ATP-citrate lyase links cellular metabolism to histone acetylation

Abstract

Histone acetylation in single-cell eukaryotes relies on acetyl coenzyme A (acetyl-CoA) synthetase enzymes that use acetate to produce acetyl-CoA. Metazoans, however, use glucose as their main carbon source and have exposure only to low concentrations of extracellular acetate. We have shown that histone acetylation in mammalian cells is dependent on adenosine triphosphate (ATP)-citrate lyase (ACL), the enzyme that converts glucose-derived citrate into acetyl-CoA. We found that ACL is required for increases in histone acetylation in response to growth factor stimulation and during differentiation, and that glucose availability can affect histone acetylation in an ACL-dependent manner. Together, these findings suggest that ACL activity is required to link growth factor-induced increases in nutrient metabolism to the regulation of histone acetylation and gene expression.

Fig. 1. ACL localizes to the nucleus and cytoplasm

(A) Subcellular localization of EGFP-tagged ACL in LN229 cells imaged by deconvolution microscopy. EGFP-ACL, visible in both nucleus and cytoplasm, is contrasted to DsRed mitochondrial marker, which is excluded from the nucleus. DAPI staining of nuclear DNA is restricted to the nucleus. (B) Western blot analysis of cytoplasmic and nuclear protein extracts from HCT116 cells. Quantitation represents mean +/- SD from 4 [ACL, AceCS1] or 8 [PARP, tubulin] nuclear and cytoplasmic samples. ACL and AceCS1 are both significantly (***, p<0.0001) enriched in the nuclear fraction as compared to tubulin.

Fig. 2. ACL is the major source of acetyl-CoA for histone acetylation in mammalian cells

In all panels, HCT116 cells were used and transfected with CTRL, ACL and/or AceCS1 siRNA as indicated for 72 hours. (A) Western blot analysis of total and acid extracts from HCT116 cells treated +/- 5mM acetate for 24 hours prior to lysis. Acid-extracted histones were visualized by Coomassie staining. Quantitation represents percent reduction of acetylated/total histone levels in siACL- and/or siAceCS1-transfected cells compared to siCTRL-transfected cells in 3 independent experiments (mean +/- SEM, *, p< 0.05; **, p< 0.01) (B) HCT116 cells were incubated with 0, 1, or 5 mM acetate for 24 hours prior to lysis. Quantitation of acetylated/total H3 levels represents triplicate samples run in parallel, mean +/- SD (*, p<0.05; ***, p<0.0005). (C) Western blot analysis of p53 acetylation in HCT116 cells treated with 0.5 μM doxorubicin for 24 hours. Phosphorylation of H2AX confirmed that the DNA damage response was activated by doxorubicin. (D) Western blot analysis of H3 and tubulin acetylation in HCT116 cells treated with 500 nM trichostatin A (TSA) for 24 hours. (E) Western blot analysis of histone acetylation in total extracts of HCT116 cells transfected for 72 hours with CTRL, ACL, and/or GCN5 siRNAs. Results are representative of 4 independent experiments. For quantitation, see Figure S2.

Fig. 3. Role of ACL and glucose availability in the acetylation of histones during cellular response to serum stimulation

Immortalized MEFs transfected with CTRL or ACL siRNAs as indicated were synchronized by serum deprivation overnight followed by serum stimulation. (A) Western blot analysis of total protein extracts from siCTRL-or siACL-treated cells at indicated time points after re-addition of serum to the culture (left panel). Ratio of acetylated histone H3 to total H3 was quantitated at each time point and fitted to a linear regression (right panel). Comparable results were obtained in each of 3 independent experiments. (B) Western blot analysis of total protein extracts from control cells at indicated times following serum re-introduction in the presence (+ = 25 mM) or absence (- = 0 mM) of glucose. (C) Western blot analysis of total protein extracts from cells transfected with siCTRL or siACL as described in Materials and Methods (33) and cultured for 48h in the presence or absence of glucose.

Fig. 4. Regulation of acetylation of histones during adipocyte differentiation by ACL and nutrient availability

3T3-L1 preadipocytes were transfected with control, ACL, and/or AceCS1 siRNAs and 2 days later stimulated to differentiate into adipocytes. (A) Total lysates and acid extracts from 3T3-L1 cells 4 days after induction of differentiation, analyzed by Western blot. (B) Acid extracts from 0 and 4 days after induction of differentiation in the presence of 0, 1, or 5 mM sodium acetate were analyzed by Western blot. (C) Cells were fixed and Oil Red O lipid staining was performed 5 days after induction of differentiation. Similar results were obtained in each of 3 independent experiments. (D) RNA was isolated 4 days after induction of differentiation in the presence or absence of 5 mM sodium acetate. Gene expression was analyzed by quantitative RT-PCR and normalized to 18S rRNA (mean +/- S.D. of triplicate samples). (E) Gene expression 4 days after induction of differentiation was quantified by Taqman quantitative RT-PCR and normalized to 18S rRNA. Data represent triplicate wells from 2 independent experiments, mean +/- S.D. (F) RNA was isolated 4 days after induction of differentiation in the presence of 0, 1, or 5 mM sodium acetate. Glut4 expression was analyzed by Taqman RT-PCR and normalized to 18S rRNA (mean +/- S.D. of triplicate samples). (G). Chromatin immunoprecipitation was performed using antibodies to Ac-H3 and Ac-H4. Immunoprecipitated Glut4 promoter sequence was analyzed by quantitative PCR. H4 genomic sequence was used as an endogenous control. The amount of immunoprecipitated H4 DNA was unchanged upon ACL silencing. Data represent mean +/- S.D. of triplicate samples from each of 2 independent experiments (H) Western blots of cells differentiated 0 or 4 days in 4 or 25 mM glucose, +/- 5 mM sodium acetate. (I) Gene expression in cells 4 days after induction of differentiation in 1, 4, or 25 mM glucose was assessed by Taqman quantitative RT-PCR and normalized to 18S rRNA. Data are the averages of 3 independent experiments (mean +/- SEM). All statistical analyses are comparisons of 2 data sets and were performed using t-tests (*, p<0.05; **, p<0.005;***, p<0.0005). C= siCTRL; A= siACL.