Making sense of vertebrate sirtuin genes
How many sirtuin genes are out there? evolution of sirtuin genes in vertebrates with a description of a new family member
Recommendation: posted 22 September 2022, validated 29 September 2022
Delsuc, F. (2022) Making sense of vertebrate sirtuin genes. Peer Community in Evolutionary Biology, 100151. https://doi.org/10.24072/pci.evolbiol.100151
Sirtuin proteins are class III histone deacetylases that are involved in a variety of fundamental biological functions mostly related to aging. These proteins are located in different subcellular compartments and are associated with different biological functions such as metabolic regulation, stress response, and cell cycle control . In mammals, the sirtuin gene family is composed of seven paralogs (SIRT1-7) grouped into four classes . Due to their involvement in maintaining cell cycle integrity, sirtuins have been studied as a way to understand fundamental mechanisms governing longevity . Indeed, the downregulation of sirtuin genes with aging seems to explain much of the pathophysiology that accumulates with aging . Biomedical studies have thus explored the potential therapeutic implications of sirtuins  but whether they can effectively be used as molecular targets for the treatment of human diseases remains to be demonstrated . Despite this biomedical interest and some phylogenetic analyses of sirtuin paralogs mostly conducted in mammals, a comprehensive evolutionary analysis of the sirtuin gene family at the scale of vertebrates was still lacking.
In this preprint, Opazo and collaborators  took advantage of the increasing availability of whole-genome sequences for species representing all main groups of vertebrates to unravel the evolution of the sirtuin gene family. To do so, they undertook a phylogenomic approach in its original sense aimed at improving functional predictions by evolutionary analysis  in order to inventory the full vertebrate sirtuin gene repertoire and reconstruct its precise duplication history. Harvesting genomic databases, they extracted all predicted sirtuin proteins and performed phylogenetic analyses based on probabilistic inference methods. Maximum likelihood and Bayesian analyses resulted in well-resolved and congruent phylogenetic trees dividing vertebrate sirtuin genes into three major clades. These analyses also revealed an additional eighth paralog that was previously overlooked because of its restricted phyletic distribution. This newly identified sirtuin family member (named SIRT8) was recovered with unambiguous statistical support as a sister-group to the SIRT3 clade. Comparative genomic analyses based on conserved gene synteny confirmed that SIRT8 was present in all sampled non-amniote vertebrate genomes (cartilaginous fish, bony fish, coelacanth, lungfish, and amphibians) except cyclostomes. SIRT8 has thus most likely been lost in the last common ancestor of amniotes (mammals, reptiles, and birds). Discovery of such previously unknown genes in vertebrates is not completely surprising given the plethora of high-quality genomes now available. However, this study highlights the importance of considering a broad taxonomic sampling to infer evolutionary patterns of gene families that have been mostly studied in mammals because of their potential importance for human biology.
Based on its phylogenetic position as closely related to SIRT3 within class I, it could be predicted that the newly identified SIRT8 paralog likely has a deacetylase activity and is probably located in mitochondria. To test these evolutionary predictions, Opazo and collaborators  conducted further bioinformatics analyses and functional experiments using the elephant shark (Callorhinchus milii) as a model species. RNAseq expression data were analyzed to determine tissue-specific transcription of sirtuin genes in vertebrates, including SIRT8 found to be mainly expressed in the ovary, which suggests a potential role in biological processes associated with reproduction. The elephant shark SIRT8 protein sequence was used with other vertebrates for comparative analyses of protein structure modeling and subcellular localization prediction both pointing to a probable mitochondrial localization. The protein localization and its function were further characterized by immunolocalization in transfected cells, and enzymatic and functional assays, which all confirmed the prediction that SIRT8 proteins are targeted to the mitochondria and have deacetylase activity. The extensive experimental efforts made in this study to shed light on the function of this newly discovered gene are both rare and highly commendable.
