Short-chain fatty acids activate acetyltransferase p300
- Wisconsin Institute for Discovery, United States
- Department of Biomolecular Chemistry, University of Wisconsin – Madison, United States
- Morgridge Institute for Research, United States
Figures
Figure 1 with 1 supplement
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) …
Figure 1—figure supplement 1
Physiological effects of SCFA treatment.
(A) Histone proteomics of HCT116 cells treated with 0.01, 0.1, or 1 mM acetate, propionate, or butyrate for 1 hr. Values are log2(fold change) over untreated control. *=p≤0.05, **=p≤0.01, …
Figure 2 with 2 supplements
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 …
Figure 2—figure supplement 1
Further SCFA metabolism.
(A) Total carnitine and CoA levels in SCFA-treated cells over 24 hr time course. (B) Acyl-carnitine labeling in HCT116, HEK293, HepG2, and MCF7 cells after 1 hr treatment with 1 mM SCFA. Values are …
Figure 2—figure supplement 2
Metabolism of SCFAs to acetyl-CoA is not necessary to induce hyperacetylation.
(A) Propionate and butyrate increase acetylation in HCT116 cells treated with non-targeting (NT) siRNA and 1 mM SCFA. (B) ACLY, ACSS2, and PCC siRNAs compared to NT siRNA + same SCFA treatment. (C) …
Figure 3 with 1 supplement
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 …
Figure 3—figure supplement 1
Recombinant p300 is actively auto-acylated.
(A) Acylation of active or denatured p300. (B) SIRT2 treatment removes endogenous acetylation on recombinant p300 without inhibiting auto-acetylation after add-back of acetyl-CoA. (C) Rate of …
Figure 4 with 1 supplement
p300 inhibition, but not HDAC inhibition, reverses SCFA-induced hyperacetylation.
(A) Acetylation of H3K27ac, H3K18ac, and p53K382ac after treatment with A485, SAHA, and 100–250 µM of propionate/butyrate for 24 hr. Values are normalized to total H3 or total p53 before calculating …
Figure 4—figure supplement 1
Representative Western blots corresponding to Figure 4.
(A) Acetylation of H3K27ac, H3K18ac, and p53K382ac after treatment with A485, SAHA, and 100–250 µM of propionate/butyrate for 24 hr. (B) Expression of HA-tagged plasmids and p300 levels in cells …
Tables
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.
| Molecule | IC50 |
|---|---|
| Propionate | 223 ± 64 µM |
| Butyrate | 52 ± 11 µM |
| Propionyl-CoA | 18 ± 7 mM |
| Butyryl-CoA | 1.13 ± 0.01 mM |
Table 2
Sites of p300 acylation.
Sites of lysine acetylation, propionylation, and butyrylation on recombinant p300 treated with acyl-CoAs. AIL: autoinhibitory loop, A: acetylation, P: propionylation, B: butyrylation.
| Lysine site(AIL lysines in bold). | Acetylation | Propionylation | Butyrylation |
|---|---|---|---|
| K1542 | A | P | |
| K1546 | A | P | |
| K1549 | A | P | |
| K1550 | A | ||
| K1551 | A | P | |
| K1554 | A | P | B |
| K1555 | A | P | B |
| K1558 | A | P | B |
| K1560 | A | P | B |
| K1568 | A | P | |
| K1569 | A | P | |
| K1570 | A | P | |
| K1583 | A | ||
| K1590 | A | ||
| K1637 | A | P |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
| HCT116 cell line (Homo-sapiens) | Colorectal carcinoma | ATCC | CCL-247 | RRID:CVCL_0291 |
| HEK-293 cell line (Homo-sapiens) | Kidney epithelial | ATCC | CRL-1573 | RRID:CVCL_0045 |
| HepG2 cell line (Homo-sapiens) | Liver epithelial | ATCC | HB-8065 | RRID:CVCL_0027 |
| MCF7 cell line (Homo-sapiens) | Adenocarcinoma epithelial | ATCC | HTB-22 | RRID:CVCL_0031 |
| PCCA mutant cell lines (Homo-sapiens) | Propionic acidemia cell lines | NIGMS Human Genetic Cell Repository, Coriell Institute | GM00371 GM00405 GM22010 GM22011 GM22123 GM22126 | |
| Transfected construct (human) | siRNA to ACSS2 (SMARTpool) | Dharmacon | L-010396-00-0005 | |
| Transfected construct (human) | siRNA to ACLY (SMARTpool) | Dharmacon | L-004915-00-0005 | |
| Transfected construct (human) | siRNA to PCCA (SMARTpool) | Dharmacon | L-008965-00-0005 | |
| Transfected construct (human) | p300 mutant constructs | Ortega et. al. 2018 | WT p300, ΔGlu p300 | |
| antibody | ACLY, rabbit | CST | 4,332 S | RRID:AB_2223744, 1:1,000 dilution |
| antibody | ACSS2, mouse | Novus Biologicals | NBP2-01269 | 1:1,000 dilution |
| antibody | FFAR2/GPR43, mouse | Novus Biologicals | MAB10082-100 | 1:1,000 dilution |
| antibody | FFAR3/GPR41, rabbit | Novus Biologicals | NBP2-14014 | 1:1,000 dilution |
| antibody | H3, mouse | Abcam | ab24834 | RRID:AB_470335, 1:5,000 dilution |
| antibody | H3 K18ac, rabbit | Abcam | ab1191 | RRID:AB_298692, 1:1,000 dilution |
| antibody | H3 K27ac, mouse | Active Motif | 39,685 | RRID:AB_2793305, 1:1,000 dilution |
| antibody | HA-Tag, rabbit | CST | 3,724 | RRID:AB_1549585, 1:1,000 dilution |
| antibody | p300 (NM11), mouse | SCBT | sc-32244 | RRID:AB_628076, 1:1,000 dilution |
| antibody | p53, mouse | CST | 2,524 | RRID:AB_331743, 1:1,000 dilution |
| antibody | p53 K382ac, rabbit | CST | 2,525 | RRID:AB_330083, 1:1,000 dilution |
| antibody | Pan anti-acetyllysine, rabbit | CST | 9,814 | 1:1,000 dilution |
| antibody | Pan anti-butyryllysine, mouse | PTM Biolabs | PTM-329 | 1:500 dilution |
| antibody | Pan anti-propionyllysine, rabbit | PTM Biolabs | PTM-201 | 1:500 dilution |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72171/elife-72171-transrepform1-v2.pdf
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Source data 1
Raw Data and Replicates for Enzyme Assays.
- https://cdn.elifesciences.org/articles/72171/elife-72171-supp1-v2.xlsx
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Source data 2
Full Western Blots and Replicate Blots.
- https://cdn.elifesciences.org/articles/72171/elife-72171-supp2-v2.zip
