Author response: Short-chain fatty acids activate acetyltransferase p300 (2021)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Short-chain fatty acids (SCFAs) acetate, propionate, and butyrate are produced in large quantities by the gut microbiome and contribute to a wide array of physiological processes. While the underlying mechanisms are largely unknown, many effects of SCFAs have been traced to changes in the cell's epigenetic state. Here, we systematically investigate how SCFAs alter the epigenome. Using quantitative proteomics of histone modification states, we identified rapid and sustained increases in histone acetylation after the addition of butyrate or propionate, but not acetate. While decades of prior observations would suggest that hyperacetylation induced by SCFAs are due to inhibition of histone deacetylases (HDACs), we found that propionate and butyrate instead activate the acetyltransferase p300. Propionate and butyrate are rapidly converted to the corresponding acyl-CoAs which are then used by p300 to catalyze auto-acylation of the autoinhibitory loop, activating the enzyme for histone/protein acetylation. This data challenges the long-held belief that SCFAs mainly regulate chromatin by inhibiting HDACs, and instead reveals a previously unknown mechanism of HAT activation that can explain how an influx of low levels of SCFAs alters global chromatin states. Editor's evaluation This study investigates the mechanism of agents like butyrate as metabolites that affect histone acetylation. The authors make the unexpected and interesting finding that such metabolites can stimulate the activity of the acetyltransferase p300 rather than the commonly accepted concept that they block histone deacetylases. The authors show evidence that p300 stimulation involves acylation of Lys residues on its autoinhibitory loop. The authors have effectively responded to prior concerns raised by the reviewers. This study should be of broad interest to the epigenetic research community. https://doi.org/10.7554/eLife.72171.sa0 Decision letter eLife's review process Introduction Short-chain fatty acids (SCFAs) play a crucial role in human health. Although SCFAs include any fatty acid with fewer than six carbons, the first three members of the family—acetate, propionate, and butyrate—are by far the most abundant physiologically (den Besten et al., 2013; Koh et al., 2016; Tan et al., 2014). These three SCFAs are produced in large quantities by bacterial fermentation of nondigestible fiber, and levels can fluctuate based on the amount and type of fiber in the diet (Bird et al., 2000; den Besten et al., 2013; Jenkins et al., 1998; Levrat et al., 1991; Marsono et al., 1993; Ríos-Covián et al., 2016; Velázquez et al., 2000). In humans, total SCFA concentrations can reach over 100 mM in the colon, in a ratio of ~60:20:20 acetate:propionate:butyrate (Cummings et al., 1987; Parada Venegas et al., 2019). SCFAs alone may provide up to 10% of daily caloric requirements in humans (Bergman, 1990). Colonocytes are especially prone to metabolize SCFAs, deriving 60–70% of their energy from SCFA oxidation (Roediger, 1982). Excess SCFAs are transported to the liver through the portal vein and are eventually released into the peripheral blood, existing at micromolar concentrations (Bloemen et al., 2009; Cummings et al., 1987). Butyrate and acetate are the most-studied members of the group. Acetate plays vital roles in cellular metabolism, especially as a building block for the central metabolite acetyl-CoA (Bose et al., 2019; Liu et al., 2018; Zhao et al., 2016). The physiological effects of butyrate are abundant: butyrate has been reported among other things to improve intestinal barrier function (Chang et al., 2014; Hamer et al., 2007; Peng et al., 2009), reduce inflammation (Chriett et al., 2019; Usami et al., 2008; Vinolo et al., 2011), improve metabolic health (Gao et al., 2009; Lin et al., 2012; Müller et al., 2019), and prevent cancer (Donohoe et al., 2012; Hague et al., 1993). The role of propionate is less clear—while it shows the same beneficial effects as butyrate in many studies, extremely high levels of propionate are associated with negative health outcomes such as propionic acidemia and autism spectrum disorders (Abdelli et al., 2019; Al-Lahham et al., 2010; Li et al., 2017). It is generally believed that SCFAs induce physiological