p300-mediated acetylation facilitates the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone

Abstract

We have used a purified recombinant chromatin assembly system, including ACF (Acf-1 + ISWI) and NAP-1, to examine the role of histone acetylation in ATP-dependent chromatin remodeling. The binding of a transcriptional activator (Gal4-VP16) to chromatin assembled using this recombinant assembly system dramatically enhances the acetylation of nucleosomal core histones by the histone acetyltransferase p300. This effect requires both the presence of Gal4-binding sites in the template and the VP16-activation domain. Order-of-addition experiments indicate that prior activator-meditated, ATP-dependent chromatin remodeling by ACF is required for the acetylation of nucleosomal histones by p300. Thus, chromatin remodeling, which requires a transcriptional activator, ACF and ATP, is an early step in the transcriptional process that regulates subsequent core histone acetylation. Glycerol gradient sedimentation and immunoprecipitation assays demonstrate that the acetylation of histones by p300 facilitates the transfer of H2A-H2B from nucleosomes to NAP-1. The results from these biochemical experiments suggest that (1) transcriptional activators (e.g., Gal4-VP16) and chromatin remodeling complexes (e.g., ACF) induce chromatin remodeling in the absence of histone acetylation; (2) transcriptional activators recruit histone acetyltransferases (e.g., p300) to promoters after chromatin remodeling has occurred; and (3) histone acetylation is important for a step subsequent to chromatin remodeling and results in the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone such as NAP-1. Our results indicate a precise role for histone acetylation, namely to alter the structure of nucleosomes (e.g., facilitate the loss of H2A-H2B dimers) that have been remodeled previously by the action of ATP-dependent chromatin remodeling complexes. Thus, transcription from chromatin templates is ordered and sequential, with precise timing and roles for ATP-dependent chromatin remodeling, subsequent histone acetylation, and alterations in nucleosome structure.

Figure 1

The binding of Gal4–VP16 to chromatin templates assembled with recombinant ACF and NAP-1 facilitates the acetylation of nucleosomal core histones by p300. (A) SDS-polyacrylamide gel analysis of purified recombinant p300, NAP-1, and ACF. Affinity-tagged proteins were expressed in Sf9 cells by infection with recombinant baculoviruses. The proteins were purified by affinity or traditional chromatography as described in Materials and Methods. (B) The binding of Gal4–VP16 facilitates the acetylation of nucleosomal core histones by p300. In lanes 1–4, free core histones were incubated with p300 and ³H-acetyl CoA in the presence or absence of the factors indicated. In lanes 5 and 6, chromatin assembled with ACF and NAP-1 was incubated with p300 and ³H-acetyl CoA in the presence or absence of the factors indicated. The samples were analyzed on 12% polyacrylamide gels and the acetylated core histones were detected by fluorography. (C) Digestion of chromatin with micrococcal nuclease (MNase) increases the acetylation of nucleosomal core histones by p300. Chromatin templates assembled with ACF and NAP-1 were incubated with increasing amounts of MNase for 10 min prior to the addition of EDTA to stop the digestion reactions. The extent of MNase digestion was determined by deproteinizing aliquots of the chromatin and analyzing the DNA fragments on ethidium bromide-stained agarose gels (top). Gal4–VP16 and p300 plus ³H-acetyl CoA were added to aliquots of digested chromatin as indicated and incubated for 1 hr (see schematic, bottom). The samples were analyzed on 12% polyacrylamide gels and the acetylated core histones were detected by fluorography (middle).

Figure 2

The acetylation of nucleosomal core histones by p300 requires Gal4-binding sites in the template and the VP16 activation domain. Plasmid DNA templates were assembled into chromatin by ACF and NAP-1. After a 4 hr incubation to allow for the completion of chromatin assembly, Gal4–VP16 or Gal4(1-147) (i.e., the Gal4 DNA-binding domain alone), as well as increasing amounts of purified recombinant p300, were added as indicated in the presence ³H-acetyl CoA. The samples were analyzed on 12% polyacrylamide gels and the acetylated core histones were detected by fluorography. (A) Experiments using a plasmid (pIE0) lacking Gal4 binding sites. (B) Experiments using a plasmid (pGIE0) containing five Gal4 binding sites.

Figure 3

Prior activator-mediated, ATP-dependent chromatin remodeling is required for the efficient acetylation of nucleosomal core histones by p300. (A) Activator-dependent acetylation of nucleosomal histones by p300. The pGIE0 plasmid was assembled into chromatin by ACF and NAP-1 in the presence ³H-acetyl CoA. After a 4 hr incubation to allow for the completion of chromatin assembly, Gal4–VP16 was added to one set of samples as indicated. At different time points relative to the start of chromatin assembly, purified recombinant p300 was added as indicated, followed by a 30 min incubation (see schematic, bottom). The samples were analyzed on 12% polyacrylamide gels and the acetylated core histones were detected by fluorography (top). (B) ATP-dependent chromatin remodeling is required for the efficient acetylation of nucleosomal core histones by p300. The pGIE0 plasmid was assembled into chromatin by ACF and NAP-1. After a 3.5 hr incubation to allow for the completion of chromatin assembly, apyrase was added as indicated. Subsequently, ³H-acetyl CoA was added, as well as p300 and Gal4–VP16 as indicated (see schematic, bottom). The samples were analyzed on 12% polyacrylamide gels and the acetylated core histones were detected by fluorography (top).

Figure 4

p300 facilitates the transfer of H2A–H2B from nucleosomes to NAP-1 in the presence of acetyl CoA. The pGIE0 plasmid was assembled into chromatin by ACF and NAP-1. After a 3 hr incubation, Gal4–VP16 (125 nm), p300 (100 nm), and unlabeled acetyl CoA (30 μm) were added as indicated, followed by incubation with the chromatin templates for 1 hr. The samples were then loaded on a 15%–40% glycerol gradient and sedimented at 60 k rpm for 3 hr at 4°C in a SW60 (Beckman) rotor. Fractions were collected from the gradients, separated on 12% polyacrylamide-SDS gels, and analyzed by Western blotting with antibodies against NAP-1, Gal4–VP16, and core histones using an ¹²⁵I protein A detection method. The presence of plasmid DNA in the fractions was analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (A) Without p300, with acetyl CoA. (B) With p300 and acetyl CoA. (C) With p300, without acetyl CoA.

Figure 5

Acetylated H2A–H2B interacts with NAP-1. Coimmunoprecipitation of NAP-1 and H2A–H2B acetylated by p300 in the presence of Gal4–VP16. The second fractions from the top of the gradients shown in Figure 4B were analyzed by coimmunoprecipitation with preimmune serum and NAP-1 antiserum. The resulting immunoprecipitates were analyzed by 12% polyacrylamide-SDS gel electrophoresis followed by Western blotting for core histones (¹²⁵I protein A detection method) and NAP-1 (enhanced chemiluminescent detection method).

Figure 6

Model for histone transfer. ATP-dependent chromatin remodeling precedes histone acetylation by p300. Post-remodeling histone acetylation facilitates the transfer of histone H2A–H2B dimers to a histone chaperone.