A novel regulator in the mammalian circadian clock has been discovered in mouse, rat, and human tissues by HMS neurobiologists. The molecule, dubbed CLOCK-interacting protein, circadian (CIPC), helps turn off a transcription factor that positively drives the clock, thereby contributing to the inhibitory component of the circadian rhythm. The findings are reported in the March issue of Nature Cell Biology.
Charles Weitz, the Robert Henry Pfeiffer professor of neurobiology at HMS, previously reported that the main circadian gene CLOCK joined with the protein called brain muscle arnt-like-1 (BMAL1) to drive the circadian clock. Few molecular contributors to the clock were known in 1998, when Weitz reported the CLOCK–BMAL1 coupling in Science. Back then, Weitz and his team did screens to identify CLOCK’s partner, and BMAL1 jumped out from about 20 other proteins that emerged from their screen. “It’s a sequence expressed in muscle and brain, but that’s all people knew about it,” said Weitz of CLOCK’s in vivo partner.
Since that finding nearly 10 years ago, new evidence has hinted that other molecular players work to inhibit CLOCK–BMAL1. In their present work, Weitz and his research team took a fresh look at the 20-odd proteins that coexisted on the lab’s screens that partnered BMAL1 with CLOCK. Aided by more recent findings that clock mechanisms have been identified in many different body tissues, the researchers had a clearer idea of what to look for. Their haystack shrank further when they factored in the suspicion that the protein of interest might bind to a fragment near CLOCK’s C-terminus, a region that can alter clock function.
Passing these proteins through an additional screen, the researchers identified CIPC (“SIP-see”) as a negative regulator of the heterodimer CLOCK–BMAL1. CIPC showed circadian activity in each examined body tissue: liver, heart, and kidney. In each of these, CIPC was under CLOCK control.
Based on these in vitro findings, the researchers studied whether CIPC and CLOCK colocalized. Tissue slices stained for CIPC and CLOCK revealed the proteins’ nuclear cohabitation in all of the examined tissue types. Using a chicken antibody—when no other antibodies worked—the researchers found that CIPC and CLOCK coprecipitated. “That was a huge step,” Weitz said. “Having CIPC and CLOCK in a complex together takes it much further toward being reasonable that CIPC is part of a clock mechanism.”
The team also evaluated CIPC’s circadian contributions by depleting it through RNA interference. Knocking down CIPC shortened the clock’s cycle by an hour, which is considered a significant effect, Weitz said.
CIPC appears to be the first circadian rhythm protein without an invertebrate homologue, since the researchers did not find an equivalent protein in C. elegans or Drosophila. The vertebrate-specific protein lends support to the theory that mammalian clocks are more complex, though the behavioral consequences of having a more complex clock are unclear.