effects by changing the cell's epigenetic state (Hamer et al., 2007; Hinnebusch et al., 2002; Krautkramer et al., 2016; Tan et al., 2014). Epigenetics describes the regulation that takes place in eukaryotic chromatin. Much of this regulation centers around histone proteins, which are heavily decorated with post-translational modifications (PTMs). Histone PTMs affect a wide array of processes, from DNA accessibility to transcription factor binding (Bannister and Kouzarides, 2011; Karch et al., 2013; Kouzarides, 2007). Histone PTMs include lysine acetylation, which opens chromatin and makes it more accessible to transcription, and lysine methylation, which performs various functions. Recently, a host of other histone PTMs have also been identified, including lysine propionylation and butyrylation (Chen et al., 2007; Dai et al., 2014; Goudarzi et al., 2016; Kebede et al., 2017). Histone PTMs, as well as the enzymes which add or remove them, are exquisitely sensitive to changes in cellular metabolism (Albaugh et al., 2011; Fan et al., 2015; Sassone-Corsi, 2013). Several central metabolites are also substrates for histone modifications: such as acetyl-CoA, which is used to acetylate histones. Histone acetylation is regulated on two fronts—first by histone acetyltransferases (HATs), which transfer acetyl groups to chromatin, and then by histone deacetylases (HDACs), which remove them (Albaugh et al., 2011). Acetate, propionate, and butyrate have all been reported to inhibit HDACs to varying extents, with butyrate being the most potent inhibitor (Bolduc et al., 2017; Candido et al., 1978; Davie, 2003; Hsu et al., 2016; Silva et al., 2018; Waldecker et al., 2008). HDAC inhibition has been proposed as a mechanism for SCFA's anti-cancer effects (Donohoe et al., 2014; Hinnebusch et al., 2002), insulin regulation (Chriett et al., 2019; Gao et al., 2009), and immunomodulation (Bolduc et al., 2017; Chang et al., 2014; Usami et al., 2008; Vinolo et al., 2011). However, there are still questions as to whether SCFAs regulate the epigenome by HDAC inhibition alone (Corfe, 2012; Donohoe et al., 2012; Gibson, 2000). It has been recently reported that the gut microbiome can affect histone modifications systemically (Krautkramer et al., 2016; Kumar et al., 2014; Romano et al., 2017; Takahashi et al., 2006; Wellen et al., 2009; Zhao et al., 2020a). Our lab has demonstrated that germ-free mice display distinct patterns of histone PTMs in multiple tissues (Krautkramer et al., 2016). This phenotype was complex, but could be generally characterized by decreased histone acetylation on multiple sites. However, acetylation levels did not depend solely on bacteria—colonized mice fed diets that produced low levels of SCFAs showed similar PTMs to germ-free mice. Finally, simply supplementing germ-free mice with SCFAs in drinking water led to changes in gene expression and histone PTMs that mimicked bacterial colonization. This data strongly suggests that SCFAs are a critical link between the microbiome and epigenetic state. Our understanding of this cross-talk is still in its infancy; thus, discovering how SCFAs interact with host cells on a mechanistic level is crucial to understanding how these systems interact as a whole. Here, we integrate proteomics and metabolomics with biochemical assays to investigate how each SCFA affects epigenetics and cell metabolism. Our results suggest that at physiologic levels, SCFAs induce histone acetylation by activating HATs instead of inhibiting HDACs. Results Propionate and butyrate induce hyperacetylation in cell culture To determine the broad effects of extracellular SCFAs on histone PTMs, we treated HCT116 cells with acetate, propionate, and butyrate individually and then performed histone proteomics (Karch et al., 2013; Krautkramer et al., 2015). This mass spectrometry-based method allows for simultaneous analysis of >70 histone PTMs, which significantly improves on traditional Western blotting (Thomas et al., 2020). While the impact of SCFAs on a few of these PTMs has been studied previously, to our knowledge this is the first study to show how SCFAs regulate a range of both canonical and non-canonical PTMs (Hinnebusch et al., 2002; Kiefer et al., 2006; Silva et al., 2018; Wang et al., 2018). HCT116 cells were initially chosen because they are derived from colon, and colon cells are in most frequent