Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate

Abstract

The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) can act intracellularly independently of its cell surface receptors through unknown mechanisms. Sphingosine kinase 2 (SphK2), one of the isoenzymes that generates S1P, was associated with histone H3 and produced S1P that regulated histone acetylation. S1P specifically bound to the histone deacetylases HDAC1 and HDAC2 and inhibited their enzymatic activity, preventing the removal of acetyl groups from lysine residues within histone tails. SphK2 associated with HDAC1 and HDAC2 in repressor complexes and was selectively enriched at the promoters of the genes encoding the cyclin-dependent kinase inhibitor p21 or the transcriptional regulator c-fos, where it enhanced local histone H3 acetylation and transcription. Thus, HDACs are direct intracellular targets of S1P and link nuclear S1P to epigenetic regulation of gene expression.

Fig. 1

Presence of SphK2 in mononucleosomes and physical association with histone H3. (A) Nuclei were isolated from MCF-7 cells transfected with vector (white bar) or SphK2 (gray bar) and incubated with [³H]sphingosine (1.5 μM) and ATP (1 mM) for 30 min. Formation of [³H]S1P was determined by differential extraction (22). Abundance of sphingoid bases sphingosine (Sph) and dihydrosphingosine (DHS), and their phosphorylated products, S1P and dihydro-S1P (DHS1P), in nuclei of vector and SphK2-expressing MCF-7 cells (7 × 10⁶) were determined by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS/MS). The data are averages of triplicate determinations and are expressed as picomoles of lipid ± SD. Asterisks indicate statistically significant differences (P < 0.05 by Student’s t test) relative to vector transfectants. (B) Association of SphK2 with chromatin. Equal amounts of cytosol, nucleoplasm, and chromatin fractions from MCF-7 cells were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with SphK2-specific antibody. Antibodies against H3 and tubulin were used as markers for chromatin and cytosol, respectively. Lysate from MCF-7 cells overexpressing SphK2 was included as a positive control (leftmost lane). (C) Association of SphK2 and histone H3. Proteins of nuclear extracts from MCF-7 cells transfected with vector, V5-SphK2, or catalytically inactive V5-SphK2^G212E were immunoprecipitated with V5-specific antibody, separated by SDS-PAGE, and probed with antibodies against V5 or H3.

Fig. 2

SphK2 and S1P enhance histone acetylation. (A) Histone acetylation in nuclear extracts from MCF-7 cells transfected with vector, V5-SphK2, or catalytically inactive V5-SphK2^G212E. Acetylation was detected by immunoblotting with antibodies to specific histone acetylation sites as indicated. (B) Effect of S1P and dihydro-S1P on histone acetylation. Purified nuclei from MCF-7 cells were treated for 5 min without (none) or with vehicle, 1 μM S1P, or 1 μM dihydro-S1P, and histone acetylation was examined by Western blotting. (C and D) Effect of depletion of SphKs. MCF-7 cells were transfected with nontargeting control siRNA, or siRNA targeted to SphK1 or SphK2. (C) Proteins from nuclear extracts were immunoblotted as indicated. Depletion of SphK1 and SphK2 was confirmed by immunoblotting with SphK1-specific and SphK2-specific antibodies, respectively. Asterisk (*) indicates nonspecific band. (D) Amounts of S1P and dihydro-S1P in purified nuclei and cytoplasm quantified by LC-ESI-MS/MS. Asterisks indicate statistically significant differences (P < 0.05 by Student’s t test) relative to control siRNA.

Fig. 3

S1P binds and inhibits HDAC1. (A and B) Effect of S1P and dihydro-S1P on HDAC1 activity. HeLa cell nuclear extracts were immunoprecipitated with control IgG or HDAC1-specific antibody. Immunoprecipitates were washed, and HDAC activity was measured in the presence of vehicle, 1 μM S1P, 1 μM dihydro-S1P (DHS1P), or 1 μM trichostatin A (TSA). HDAC activities are averages of triplicate determinations ± SD and expressed as arbitrary fluorescence units (AFU). (Inset) Immunoprecipitated proteins were separated by SDS-PAGE and analyzed by Western blotting with HDAC1-specific antibody. (B) Activity of recombinant HDAC1 (250 ng) determined in the absence or presence of two concentrations of S1P or dihydro-S1P (0.5 or 5 μM), sphingosine (5 μM), LPA (5 μM), or TSA (1 μM). (C) Association of HDAC with immobilized S1P. Nuclear extracts (NE) from MCF-7 cells were incubated with control (no lipid), LPA, or S1P affinity matrices (as indicated), then washed and bound proteins were resolved by SDS-PAGE and analyzed by Western blotting with antibodies against HDAC1 or HDAC2. (D) Detection of sphingoid bases. Extracted ion chromatogram for LC-MS/MS reverse-phased separation of sphingoid bases in nuclear extracts (left), or HDAC1-specific antibody (center), and control IgG immunoprecipitates (right). Quantification of sphingosine (Sph) and dihydrosphingosine (DHS), and their phosphorylated products, S1P and dihydro-S1P (DHS1P), is shown in table S1.

Fig. 4

SphK2 binding to p21 and c-fos promoters enhances acetylation of histone H3. (A and B) MCF-7 cells transfected with vector, V5-SphK2, or V5-SphK2^G212E were treated with vehicle or PMA (100 nM, hatched bars) for 3 hours (A) or 30 min (B) and subjected to ChIP analyses with antibodies to V5, HDAC1, H3-K9ac, or normal rabbit IgG, as indicated. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the p21 and c-fos genes. Relative binding to the promoter is expressed as the percentage of input. Data are means ± SD. **P < 0.01, relative to vector transfectants, *P < 0.01, relative to PMA-treated vector transfectants, by Student’s t test. (C and D) MCF-7 cells transfected with control siRNA or siSphK2 were treated with vehicle or PMA (100 nM, hatched bars) for 3 hours (C) or 30 min (D) and subjected to ChIP analyses with antibody against H3-K9ac or with normal rabbit IgG. *P < 0.01, relative to PMA-treated siControl. (E) Model for regulation of histone acetylation and gene transcription by nuclear SphK2 and S1P. PMA stimulates nuclear SphK2, which is associated with specific promoter regions, such as those for p21 and c-fos genes, and increases production of S1P. S1P in turn inhibits HDAC1 and HDAC2, which results in increased acetylation (Ac) of histone(s) and leads to enhanced gene transcription.