Histone H4 acetylation differentially modulates arginine methylation by an in Cis mechanism

Abstract

Histone H4 undergoes extensive post-translational modifications (PTMs) at its N-terminal tail. Many of these PTMs profoundly affect the on and off status of gene transcription. The molecular mechanism by which histone PTMs modulate genetic and epigenetic processes is not fully understood. In particular, how a PTM mark affects the presence and level of other histone modification marks needs to be addressed and is essential for better understanding the molecular basis of histone code hypothesis. To dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. Although certain acetylations present a repressive impact on PRMT1-mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases the activity of PRMT1 but increases that of PRMT5. Furthermore, circular dichroism study and computer simulation demonstrate that hyperacetylation increases the content of ordered secondary structures at the H4 tail region. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation and unravel the complex intercommunications that exist between different the PTM marks in cis. The divergent activities of PRMT1 and PRMT5 with respect to different acetyl-H4 substrates suggest that type I and type II protein-arginine methyltransferases use distinct molecular determinants for substrate recognition and catalysis.

FIGURE 1.

Methylation of H4R3 by PRMT1 and PRMT5.

FIGURE 2.

A typical NMR spectrum used for calibration of H4 peptide concentration. The NMR solution contained 4.5 mm H4K16ac peptide and 1 mm DSS in D₂O.

FIGURE 3.

Effects of lysine acetylation on Arg-3 methylation by PRMT1. The reaction buffer contained 50 mm Hepes, pH 8.0, 10 mm NaCl, and 1 mm DTT. The concentrations of PRMT1, [¹⁴C]AdoMet, and H4–20 were 0.1, 20, and 100 μm, respectively. The reaction time was 10 min.

FIGURE 4.

Effects of acetylation on Arg-3 methylation catalyzed by PRMT5. Reaction buffer contained 50 mm Hepes, pH 8.0, 10 mm NaCl, and 1 mm DTT. The concentrations of PRMT5, [¹⁴C]AdoMet, and H4 peptide were 0.1, 30, and 200 μm respectively. The reaction time was 1 h.

FIGURE 5.

Acetylation on H4 protein inhibits its methylation by PRMT1, although it promotes its methylation by PRMT5. A, p300 acetylation inhibits H4 methylation by PRMT1. H4 protein was incubated with acetyl-CoA in the absence or presence of p300 before submission to PRMT1 methylation with [¹⁴C]AdoMet. Methylated H4 bands were separated by 15% SDS-PAGE and visualized by storage phosphor scan, which is the same method as for B–D. B, p300 acetylation promotes H4 methylation by PRMT5. H4 protein was incubated with acetyl-CoA in the absence or presence of p300 before submission to PRMT5 methylation. C, MOF acetylation inhibits H4 methylation by PRMT1. H4 protein was incubated with acetyl-CoA in the absence or presence of MOF before submission to PRMT1 methylation. D, MOF acetylation promotes H4 methylation by PRMT5. H4 protein was incubated with acetyl-CoA in the absence or presence of MOF before submission to PRMT5 methylation.

FIGURE 6.

Secondary structure analysis of unacetylated H4 peptide (A) and tetraacetylated H4 peptide (P). CD spectra of unacetylated H4 (A) and tetraacetylated H4 (B) were measured at different concentrations of TFE (0–80%) in 20 mm Tris-HCl buffer, pH 7.4. Black, 0% TFE; blue, 5% TFE; orange, 20% TFE; green, 40% TFE; red, 80% TFE. The CD data were analyzed by Dichroweb to calculate secondary structure compositions. C, column graph showing the distribution of secondary structures for the two peptides at 20% of TFE. D, changes in the composition of ordered structures (i.e. helix and strand) for each peptide with TFE concentration.

FIGURE 7.

Simulated structural changes upon tetraacetylation of the N-terminal H4 tail. a, probability distribution of the radius of gyration of the unacetylated and tetra-acetylated H4 peptides. b, probability distribution of the pairwise backbone root mean square deviation (RMSD) of the structures of the equilibrium population.

FIGURE 8.

Clustering of the equilibrium population of the unacetylated (left column) and tetraacetylated (right column) H4 peptides. Rows a–c show the representative conformations of clusters 1–3, respectively. The color code represents different secondary structural elements as identified throuh a DSSP analysis (48). Purple, α-helix; blue, 3₁₀-helix; green, turn; orange, random coil.

FIGURE 9.

Summary of the effects of lysine acetylation on Arg-3 methylation in H4. Acetylation of the N-terminal H4 tail reciprocally affects PRMT1-mediated Arg-3 methylation and PRMT5-mediated Arg-3 methylation. A solid line means that the effect of acetylation is appreciably strong, and a dotted line means that the effect of acetylation is relatively weak.