contact with bacterially produced SCFAs. Propionate and butyrate induced rapid and dose-dependent increases in histone acetylation but did not affect histone methylation (Figure 1A). In contrast, acetate had no significant effect on histone PTMs at any dose or time point tested. These results are consistent with previous reports (Hinnebusch et al., 2002; Kiefer et al., 2006; Silva et al., 2018). The hyperacetylation phenotype with propionate and butyrate was rapid and stable, persisting for at least 24 hr (Figure 1B). We also detected changes in both histone propionylation and butyrylation on multiple sites, including significant increases in K18/K23 propionylation after both propionate and butyrate treatment. In general, changes in histone propionylation and butyrylation were less consistent across experiments compared to the robust changes in histone acetylation, likely due in part to the technical challenges of measuring low-abundance propionylation/butyrylation. Based on these experiments, we chose a standard treatment of 1 mM SCFA for 1 hr, which we used for further experiments unless otherwise indicated. This dose did not affect media pH, media glucose, or cell viability for at least 24 hr (Figure 1—figure supplement 1). The 1 mM dose is within the physiological concentrations in the colon (which range from 0.5 to 15 mM), but is higher than reported concentrations in other parts of the body (which range from 0 to 150 µM) (Cummings et al., 1987; Parada Venegas et al., 2019). Figure 1 with 1 supplement see all Download asset Open asset Extracellular propionate and butyrate induce histone hyperacetylation. (A) Histone proteomics of HCT116 cells treated with 1 mM acetate, propionate, or butyrate for 1 hr. (B) Histone acylation over a time course of 1 mM acetate, propionate, or butyrate treatment. (C) SCFA treatment (1 mM, 1 hr). of HEK293, MCF7, and HepG2 cell lines. All values are log2(fold change) over untreated, time-matched controls. *=p≤0.05, **=p≤0.01, ***=p≤0.001. n=3 per condition. To determine whether this effect was unique to HCT116 cells, we also treated three other cell lines derived from diverse human tissues. HEK293, HepG2, and MCF7 cells showed remarkably similar hyperacetylation phenotypes after propionate and butyrate treatment (Figure 1C). Thus, the ability of propionate and butyrate to increase histone acetylation, but not methylation, can be generalized to multiple cell types. Propionate and butyrate are rapidly metabolized into acyl-CoAs This experiment raised several intriguing questions. First, what differentiates propionate and butyrate from acetate? It is general knowledge that butyrate can act as an HDAC inhibitor, slowing the removal of acetate from chromatin (Davie, 2003). This could explain the difference between acetate and butyrate, but it does not necessarily explain the difference between acetate and propionate. To test whether propionate could act as an HDAC inhibitor in HCT116 cells, we performed HDAC assays on nuclear extract and determined the apparent IC50 values for these SCFAs and their corresponding acyl-CoA forms. Table 1 lists the IC50 values of HDAC inhibition for propionate, butyrate, and corresponding acyl-CoAs. These IC50 values are on the low end of previously published values, which range from 50 to 300 µM in nuclear extract and 1–10 mM in whole cells (Huber et al., 2011; Silva et al., 2018; Vinolo et al., 2011; Waldecker et al., 2008). While SCFA concentrations in media were theoretically higher than our determined IC50 values (Figure 2C), it has been previously reported that intracellular concentrations of SCFAs are much lower than extracellular concentrations, which could have bearing on this mechanism (Donohoe et al., 2012). Figure 2 with 2 supplements see all Download asset Open asset Metabolism of extracellular short-chain fatty acids (SCFAs). (A) Overall scheme of SCFA metabolism. (B) Proportion of 13C on histones after 4 hr treatment with labeled SCFAs. (C) Concentration of SCFAs in media (µM) and in cells (femtomole/cell) over a 24 hr time course. (D, E) 13C labeling of TCA cycle metabolites (D) and acyl-carnitines (E) over a 24-hr time course. Values are average and standard deviation of normalized signal intensity of ≥3 biological replicates. Table 1 IC50 of HDAC inhibition for SCFAs and acyl-CoAs. IC50 values of HDAC inhibition for propionate, butyrate, propionyl-CoA, and