Molecular Cell

Previews Greetings from the Planet Croton Allyson Evans1,* 1Molecular Cell, Cell Press, 600 Technology Square, 5th Floor, Cambridge, MA 02139, USA *Correspondence: [email protected]

In this issue of Molecular Cell, Sabari et al. (2015) discover that levels of intracellular crotonyl-CoA impact the histone acylation landscape, providing deeper insight into the exotic histone modification, crotonylation, and exploring new avenues by which cellular metabolism can influence gene expression. When I first heard of crotonylation, I was huddled in a frigid hotel conference room on a steamy Caribbean island. My ears began to thaw as the first author told this story. I was struck by how the work not only characterized this relatively uncharted histone modification, but explored new territories by which the byproducts of metabolic reactions could directly influence gene expression. Histone modifications come in all shapes and sizes, and our desks at Molecular Cell are no strangers to papers that examine everything from histone methylation and acetylation to propionylation, ADP ribosylation, and sumoylation. Histone marks occur on a variety of amino acids, either within the histone globular domain or on the tails, and can influence the packaging, condensation, or protein binding to chromatin to exquisitely regulate processes from gene expression to genomic stability (Kouzarides, 2007). But what is crotonylation? Croton sounded like an alien planet where one might find Superman’s alter ego. A quick Google search from the back of the conference room told me that Croton wasn’t a planet at all—it was a plant. It is derived from the Greek ‘‘Kroton’’ and describes a genus of a flowering species. The plant has been long touted for its healing properties in Chinese medicine, and if you are a cocktail drinker, you may appreciate its contributions in flavoring Campari. The oil from these plants can also produce 12-O-tetradecanoylphorbol-13-acetate (TPA)—yes, that same nasty phorbol ester used to induce squamous cell carcinoma in our friendly laboratory mouse. It was also thought that C25H40N7O17P3S could be derived from croton oil, so the name crotonyl-CoA stuck. But how does this particular acyl-CoA differ from others, like acetyl-CoA and

butyryl-CoA? How is crotonyl-CoA produced in the cell? Is it just a metabolic byproduct, or is it important in regulating critical biological processes? A 2011 article identifying new histone marks from Yingming Zhao’s group began to answer some of these questions when they first reported lysine crotonylation. They found crotonylation on histones H2A, H2B, H3, H4, and linker histone H1. Their work showed that these marks were conserved from yeast to humans and suggested that it is associated with enhancers and active transcription start sites (Tan et al., 2011). In this issue, Sabari et al. (2015) pick up where Zhao’s group left off, leading the field to a deeper understanding of how the enzymes and intermediary metabolites that regulate acylCoAs may impact chromatin and gene expression. Sabari et al. begin by searching for enzymes capable of crotonylating histones. When they purify crotonyltransferase activity from nuclear extracts, they see that both histone crotonylase activity and histone acetylase activity co-elute. This leads them to p300, which they find is not only capable of acetylating histones, but can also crotonylate them. The authors go on to show that in transcription assays, p300-mediated crotonylation can enhance transcription, and even see that crotonyl-CoA stimulates transcription more potently than acetyl-CoA. In cells, they show that crotonylated H3K18 correlates with levels of transcription and is induced at genes where p300 is recruited in response to LPS stimulation. Despite this, the blunt methods of mutating lysines to neutralize charge or mimic acetylation currently in our toolbox aren’t able to distinguish among what might be subtle differences between acyl modifications. More studies will also be needed to un-

derstand the mechanisms by which crotonylation can affect transcription. It is possible that readers of acetylated chromatin might differentially detect the more rigid and bulky crotonyl group, or not detect them at all. Another possibility is that crotonylated lysines could be less readily de-acylated, thereby affecting the stability of the mark and influencing gene expression. Although Sabari et al. find that a histone acetyltransferase can add both acetyl and crotonyl groups, future studies will be needed to determine if histone de-acetylases also have as broad a range of substrates. Given Sabari et al.’s findings that point to crosstalk between the metabolic status of the cell and crotonylation, it is intriguing to speculate a role for the nutrient-sensing, NAD+-dependent sirtuin class of deacetylases (Guarente, 2011) in this process. Enzymatic regulation by p300 isn’t the only means by which crotonylation can be regulated. Sabari et al.’s work hints that pathways that influence the concentration of crotonyl-CoA can also modify histone crotonylation. Due to its ability to charge acetate with free CoA-SH as well as form longer-chains SCFAs, acyl-CoA synthetase (ACSS2) is a good candidate for converting crotonate to crotonylCoA. Consistent with this, when Sabari et al. knock down ACSS2, they see a reduction in crotonyl-CoA levels and a decrease in global crotonylated H3K18, suggesting that ACSS2 may be important in converting crotonate to crotonyl-CoA in cells. The implications for this are especially intriguing given recent evidence that mice deficient for ACSS2 have reduced tumor burden (Comerford et al., 2014). While ACSS2’s role in converting acetate to acetyl-CoA is likely important in a cancer context where more typical citrate-derived sources of acetyl-CoA

