active pcaf hat domain  (Upstate Biotechnology Inc)

Journal:

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

doi: 10.1038/sj.emboj.7600768

Figure Lengend Snippet: 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.

Article Snippet: Materials Antibodies to H3K14ac, H3S10p, and phosphoacetylated H3 and active PCAF HAT domain were from Upstate Biotechnology.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Incubation, SDS Page, Purification

Journal:

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

doi: 10.1038/sj.emboj.7600768

Figure Lengend Snippet: 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.

Article Snippet: Materials Antibodies to H3K14ac, H3S10p, and phosphoacetylated H3 and active PCAF HAT domain were from Upstate Biotechnology.

Techniques: Activity Assay, Recombinant, Staining, Incubation, Mobility Shift, Western Blot, Concentration Assay, Radioactivity

Journal:

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

doi: 10.1038/sj.emboj.7600768

Figure Lengend Snippet: 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.

Article Snippet: Materials Antibodies to H3K14ac, H3S10p, and phosphoacetylated H3 and active PCAF HAT domain were from Upstate Biotechnology.

Techniques: Concentration Assay, Staining, Incubation, Recombinant, Binding Assay