butyryl-CoA. Values are calculated from dose curves using n=2 technical replicates, raw data can be found in Source data 1. MoleculeIC50Propionate223 ± 64 µMButyrate52 ± 11 µMPropionyl-CoA18 ± 7 mMButyryl-CoA1.13 ± 0.01 mM To determine intracellular SCFA concentrations before and after the extracellular addition of SCFAs, we treated cells with fully labeled 13C-acetate, propionate, or butyrate and ran targeted LC-MS/MS metabolomics to track SCFAs through cellular metabolism (Figure 2A). After treatments, SCFA concentrations in media declined from 1 mM to 0.5 mM over 24 hr. SCFA levels measured in cellular extractions were similar among the three SCFAs (max ~3-fold difference), with all showing a decline by 24 hr (Figure 2C). From levels determined in the cellular extractions, we estimated intracellular concentrations of ~40 µM for butyrate and ~100 µM for propionate and acetate, which are consistent with previous reports (Donohoe et al., 2012). In additional control experiments, SCFA concentrations in media without cells incubated at 37°C declined at almost an identical rate as that observed in media incubated with cells, suggesting that the bulk of the decrease in media SCFA concentration was due to volatility and not to cellular consumption (Figure 2—figure supplement 1). The calculated values suggest that free SCFAs are rapidly metabolized instead of building up in cells (Boets et al., 2017; Clausen and Mortensen, 1994; Clausen and Mortensen, 1995). With this in mind, we searched for labeled carbons in downstream TCA cycle metabolites. However, we found no significant labeling of TCA cycle metabolites after propionate and butyrate treatment (Figure 2D). Instead, labeled carbons built up as acyl-carnitine species, appearing in as little as 10 min and remaining stable for 24 hrs (Figure 2E). Intriguingly, this buildup did not occur in acetate-treated cells. The same trend occurred in acyl-CoA levels, although these species degrade rapidly during sample processing and are thus difficult to detect by mass spectrometry (Figure 2—figure supplement 1). Accumulation of acyl-CoAs after SCFA treatment has been previously reported (Basu et al., 2011). In that study, increases in acyl-CoA lead to a decrease of free coenzyme A. Under our conditions, however, free carnitine and free CoA levels did not significantly change (Figure 2—figure supplement 1). To confirm that these metabolic changes were consistent across cell lines, we also performed targeted LC-MS/MS metabolomics in HEK293, HepG2, and MCF7 cells. All cell lines showed the same upregulation of acyl-carnitines after butyrate and propionate treatment (Figure 2—figure supplement 1). Ultimately, these data suggest that histone hyperacetylation may not be due to the presence of intracellular propionate or butyrate, as the intracellular levels of these metabolites are low, but to the presence of propionyl- or butyryl-CoA. Since propionyl- and butyryl-CoA do not inhibit HDAC at physiological concentrations, this raises the possibility that the histone hyperacetylation phenotype may not be due to HDAC inhibition, but to a different mechanism (Table 1; Vogelauer et al., 2012). The intracellular levels of SCFAs do not support robust HDAC inhibition at micromolar treatment concentrations. However, we and others have measured histone hyperacetylation in response to treatment concentrations as low as 10 µM (Figure 1—figure supplement 1; Biermann et al., 2011; Donohoe et al., 2012; Kespohl et al., 2017). At these concentrations, HDACs would be inhibited <1%. Alternatively, it has been proposed that hyperacetylation under SCFA treatment is due to a direct mechanism, in which acetyl-CoA generated by SCFAs is added directly onto histones (Donohoe et al., 2012). To test whether this occurred in our system, we performed histone proteomics after a 4-hr treatment with 13C-labeled SCFAs. This treatment only resulted in 1–5% 13C-labeling on histones, which cannot account for the two- or threefold increases in histone acetylation seen after SCFA treatment (Figure 2B). This indicates that the majority of new acetyl groups (~90–95%) were not generated from labeled SCFAs. To confirm that SCFAs do not need to be metabolized into acetyl-CoA to induce hyperacetylation, we knocked down several enzymes involved in acetyl-CoA generation from SCFAs (Figure 2A). siRNA knockdown or