In the last post, I covered how proteins PER and CRY heterodimerize to inhibit CLOCK and BMAL1 heterodimers from forming in the nucleus. This, currently, is the best understood transcriptional-translational feedback loop of mammalian molecular clocks. There are, however, other levels of regulation that I’ll cover here.
There is an “accessory loop” that aids in the aforementioned process, involving a protein called Rev-Erb alpha (α). One of the genes that the BMAL1-CLK/NPAS2 heterodimer upregulates is the NR1D1 gene – the gene that encodes for the Rev-Erbα protein. This follows the same mechanism that we saw with per and cry genes in mammals and per and tim genes in D. melanogaster. The BMAL1-CLK/NPAS2 heterodimer will be highly active as PER and CRY protein levels are low (from night, leading into midday). As their levels reach peak concentrations, and their rate of heterodimer formation is maximal, their translocation into the nucleus will be at its highest. They will then sequester away BMAL1, CLOCK, and NPAS2 proteins, preventing their ability to heterodimerize, thus preventing their ability to bind to the E-boxes of a very large amount of target genes. And so, Rev-Erbα oscillates with PER and CRY over a 24-hour period, aiding in this process.
Rev-Erbα is a transcription factor, much like CLK, NPAS2 CYC, and BMAL1. And, if you recall from my introductory post, a transcription factor is a protein that scans along DNA, search for an appropriate place to bind. It will bind to a specific sequence of a gene’s control region and modulate its expression. The previous transcription factors we’ve gone over bind to the E-box of their target genes. Rev-Erb α binds to Rev response elements (RevREs) with a specific sequence of DNA that’s rich in adenosines and thymines, followed by the sequence AGGTCA. Unlike the BMAL1-CLK/NPAS2 heterodimer in mammals or CLK/CYC heterodimer in D. melanogaster which enhanced the transcription of their target genes, Rev-Erb α tends to repress its target’s transcription. One such example that has a RevRE and is repressed by Rev-Erb α binding is Bmal1 gene.
When Rev-Erbα levels are high (around midday), it will repress transcription of the Bmal1 gene, thereby lowering BMAL1 protein levels. Also around this time, PER/CRY heterodimers are entering into the nucleus of the cell, sequestering BMAL1-CLK/NPAS2 heterodimers and preventing the genes that contain E-boxes from being activated. Rev-Erbα lends to the efficiency of this process. I started this post off describing Rev-Erbα as being an accessory player in transcriptional-translational feedback loop we previously discussed – you can see just that:
It might be a little tough with a two-dimensional image but, as with any cell signaling or physiology cartoons, try to envision it in three dimensions. All of these proteins are floating around in the nucleus – their interaction is largely determined by their concentration levels. It is possible then, that not all BMAL1 is “caught” by PER/CRY heterodimers. BMAL1 can be thought of as the limiting factor in heterodimer formation as either CLK or NPAS2 can pair with it.
By lowering BMAL1’s concentration by repressing its gene’s transcription, the chance of preventing heterodimer formation increases greatly. Rev-Erbα then, is another layer of regulation, acting as somewhat as a safeguard.
Most cellular mechanisms have multiple routes of regulation, varying in their degree of repression and activation. We’ve seen two different ways for Bmal1 transcription to be repressed, but what about its activation? In the above image, there’s a new gene and protein we’ve yet to discuss. The gene NR1F1 (also called RORα ) encodes for the protein retinoic acid-related orphan receptor α (RORα) that competes for binding with Rev-Erb α for Bmal1’s response element and activates its transcription. RORα is one of many retinoic acid related orphan receptors which, in addition to circadian rhythm, play a role in metabolism, inflammation, brain function, and more. In this context, RORα induces Bmal1 transcription We should also modify the name of our previous Rev response element (RevRE) to the more appropriate, ROR/Rev response element or RRE. And so, RORα is a necessary component of Bmal1 expression, regulated by our body’s master clock, the suprachiasmatic nucleus. This small part of the hypothalamus will be the topic of the next article in this ongoing series.
Cook, D. N., Kang, H. S., & Jetten, A. M. (2015). Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism. Nuclear receptor research, 2, 101185. https://doi.org/10.11131/2015/101185
Ramakrishnan SN, Lau P, Burke LJ, Muscat GE. Rev-erbbeta regulates the expression of genes involved in lipid absorption in skeletal muscle cells: evidence for cross-talk between orphan nuclear receptors and myokines. J Biol Chem. 2005;280(10):8651‐8659. doi:10.1074/jbc.M413949200
Zhang, Y., Luo, X. Y., Wu, D. H., & Xu, Y. (2015). ROR nuclear receptors: structures, related diseases, and drug discovery. Acta pharmacologica Sinica, 36(1), 71–87. https://doi.org/10.1038/aps.2014.120