Scientists have known for several decades that mutations in the period gene which governs rhythmic transcription in mammals can wreak havoc on their circadian, or 24-hour, rhythms, disrupting the biological clocks present throughout the body that control multiple physiological and behavioral processes, from sleep-wake cycles to hormone release and metabolism. They’ve also long known why — in essence, because proteins produced by the period gene, known as PER, can bind another protein key to gene expression, CLK:BMAL1, effectively repressing it by removing it from an organism’s DNA.
Thanks to Texas A&M University biologists in the Center for Biological Clocks Research (CBCR), scientists now know the how behind the why, courtesy of recent research published January 25 in the Proceedings of the National Academy of Sciences that points to a new protein, the heat shock protein HSP68, which is also crucial to the tumultuous mix.
The groundbreaking study takes into account the past and present work of three Texas A&M biologists — Christine Merlin, Jerome Menet and Paul Hardin — and their respective pooled insights across three model organisms: monarch butterflies, mice and fruit flies.
“We used the monarch butterfly as a model system because it harbors a simplified version of the CLK:BMAL1-activated circadian clock present in mammals,” said Merlin, a 2017 Klingenstein-Simons Fellow in Neuroscience and 2020 Presidential Impact Fellow at Texas A&M. “We found that the protein domain encoded by a region homologous to mouse CLOCK exon 19 and the histone methyltransferase TRITHORAX (TRX) are both necessary for monarch CLK:BMAL1-mediated transcriptional activation, CLK-PER interaction and PER repression.
“Surprisingly, we found that TRX catalytic activity is essential for CLK-PER interaction and PER repression via the methylation of a single arginine methylation site (R45) on a newly identified player, the heat shock protein HSP68. Since PER and CLK are a highly disordered proteins, it is possible that HSP, which traditionally functions in protein folding, plays a critical role in PER and/or CLK folding to permit progression through the circadian cycle.”
The team used a variety of molecular and biochemical assays to draw their conclusions, which Merlin describes as a “multi-year labor of love from Texas A&M postdoctoral research associate and first author Ying Zhang” that builds upon the previous work of her CBCR colleagues Hardin and Menet.
“My two primary collaborators happen to be the ones who contributed in large part to our knowledge regarding PER function in repression in fruit flies as members of Michael Rosbash’s lab at Brandeis,” Merlin said. “Paul Hardin showed that PER was a circadian repressor and discovered the concept of the feedback loop which later led to the 2017 Nobel Prize, and Jerome Menet showed that PER was responsible for removing CLK:BMAL1 off-DNA. What none of us anticipated was that the Monarch butterfly would be the one unveiling new secrets behind PER mode of action in the clockwork.”
As for the team’s next steps, Merlin says they plan to analyze the temporal binding dynamics of repressive complexes onto CLK:BMAL1 and also investigate whether HSP68 plays a role in the regulation of CLK and PER conformational states.
“Proper circadian rhythms sustain health,” Merlin said. “However, living in our modern society means being exposed to a variety of factors, such as artificial light, shift work and at-will food consumption, that disrupt our rhythms and in turn increase disease risk. This underscores the need to understand the molecular mechanisms generating rhythms, as clock components could be, for example, targeted by drugs for treatment.”
The team’s PNAS paper, “TRITHORAX-dependent arginine methylation of HSP68 mediates circadian repression by PERIOD in the monarch butterfly,” can be viewed online along with related figures and captions.
Their research was funded in part by the National Institutes of Health (Grant Number GM124617) and Merlin’s 2017 Klingenstein-Simons Fellowship in Neuroscience.
For additional information Merlin and her research, go to http://www.merlinlab.org.
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