genetic knockout of ACLY (an enzyme involved in transport of acetyl-CoA out of the mitochondria), ACSS2 (the cytoplasmic acetyl-CoA ligase), and PCC (an enzyme in the pathway that metabolizes propionyl-CoA to succinyl-CoA) had no significant effect on propionate or butyrate-induced histone acetylation (Figure 2—figure supplement 2). This data indicates that acyl-CoAs are not metabolized to acetyl-CoA in sufficient quantities to explain the induced hyperacetylation, indicating a different mechanism is at play. Propionyl- and butyryl-CoA activate histone acetylation Next, we tested whether the rapidly formed propionyl- and butyryl-CoA from propionate and butyrate treatment could directly activate HATs (Table 1). HAT assays demonstrated that propionyl- and butyryl-CoA induced dose-dependent increases in the HAT activity of nuclear extract (Figure 3A). Free propionate and butyrate had no effect. However, since this assay simply measures the release of CoA, the apparent increase in HAT activity could have resulted from increased propionyl- or butyryl-transfer reactions. In addition, these results do not indicate which HAT enzymes may be activated. Figure 3 with 1 supplement see all Download asset Open asset Acyl-CoAs activate p300. (A) HAT activity of nuclear extract treated with a dose curve of SCFAs and acyl-CoAs. (B) Rate of p300 auto-acylation with acetyl-CoA, propionyl-CoA, and butyryl-CoA. Values are average and standard deviation, n=4 per condition. (C) Rate of histone acetylation by acetylated, propionylated, or butyrylated p300 using radioactive acetyl-CoA. Values are average and standard deviation, n=3 per condition. Replicate results from this assay performed with different enzyme preparations on different days are shown in Figure 3—figure supplement 1. Quantification for all replicates including Western blots and radioactive assays available in Source data 1. HAT, histone acetyltransferase; SCFA, short-chain fatty acid. HATs can be categorized into three main families—GNAT, MYST, and p300/CBP (Albaugh et al., 2011; Berndsen and Denu, 2008; Fan et al., 2015). Most HATs prefer specific histone lysines and catalyze acetyl-transfer to a handful of histone substrates (Roth et al., 2001). However, p300/CBP acetylates a large number of lysine sites in vitro, and is known to target several of our measured hyperacetylation sites in vivo (Henry et al., 2013). p300/CBP are two closely related transcriptional co-activators—large proteins consisting of several domains and even more binding partners and acetylation targets (Chan and La Thangue, 2001; Dancy and Cole, 2015; Goodman and Smolik, 2000). Both proteins encode an autoinhibitory loop (AIL, also called the autoregulatory or activation which be to stimulate HAT activity et al., 2006; et al., have identified sites of propionylation and butyrylation on including on several however, how these modifications are added and whether they have a on p300 activity are not known (Chen et al., 2007; et al., 2009; et al., 2017). First, we determined whether p300 can catalyze auto-acylation using propionyl- or butyryl-CoA. we found that p300 performed auto-acylation with propionyl- or butyryl-CoA at from with acetyl-CoA (Figure was as p300 did not show increases in acylation under the same (Figure 3—figure supplement 1A). analysis the of propionylation and butyrylation on several lysine sites under these (Table 2). Table 2 of p300 of lysine acetylation, and butyrylation on p300 treated with acyl-CoAs. autoinhibitory loop, acetylation, lysines in Next, we auto-acylation p300 histone acetylation. To do we compared and of p300 in radioactive HAT assays to acetylation on a All of p300 propionylated, and were significantly more than an control (Figure In addition, there were no significant in the activity of propionylated, or butyrylated which all showed increase in their compared to p300. We these results by Western HAT activity assays with histone which are a more physiological than a (Figure 3—figure supplement these data showed an increase in activity for all three of p300. In these data that p300 and propionylation on sites, and that auto-acylation the enzyme in a similar to To investigate whether this previously mechanism in we used of each To any effects from HDAC inhibition in these experiments, we used treatment concentrations from 100 to which should lead to no more than HDAC