The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro

Abstract

An in vitro reconstitution system for the analysis of replication-coupled nucleosome assembly is described. In this "two-step system," nucleosome assembly is performed in a separate reaction from DNA replication, wherein purified newly replicated DNA remains noncovalently marked for subsequent chromatin assembly factor-1 (CAF-1)-dependent nucleosome assembly. Because the nucleosome assembly is performed separately from the DNA replication step, this system is more versatile and biochemically tractable when compared with nucleosome assembly during simian virus 40 (SV40) DNA replication. The N-terminal domains of histones H3 and H4 play an important but redundant function in nucleosome assembly in the budding yeast, Saccharomyces cerevisiae. It had been proposed that at least one tail of histone H3 or H4 is required for replication-coupled nucleosome assembly. However, we demonstrate that the N-terminal domains of both histone H3 and H4 are dispensable for CAF-1-mediated formation of nucleosome cores onto newly replicated DNA in vitro. CAF-1 and each of its individual subunits stably bound to recombinant (H3.H4)(2) tetramers lacking the N-terminal domains of both H3 and H4. Therefore, the N-terminal tails of histone H3 and H4 that contain the specific acetylation sites are not necessary for CAF-1-dependent nucleosome assembly onto replicated DNA. We suggest that the histone acetylation may be required for a CAF-1 independent pathway or function after deposition, by marking of newly replicated chromatin.

Figure 1

Scheme of the two-step system for the analysis of replication-coupled nucleosome assembly. Newly replicated DNA purified by spin-column chromatography is competent for replication-dependent nucleosome assembly by CAF-1. In this vitro system, replication-dependent nucleosome assembly is analyzed in a completely separate reaction from the DNA replication reaction. The DNA replication products are first separated from the replication proteins, and then a second nucleosome assembly reaction is performed. To allow nucleosome assembly to proceed, the effluent from the spin column containing DNA is incubated with fresh S100 extract, CAF-1, and topoisomerase activities for various periods of time, followed by additional incubation with H2A.2B for 15 min. Because this is a reconstitution system, S100 extract can be replaced by H3.H4-depleted S100 extract and recombinant H3.H4 tetramers in some experiments (see Fig. 3B).

Figure 2

Biochemical analysis of CAF-1-dependent nucleosome assembly. (A) Nucleosome assembly in the two-step system. Replicated and semipurified DNA was subjected to supercoiling reactions with increasing amounts of CAF-1 either in the presence or the absence of S100 (56 μg), as indicated. (B) Nucleosome assembly during SV40 DNA replication. SV40 DNA replication reactions were performed with a large SV40 T-antigen and S100 (87.5 μg) in the presence of increasing amounts of CAF-1 for 45 min. (Upper) An autoradiograph and (Lower) an ethidium bromide-stained gel showing bulk DNA. Molar ratio between CAF-1 and total DNA (not replicated DNA) is indicated in A and B. Migration of relaxed, covalently closed circular DNA (form Io), nicked circular DNA (form II), and negatively supercoiled DNA (form I) are indicated.

Figure 3

N termini of H3.H4 are not required for CAF-1-dependent nucleosome assembly. (A) A Coomassie blue-stained gel. Four different H3.H4 tetramers (40 or 160 pmol) were loaded on 18% polyacrylamide gel, and stained with Coomassie blue. (B) CAF-1-dependent nucleosome assembly with recombinant (H3.H4)2. A large-scale SV40 DNA replication was performed with H3.H4-depleted human cell extract S100(−). After separation of DNA, supercoiling assay was performed with CAF-1 (50 fmol), S100(−) (48 μg), and increasing amounts of each recombinant (H3.H4)2, as indicated by + or −. However, each recombinant (H3.H4)₂ was preincubated at 4°C for 15 min with CAF-1, and subsequently with S100(−) at 37°C for 15 min. (Upper) An autoradiography and (Lower) an ethidium bromide-stained gel. Migrations of form I, Io, or II DNA are indicated as in Fig. 1.

Figure 4

N termini of H3.H4 are not required for association between CAF-1 and H3.H4. (A) Binding between histone H3.H4 and CAF-1. A total of 60 pmol of either wild-type or mutant (H3.H4)₂ tetramers was immunoprecipitated by anti-p60 antibody either in the presence or the absence of recombinant CAF-1 (30 pmol), as indicated by + or −. Precipitated proteins were loaded onto an 18% acrylamide gel, and analyzed by silver staining. (B) A competitive binding assay. A total of 300 pmol of wild-type or mutant (H3.H4)₂ tetramers were immunoprecipitated by anti-CAF-1 antibodies (anti-p150 antibody for lanes 2 and 3 or anti-p60 antibody for lanes 4 and 5), either in the presence or the absence of CAF-1 (150 pmol), as indicated by + or −. Precipitated proteins were analyzed in an 18% polyacrylamide gel and stained with Coomassie blue. 12.5% of input histones were loaded in lanes I. Three subunits of CAF-1 (p150, p60, and p48), Ig heavy chain (IgH) and light chain (IgL), and each histone H3 and H4 are indicated.

Figure 5

The N termini of H3.H4 are not required for association of p150 or p60 with H3.H4. (A) Binding of p60 to histones. A total of 50 pmol of recombinant p60 was incubated with excess (≈5 μg) GST-histones or GST-RFC (37-kDa subunit of human RFC) and pulled down with glutathione beads. Precipitated proteins were loaded on an 18% polyacrylamide gel and analyzed by Western blotting with anti-p60 monoclonal antibody. 25% of input histones (I) were loaded in lanes 1. (B) Competitive binding assay. A total of 50 pmol of wild-type or mutant (H3.H4)₂ tetramers were immunoprecipitated by anti-CAF-1 antibodies (anti-p150 antibody for lanes 2, 3, 8, and 9 or anti-p60 antibody for lanes 5 and 6), either in the presence or absence of recombinant p150, p150/p60 (25 pmol), or p60 (50 pmol), as indicated by + or −. Precipitated proteins were resolved in an 18% polyacrylamide gel and silver stained. 12.5% of input histones (I) were loaded in lane 1. p150, p60, and p48, Ig heavy chain (IgH) and light chain (IgL), and individual histone H3 and H4 are indicated.