Overall, this work by Opazo and collaborators  provides a comprehensive phylogenomic study of the sirtuin gene family in vertebrates based on detailed evolutionary analyses using state-of-the-art phylogenetic reconstruction methods. It also illustrates the power of adopting an integrative comparative approach supplementing the reconstruction of the duplication history of the gene family with complementary functional experiments in order to elucidate the function of the newly discovered SIRT8 family member. These results provide a reference phylogenetic framework for the evolution of sirtuin genes and the further functional characterization of the eight vertebrate paralogs with potential relevance for understanding the cellular biology of aging and its associated diseases in human.
 Vassilopoulos A, Fritz KS, Petersen DR, Gius D (2011) The human sirtuin family: Evolutionary divergences and functions. Human Genomics, 5, 485. https://doi.org/10.1186/1479-7364-5-5-485
 Yamamoto H, Schoonjans K, Auwerx J (2007) Sirtuin Functions in Health and Disease. Molecular Endocrinology, 21, 1745–1755. https://doi.org/10.1210/me.2007-0079
 Morris BJ (2013) Seven sirtuins for seven deadly diseases ofaging. Free Radical Biology and Medicine, 56, 133–171. https://doi.org/10.1016/j.freeradbiomed.2012.10.525
 Bordo D Structure and Evolution of Human Sirtuins. Current Drug Targets, 14, 662–665. http://dx.doi.org/10.2174/1389450111314060007
 Opazo JC, Vandewege MW, Hoffmann FG, Zavala K, Meléndez C, Luchsinger C, Cavieres VA, Vargas-Chacoff L, Morera FJ, Burgos PV, Tapia-Rojas C, Mardones GA (2022) How many sirtuin genes are out there? evolution of sirtuin genes in vertebrates with a description of a new family member. bioRxiv, 2020.07.17.209510, ver. 5 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2020.07.17.209510
 Eisen JA (1998) Phylogenomics: Improving Functional Predictions for Uncharacterized Genes by Evolutionary Analysis. Genome Research, 8, 163–167. https://doi.org/10.1101/gr.8.3.163
The recommender in charge of the evaluation of the article and the reviewers declared that they have no conflict of interest (as defined in the code of conduct of PCI) with the authors or with the content of the article. The authors declared that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article.
Reviewed by Nicolas Leurs, 24 Aug 2022
Reviewed by anonymous reviewer 1, 20 Aug 2022
Reviewed by Filipe Castro, 19 Aug 2022
Evaluation round #1
DOI or URL of the preprint: https://doi.org/10.1101/2020.07.17.209510
Version of the preprint: 3
Author's Reply, 12 Aug 2022
Decision by Frédéric Delsuc, posted 07 Jul 2022
Dear Drs. Juan Opazo and Gonzalo Mardones,
I now have received three reviews from colleagues that have carefuly read your preprint entitled "How many sirtuin genes are out there? Evolution of sirtuin genes in vertebrates with a description of a new family member" submitted for evaluation by Peer Community in Evolutionary Biology.
As you will see in their detailed review reports, all three reviewers found the manuscript interesting and clearly written. I agree with them that your study provides a comprehensive study of this gene family based on detailed evolutionary analyses and I share their appreciation of the efforts you deployed to shed light on the potential function of the newly discovered sirtuin SIRT3-like paralog through functional experiments. All three reviewers also provide valuable suggestions that I think will improve the clarity of the study.
The following points are particularly worth addressing in your revision:
1. More details are needed regarding the construction of the alignment. In particular, the length of the alignment and whether or not it has been processed with a site filtering method before phylogenetic analysis should be indicated. If site filtering has not been performed, as suggested by one of the reviewer, the effect of applying a method such as HMMCleaner on phylogenetic inference might be worth exploring.
2. Two reviewers required additional explanation/clarification regarding the phylogenetic position of the lamprey sequence to the SIRT3 clade and the potential reasons why this sequence is particularly difficult to place in the tree. I agree with them that the potential effect of the compositional bias of the lamprey sequence should be further explored and discussed.
3. Finally, two reviewers had questions concerning the name of the newly identified SIRT3-like paralog and suggest renaming the gene independently of its similarity to SIRT3 by perhaps simply calling it SIRT8. It would be good to hear your informed opinion on this nomenclatural issue.
I am looking forward to read the revised version of the manuscript.
Frédéric Delsuc on the behalf of PCI Evol Biol