inhibition (Table 1). First, we treated cells with a potent inhibitor of both histone acetylation and of and thus should inhibit histone hyperacetylation due to p300 activation et al., 2017). treatment hyperacetylation on p300 targets and (Figure Figure supplement 1A). treatment did not hyperacetylation on histone substrates of but also on the p300 we treated cells with the potent HDAC inhibitor, Both and butyrate are to be of HDACs, although the IC50 of is significantly lower and 2007; et al., 2007). with 10 µM of this potent inhibitor should effectively HDAC inhibition, at least of and HDACs, which are known to target these sites et al., 2019; et al., 2018; et al., 2018). Thus, propionate and butyrate act solely as HDAC they should not induce further hyperacetylation under these However, 100 µM treatment of propionate or butyrate with still led to significant increases in acetylation over a control (Figure Figure supplement 1A). these results suggest that propionate and butyrate induce histone hyperacetylation by an mechanism to HDAC inhibition, likely through the of p300 by rapidly formed acyl-CoAs. Figure 4 with 1 supplement see all Download asset Open asset p300 inhibition, but not HDAC inhibition, hyperacetylation. (A) of and after treatment with and µM of for 24 hr. Values are normalized to total or total before changes to the (B) of and in cells with or p300 Values are change over cells with the same (C) of p300 after treatment with µM of propionate or is measured with a radioactive assay and normalized to concentration of p300 in each *=p≤0.05, **=p≤0.01, ***=p≤0.001. per condition. histone SCFA, short-chain fatty acid. To this mechanism of SCFA on chromatin, we HCT116 cells with two p300 p300 or an in which 11 of the lysines were to a et al., 2018). The changes the on sites, which in auto-acylation of the and p300 µM propionate treatment induced significant increases in histone acetylation after and (Figure Figure supplement However, cells with the showed significantly increases in acetylation, of that seen in cells. In addition, we that even the increases in acetylation in the were due to of p300 in cells, or acylation of lysine sites on the these data strongly suggest that the lysines of p300 are to induce the of histone hyperacetylation after propionate treatment. Finally, we performed of p300 from HCT116 cells treated with µM propionate or p300 from propionate and cells showed but significant increases in activity in a radioactive HAT assay (Figure blots also identified increased propionylation and butyrylation of p300 under these (Figure supplement In our results that p300 rather than HDAC inhibition, plays a critical role for propionate and butyrate-induced chromatin hyperacetylation at low concentrations. Discussion butyrate and ability to alter chromatin have been to HDAC inhibition et al., However, the IC50 values for these two are with reported values between 50 and 300 µM in nuclear extract and between 1 and 10 mM in whole cells (Huber et al., 2011; Silva et al., 2018; Vinolo et al., 2011; Waldecker et al., 2008). While it is to reach concentrations in the gut or portal few other would reach these In addition, our previous data suggests that SCFAs can induce histone hyperacetylation in colon, and which SCFA concentrations that by of (Krautkramer et al., 2016). in the intracellular concentrations of SCFAs could be significantly lower than concentrations (Donohoe et al., 2012; et al., Thus, the that increased histone acetylation is by HDAC inhibition In this study, we a previously mechanism and provide evidence that butyrate and propionate activate p300 at low levels, through the rapid to propionyl- and and activation of p300. While the IC50 values for HDAC inhibition are in the high µM to mM values for p300 in the to low µM range et al., 2006; Liu et al., 2008). induced by high of butyrate are almost after butyrate and Wang et al., 2018). In contrast, of p300 increases its from to hr et al., 2012). Thus, even after extracellular SCFAs have been fully p300 could still theoretically induce hyperacetylation. of p300 may thus a mechanism in physiological systems SCFA concentrations are It is to that acetate had effect on histone acetylation in the systems in this Acetate treatment has been previously reported to increase histone acetylation, but only under such as et al., 2017; Gao et al., or after genetic