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Molecular Cell

Previews are compromised by the Warburg effect, it will now also be interesting to examine whether crotonyl-CoA and histone crotonylation levels are also impacted in ACSS2 deficient mice and whether this plays a role in the tumor progression. Although Sabari et al. (2015) show that enzymes involved in crotonate to crotonyl-CoA conversion can influence histone crotonylation, they also find that factors other than the levels of crotonyl-CoA can influence crotonylation. When they measure crotonyl-CoA levels in the cell, they find it is much less abundant than acetyl-CoA. However, when they simply reduce the concentration of acetyl-CoA by knocking down ATP citrate lyase (ACL) (which can convert citrate to acetyl-CoA in the cytoplasm and nucleus), this not only decreased acetylation of HK18 as expected (Wellen et al., 2009), but actually increased global H3K18 crotonylation. In addition, when they knock down PDH, which was recently shown to synthesize acetyl-CoA in the nucleus for histone acetylation (Sutendra et al., 2014), this also caused an increase in H3K18 crotonylation. These results suggest that crotonyl-CoA can compete with acetyl-CoA in p300-catalyzed reactions. This adds another dimension to previous work that proposed that metabolic pathways influencing acetyl-CoA concentrations could impact histone modification and gene expression (Sutendra et al., 2014; Cai et al., 2011). In nutrientreplete conditions, increasing acetyl-

CoA levels led to histone acetylation, which was important for growth (Cai et al., 2011). But what about nutrientdepleted conditions in which acetyl-CoA levels should dramatically decrease? Although not examined, Sabari’s work might predict an increased percentage of crotonylated histones whose function may be to preserve transcription of critical genes during starvation. In summary, this work sheds light on how metabolic pathways influence gene expression through histone crotonylation. But what about the possibility that nonhistone proteins could be crotonylated, and that this could in turn regulate cellular metabolism? Precedence for this comes from work of the Guan lab, which revealed acetylation on almost every metabolic enzyme in the cell (Zhao et al., 2010). Follow-up studies have shown protein acetylation to have a dramatic effect on regulation of various metabolic enzymes and pathways. The mitochondrion, for example, is a hotspot for acyl modifications that can impact metabolic function (Kim et al., 2006; Park et al., 2013). Future work will determine whether non-histone crotonylation occurs and how it might affect protein and cellular function. To address these and other questions, one difficulty to overcome is the ability to measure the concentration and localization of crotonyl-CoA pools. More sophisticated techniques will be needed to quantify the ratios of acyl-CoAs on a subcellular or even more local levels and to determine

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how these ratios change in different cell types or cellular environments and influence their interplay and their effects on protein regulation. REFERENCES Cai, L., Sutter, B.M., Li, B., and Tu, B.P. (2011). Mol. Cell 42, 426–437. Comerford, S.A., Huang, Z., Du, X., Wang, Y., Cai, L., Witkiewicz, A.K., Walters, H., Tantawy, M.N., Fu, A., Manning, H.C., et al. (2014). Cell 159, 1591–1602. Guarente, L. (2011). Cold Spring Harb. Symp. Quant. Biol. 76, 81–90. Kim, S.C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., et al. (2006). Mol. Cell 23, 607–618. Kouzarides, T. (2007). Cell 128, 693–705. Park, J., Chen, Y., Tishkoff, D.X., Peng, C., Tan, M., Dai, L., Xie, Z., Zhang, Y., Zwaans, B.M., Skinner, M.E., et al. (2013). Mol. Cell 50, 919–930. Sabari, B.R., Tang, Z., Huang, H., Yong-Gonzalez, V., Molina, H., Kong, H.E., Dai, L., Shimada, M., Cross, J.R., Zhao, Y., et al. (2015). Mol. Cell 58, this issue, 203–215. Sutendra, G., Kinnaird, A., Dromparis, P., Paulin, R., Stenson, T.H., Haromy, A., Hashimoto, K., Zhang, N., Flaim, E., and Michelakis, E.D. (2014). Cell 158, 84–97. Tan, M., Luo, H., Lee, S., Jin, F., Yang, J.S., Montellier, E., Buchou, T., Cheng, Z., Rousseaux, S., Rajagopal, N., et al. (2011). Cell 146, 1016–1028. Wellen, K.E., Hatzivassiliou, G., Sachdeva, U.M., Bui, T.V., Cross, J.R., and Thompson, C.B. (2009). Science 324, 1076–1080. Zhao, S., Xu, W., Jiang, W., Yu, W., Lin, Y., Zhang, T., Yao, J., Zhou, L., Zeng, Y., Li, H., et al. (2010). Science 327, 1000–1004.

Greetings from the planet croton.

In this issue of Molecular Cell, Sabari et al. (2015) discover that levels of intracellular crotonyl-CoA impact the histone acylation landscape, provi...
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