Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3

Abstract

The acetylation levels of lysine residues in nucleosomes, which are determined by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1-/- cells are lower than in Hmgn1+/+ cells. Induced expression of wild-type HMGN1, but not of a mutant that does not bind to chromatin, in Hmgn1-/- cells elevates the levels of H3K14ac. In vivo, HMGN1 elevates the levels of H3K14ac by enhancing the action of HAT. In vitro, HMGN1 enhances the ability of PCAF to acetylate nucleosomal, but not free, H3. Thus, HMGN1 modulates the levels of H3K14ac by binding to chromatin. We suggest that HMGN1, and perhaps similar architectural proteins, modulates the levels of acetylation in chromatin by altering the equilibrium generated by the opposing enzymatic activities that continuously modify and de-modify the histone tails in nucleosomes.

Figure 1

HMGN1 stimulates the acetylation of H3K14. (A) Western blotting of histones extracted from growing Hmgn1^+/+ or Hmgn1^−/− MEFs, resolved by SDS–PAGE, and developed with either anti-H3 (α-H3) or anti-H3K14ac. The relative signal intensities of H3K14ac, normalized to the intensities of total H3, shown in the bar graphs below the Westerns indicate lower levels of H3K14ac in the Hmgn1^−/− cells. (B) Expression of wild type, but not mutant, HMGN1 protein elevates the levels of H3K14ac. Western analysis of extracts from Hmgn1^−/− cells, stably transfected with inducible plasmids expressing either HMGN1 or S20,24E HMGN1 mutant protein, which does not bind chromatin, under the control of the Tet promoter. Dox-induced expression of HMGN1, but not of S20,24E HMGN1 (verified by Western with anti-HMGN1 antibodies) raises the levels of H3K14ac. The relative signal intensities of H3K14ac, normalized to the intensities of total H3, are shown in the bar graphs below the Westerns.

Figure 2

HMGN1 elevates the levels of H3K14ac by stimulating HAT activity. (A) Kinetics of H3K14 acetylation in Hmgn1^−/− and Hmgn1^+/+ cells grown in the presence of the HDAC inhibitor TSA. Hmgn1^+/+ and Hmgn1^−/− fibroblasts were grown in the presence of TSA for various times. Western analyses of the histones extracted from these cells are shown on the right. The graph depicts the relative signal intensities of H3K14ac normalized to the intensities of total H3. Non-TSA-treated Hmgn1^+/+ (NT in the graph) cells are used as controls. The broken lines extrapolate the initial slopes of the curves. (B) Similar levels of HAT activities in Hmgn1^−/− and Hmgn1^+/+ cells. Cell extracts were immunoprecipitated with antibodies to either PCAF or p300, and the immunoprecipitates incubated with histone H3 and [¹⁴C]acetyl-CoA. The reaction mixtures were fractionated on 15% SDS–PAGE and the gels autoradiographed. Control reaction of H3 incubated with [¹⁴C]acetyl-CoA and with either purified p300, purified PCAF HAT domain, no enzymes, or immunoprecipitated with normal, non-immune antibody (N-IgG) are included. (C) The relative signal intensities of ¹⁴C-H3, normalized to the intensities of total H3, are shown in the bar graphs below.

Figure 3

HMGN1 enhances the acetylation of nucleosomal H3K14. (A) HMGN1 enhances the activity of the recombinant HAT domain of PCAF (r PCAF-HAT) Coomassie blue-stained 15% polyacrylamide SDS-containing gels, and corresponding autoradiograms (¹⁴C), of reaction mixtures containing equal amounts of chicken erythrocyte CPs, reconstituted with increasing amounts of HMGN1, and incubated with [¹⁴C]acetyl-CoA and r PCAF-HAT. Note the direct correlation between HMGN1 input and acetylation. The EtBr-stained panel depicts mobility shift assays of the reaction mixtures, on native 4% polyacrylamide gels in 2 × TBE. Note that, under these reaction conditions, HMGN1 bound cooperatively to CPs and formed a complex (CP+2HMGN1). (B) Western analysis of reaction mixtures performed as in (A) indicates that HMGN1 enhances the acetylation of H3K14. (C) HMGN1 stimulates the activity of PCAF complex. Reaction performed as in (A). (D) HMGN1 enhances the rPCAF-mediated acetylation of H3. Time course of the reaction of nucleosomes incubated either without HMGN1, or with a four-fold molar excess of HMGN1. (E) PCAF is the only active HAT in the complex. PCAF complex incubated with 0–250 μM concentration of H3-CoA-20, a specific inhibitor on PCAF. Note that the inhibitor abolished the acetylation of H3 in either the presence or absence of HMGN1. (F) Quantification of the radioactivity incorporated into H3 at various concentrations of inhibitor.

Figure 4

Effect of HMGN1 on PCAF-mediated H3 acetylation (A). Lineweaver–Burk plot showing the effect of HMGN1 on pCAF-mediated acetylation of nucleosome cores at a fixed concentration of [¹⁴C]acetyl-CoA (100 μM) and various concentrations of nucleosomes (0.006–1 μM) in either the presence or absence of HMGN1. For each point, the concentration of HMGN1 was adjusted to maintain a constant HMGN1:core nucleosome molar ratio. (B) Lineweaver–Burk plot showing the effect of HMGN1 on r-PCAF-mediated acetylation of H3 in nucleosome cores. HAT assays were carried out with a fixed concentration of nucleosomes (1 μM) and increasing concentrations of [¹⁴C]acetyl-CoA, in either the presence (4 μM) or absence of HMGN1. (C) Coomassie blue-stained 15% polyacrylamide SDS-containing gels and corresponding Westerns of reaction mixtures containing equal amounts of histone H3 incubated with increasing amounts of HMGN1, followed by incubation with [¹⁴C]acetyl-CoA and recombinant HAT domain of PCAF. Note that HMGN1 does not stimulate acetylation. (D) Binding HMGN1 to CP enhances H3 acetylation by either PCAF HAT domain (•) or PCAF complex (▪). HMGN1 does not enhance the acetylation of free histone H3 (▴). The relative acetylation levels were determined by quantifying the data in panels A and C in Figure 3 and panel C in Figure 4.

Figure 5

HMGN1 does not affect global acetylation and phosphoacetylation of histone H3 during IE gene induction. (A) HMGN1 enhances the levels of H3 phosphoacetylation in growing but not quiescent mouse fibroblasts. (B) Western analysis of the histone modifications analyzed with the antibodies indicated on the left during the course of anisomysin treatment of Hmgn1^+/+ and Hmgn1^−/− cells. (C) Plots depicting the time course of phosphoacetylation and phosphorylation of H3 in Hmgn1^+/+ or Hmgn1^−/− cells during anisomysin treatment.

Figure 6

Loss of HMGN1 alters the expression and modification levels of junD but not c-jun. (A) Real-time RT–PCR analysis of junD and c-jun expression during anisomysin treatment of Hmgn1^+/+ or Hmgn1^−/− cells. (B) HMGN1 enhances post-translational modification levels in junD, but not in c-jun. The relative enrichment of junD and c-jun sequences in the chromatin of anisomysin-treated Hmgn1^+/+ or Hmgn1^−/− cells is shown, which were immunoprecipitated with antibodies to either phosphoacetylated-H3 or H3K14ac. The immunoprecipitates were analyzed by quantitative real-time PCR with primers specific to the regions indicated in the schematic diagram of the gene. All time points refer to duration of anisomysin exposure.