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H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo.

Ejlassi-Lassallette A, Mocquard E, Arnaud MC, Thiriet C - Mol. Biol. Cell (2010)

Bottom Line: We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed.Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly.These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.

View Article: PubMed Central - PubMed

Affiliation: UMR-CNRS 6204, Dynamique de la chromatine et épigénétique, Faculté des sciences et des techniques, Université de Nantes, 44322 Nantes, France.

ABSTRACT
While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.

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Purification and analysis of the replication complex. (A) Experimental scheme of the procedure of purification of replication complex. (B) Silver-stained SDS–PAGE of input (Input), immunoprecipitated material (IP), and immunoprecipitated material in the presence of competitor bacterial DNA (Compet), respectively. The molecular mass markers are 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa, respectively. (C) Analysis of HAT1 in replication complex. Antibodies to HAT1 are used to reveal Western blot of untreated cell fractions (Untreated), bound (IP) and unbound (UN) fractions from chromatin untreated with BrdU (No BrdU), BrdU immunoprecipitated chromatin (IP BrdU), and PCNA immunoprecipitated chromatin in conditions identical to IP BrdU (IP PCNA), respectively. Densitometric profiles of immunostained HAT1 band are shown.
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Figure 5: Purification and analysis of the replication complex. (A) Experimental scheme of the procedure of purification of replication complex. (B) Silver-stained SDS–PAGE of input (Input), immunoprecipitated material (IP), and immunoprecipitated material in the presence of competitor bacterial DNA (Compet), respectively. The molecular mass markers are 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa, respectively. (C) Analysis of HAT1 in replication complex. Antibodies to HAT1 are used to reveal Western blot of untreated cell fractions (Untreated), bound (IP) and unbound (UN) fractions from chromatin untreated with BrdU (No BrdU), BrdU immunoprecipitated chromatin (IP BrdU), and PCNA immunoprecipitated chromatin in conditions identical to IP BrdU (IP PCNA), respectively. Densitometric profiles of immunostained HAT1 band are shown.

Mentions: To verify whether Hat1 was only involved in nuclear import of H3/H4 or was also critical for supplying newly synthesized histones to assembly factors at replication forks, we developed a novel approach for preparing proteins in the vicinity of the replication forks. Based on the principle that factors involved in replication might be immunoprecipitated with newly synthesized DNA, we carried out short bromodeoxyuridine (BrdU) pulses of Physarum cells at the beginning of S-phase. Nuclei were then prepared and fixed with formaldehyde before shearing chromatin by sonication, similarly to chromatin immunoprecipitation experiments (Figure 5A). Soluble chromatin was irradiated with UV to induce DNA breaks and to make more accessible BrdU to specific antibody. Recently, Dejardin and Kingston (2009) have reported a related approach for examining the protein composition in telomeric chromatin using specific sequences for IP, although we focused not on specific sequences but on newly replicated DNA. To ensure that the IPs were specific for replicating chromatin, we carried out experiments of IP in the presence of an excess of competitor BrdU–labeled bacterial DNA. Clearly, the results revealed that immunoprecipitated peptides were not recovered in the presence of competitor BrdU–containing DNA, demonstrating that visualized peptides in SDS–PAGE were associated with BrdU-labeled chromatin (Figure 5B). Then we wanted to examine whether this approach allowed the recovery of known marks of replication. Western blot analyses of immunoprecipitated replicating chromatin revealed, as expected, the presence of a ∼30-kDa band with anti–proliferating cell nuclear antigen (PCNA) antibody (unpublished data). To determine whether Hat1 was associated with BrdU-containing chromatin, we repeated the IP experiments and analyzed by Western blot with anti-Hat1 antibodies (Figure 5C). A band of the expected molecular mass and corresponding to Hat1 was visualized in BrdU chromatin IP, which was not detected in control IP untreated with the thymidine analogue. However, the appearance was fuzzy, compared with Hat1 signal from nuclear fractions, most likely due to the chemical and heat treatments. Similar IP experiments carried out with anti-PCNA antibody showed that Hat1 coimmunoprecipitated with this replication factor (Figure 5C). These results showed that Hat1 can localize in the vicinity of replication sites during S-phase, possibly for delivering newly synthesized histones to chromatin assembly factors.FIGURE 5:


H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo.

