhythms are tamed by a master clock in the brain, which coordinates tissue-specific rhythms according to light input it receives from the outside world.

Association of BRCA1 with Rad51 in Mitotic and Meiotic Cells

BRCA1 immunostaining reveals discrete, nuclear foci during S phase of the cell cycle. Human Rad51, a homolog of bacterial RecA, behaves similarly. The two proteins were found to colocalize in vivo and to coimmunoprecipitate. BRCA1 residues 758-1064 alone formed Rad51-containing complexes in vitro. Rad51 is also specifically associated with developing synaptonemal complexes in meiotic cells, and BRCA1 and Rad51 were both detected on asynapsed (axial) elements of human synaptonemal complexes. These findings suggest a functional interaction between BRCA1 and Rad51 in the meiotic and mitotic cell cycles, which, in turn, suggests a role for BRCA1 in the control of recombination and of genome integrity.

mCRY1 and mCRY2 Are Essential Components of the Negative Limb of the Circadian Clock Feedback Loop

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

Cloning and Characterization of a Mammalian Melatonin Receptor That Mediates Reproductive and Circadian Responses

The pineal hormone melatonin regulates seasonal reproductive function and modulates circadian rhythms in mammals. We now report the cloning and characterization of a high affinity receptor for melatonin from the sheep and human. The receptor cDNAs encode proteins that are members of a newly discovered group within the G protein-coupled receptor family. Expression of the sheep and human receptors in COS-7 cells results in high affinity 2-[125I]iodomelatonin binding and pharmacological characteristics similar to endogenous high affinity receptors. Functional studies of NIH 3T3 cells stably expressing the sheep receptor show that the mammalian melatonin receptor is coupled to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism. In situ hybridization studies of melatonin receptor mRNA in several mammals reveal hybridization signals in the hypophyseal pars tuberalis and hypothalamic suprachiasmatic nucleus. The cloned high affinity receptor likely mediates the reproductive and circadian actions of melatonin in mammals.

Three period Homologs in Mammals: Differential Light Responses in the Suprachiasmatic Circadian Clock and Oscillating Transcripts Outside of Brain

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

A Molecular Mechanism Regulating Rhythmic Output from the Suprachiasmatic Circadian Clock

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

Two period Homologs: Circadian Expression and Photic Regulation in the Suprachiasmatic Nuclei

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

Differential Functions of mPer1, mPer2, and mPer3 in the SCN Circadian Clock

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.

Molecular cloning of the rat A2 adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity (Kd = 38.6 nM) and saturable binding of the A2 adenosine receptor-selective ligand [3H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the A2 subtype (NECA greater than (R)-PIA greater than CPA greater than (S)-PIA). In situ hybridization demonstrated that rat A2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D2 dopamine receptor mRNA, and never co-expressed with striatal D1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A2 subtype of adenosine receptor in the forebrain. The co-expression of D2 dopamine and A2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

Molecular Dissection of Two Distinct Actions of Melatonin on the Suprachiasmatic Circadian Clock

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.