Ejlassi-Lassallette A, Mocquard E, Arnaud MC, Thiriet C - Mol. Biol. Cell (2010)

Purification and analysis of the replication complex. (A) Experimental scheme of the procedure of purification of replication complex. (B) Silver-stained SDS–PAGE of input (Input), immunoprecipitated material (IP), and immunoprecipitated material in the presence of competitor bacterial DNA (Compet), respectively. The molecular mass markers are 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa, respectively. (C) Analysis of HAT1 in replication complex. Antibodies to HAT1 are used to reveal Western blot of untreated cell fractions (Untreated), bound (IP) and unbound (UN) fractions from chromatin untreated with BrdU (No BrdU), BrdU immunoprecipitated chromatin (IP BrdU), and PCNA immunoprecipitated chromatin in conditions identical to IP BrdU (IP PCNA), respectively. Densitometric profiles of immunostained HAT1 band are shown.
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Related In: Results  -  Collection

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Figure 5: Purification and analysis of the replication complex. (A) Experimental scheme of the procedure of purification of replication complex. (B) Silver-stained SDS–PAGE of input (Input), immunoprecipitated material (IP), and immunoprecipitated material in the presence of competitor bacterial DNA (Compet), respectively. The molecular mass markers are 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa, respectively. (C) Analysis of HAT1 in replication complex. Antibodies to HAT1 are used to reveal Western blot of untreated cell fractions (Untreated), bound (IP) and unbound (UN) fractions from chromatin untreated with BrdU (No BrdU), BrdU immunoprecipitated chromatin (IP BrdU), and PCNA immunoprecipitated chromatin in conditions identical to IP BrdU (IP PCNA), respectively. Densitometric profiles of immunostained HAT1 band are shown.
Mentions: To verify whether Hat1 was only involved in nuclear import of H3/H4 or was also critical for supplying newly synthesized histones to assembly factors at replication forks, we developed a novel approach for preparing proteins in the vicinity of the replication forks. Based on the principle that factors involved in replication might be immunoprecipitated with newly synthesized DNA, we carried out short bromodeoxyuridine (BrdU) pulses of Physarum cells at the beginning of S-phase. Nuclei were then prepared and fixed with formaldehyde before shearing chromatin by sonication, similarly to chromatin immunoprecipitation experiments (Figure 5A). Soluble chromatin was irradiated with UV to induce DNA breaks and to make more accessible BrdU to specific antibody. Recently, Dejardin and Kingston (2009) have reported a related approach for examining the protein composition in telomeric chromatin using specific sequences for IP, although we focused not on specific sequences but on newly replicated DNA. To ensure that the IPs were specific for replicating chromatin, we carried out experiments of IP in the presence of an excess of competitor BrdU–labeled bacterial DNA. Clearly, the results revealed that immunoprecipitated peptides were not recovered in the presence of competitor BrdU–containing DNA, demonstrating that visualized peptides in SDS–PAGE were associated with BrdU-labeled chromatin (Figure 5B). Then we wanted to examine whether this approach allowed the recovery of known marks of replication. Western blot analyses of immunoprecipitated replicating chromatin revealed, as expected, the presence of a ∼30-kDa band with anti–proliferating cell nuclear antigen (PCNA) antibody (unpublished data). To determine whether Hat1 was associated with BrdU-containing chromatin, we repeated the IP experiments and analyzed by Western blot with anti-Hat1 antibodies (Figure 5C). A band of the expected molecular mass and corresponding to Hat1 was visualized in BrdU chromatin IP, which was not detected in control IP untreated with the thymidine analogue. However, the appearance was fuzzy, compared with Hat1 signal from nuclear fractions, most likely due to the chemical and heat treatments. Similar IP experiments carried out with anti-PCNA antibody showed that Hat1 coimmunoprecipitated with this replication factor (Figure 5C). These results showed that Hat1 can localize in the vicinity of replication sites during S-phase, possibly for delivering newly synthesized histones to chromatin assembly factors.FIGURE 5:

Bottom Line: We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed.Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly.These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.

View Article: PubMed Central - PubMed

Affiliation: UMR-CNRS 6204, Dynamique de la chromatine et épigénétique, Faculté des sciences et des techniques, Université de Nantes, 44322 Nantes, France.

ABSTRACT
While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.

Show MeSH