Limits...
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.

Show MeSH
Histone H4 acetylation is required for nuclear import and facilitates chromatin assembly. (A) Mutations of lysine residues 5 and 12 of H4. Scheme represents the substitutions of lysines 5 and 12 to glutamine (Q) to mimic acetylation and to arginine (R) to prevent acetylation. (B) Cellular localization of the different histone complexes. Exogenous histones were incorporated for 1 h in early S-phase, similarly to Figure 1, and cells were fractionated before electrophoretic analyses of the fractions. (C) Chromatin assembly analyses of exogenous mutant complexes. Chromatin is prepared from nuclear fractions obtained after exogenous histone complex treatment, H3/FH4, H3/FH4-Q5/12, and H3/FH4-R5/12, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3020919&req=5

Figure 3: Histone H4 acetylation is required for nuclear import and facilitates chromatin assembly. (A) Mutations of lysine residues 5 and 12 of H4. Scheme represents the substitutions of lysines 5 and 12 to glutamine (Q) to mimic acetylation and to arginine (R) to prevent acetylation. (B) Cellular localization of the different histone complexes. Exogenous histones were incorporated for 1 h in early S-phase, similarly to Figure 1, and cells were fractionated before electrophoretic analyses of the fractions. (C) Chromatin assembly analyses of exogenous mutant complexes. Chromatin is prepared from nuclear fractions obtained after exogenous histone complex treatment, H3/FH4, H3/FH4-Q5/12, and H3/FH4-R5/12, respectively.

Mentions: It has been well documented that newly synthesized H3/H4 histones exhibit an evolutionarily conserved pattern of acetylation, especially histone H4, which is acetylated on lysines 5 and 12 (Sobel et al., 1995; Annunziato and Hansen, 2000). Interestingly, we found that lacking the H4 tail domain of H3/H4 impedes the nuclear recovery of the complex. We assumed that the absence of accumulation of the complex within the nucleus might be caused by the loss of replication-dependent acetylable residues within the H3/H4n complex. To verify this hypothesis, we generated histone mutants by substituting K5 and K12 for glutamine (FH4-Q5/12) and for arginine (FH4-R5/12), which are commonly used for mimicking acetylated lysine and unacetylated lysine, respectively (Figure 3A) (Megee et al., 1990; Blackwell et al., 2007; Wang and Hayes, 2008). Histone mutant complexes were then purified and incorporated into Physarum at the beginning of S-phase for 1 h (Figure 3B). Importantly, incorporation analyses revealed that mimicking replication-dependent acetylation at positions 5 and 12 of H4 (FH4-Q5/12) improved nuclear import of H3/H4 complex compared with the wild type. In contrast, preventing acetylation of H4 at lysines 5 and 12 (FH4-R5/12) inhibited nuclear import of the histone complex. Furthermore, the failure to recover the exogenous FH4-R5/12 by Western blot following incorporation and by anti-FLAG IP of the cytoplasmic and nuclear soluble fractions suggested that the FH4-R5/12–containing complex was degraded (Supplemental Figure S2). Chromatin assembly was then examined to determine whether acetylation at positions 5 and 12 of H4 affects histone deposition into chromatin (Figure 3C). The results showed that assembly of histone mutants reflects the efficiency of nuclear import of these histone complexes. These results strongly suggested that acetylation at positions 5 and 12 facilitates nuclear import of histones and chromatin assembly in vivo.FIGURE 3.


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)

Histone H4 acetylation is required for nuclear import and facilitates chromatin assembly. (A) Mutations of lysine residues 5 and 12 of H4. Scheme represents the substitutions of lysines 5 and 12 to glutamine (Q) to mimic acetylation and to arginine (R) to prevent acetylation. (B) Cellular localization of the different histone complexes. Exogenous histones were incorporated for 1 h in early S-phase, similarly to Figure 1, and cells were fractionated before electrophoretic analyses of the fractions. (C) Chromatin assembly analyses of exogenous mutant complexes. Chromatin is prepared from nuclear fractions obtained after exogenous histone complex treatment, H3/FH4, H3/FH4-Q5/12, and H3/FH4-R5/12, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020919&req=5

Figure 3: Histone H4 acetylation is required for nuclear import and facilitates chromatin assembly. (A) Mutations of lysine residues 5 and 12 of H4. Scheme represents the substitutions of lysines 5 and 12 to glutamine (Q) to mimic acetylation and to arginine (R) to prevent acetylation. (B) Cellular localization of the different histone complexes. Exogenous histones were incorporated for 1 h in early S-phase, similarly to Figure 1, and cells were fractionated before electrophoretic analyses of the fractions. (C) Chromatin assembly analyses of exogenous mutant complexes. Chromatin is prepared from nuclear fractions obtained after exogenous histone complex treatment, H3/FH4, H3/FH4-Q5/12, and H3/FH4-R5/12, respectively.
Mentions: It has been well documented that newly synthesized H3/H4 histones exhibit an evolutionarily conserved pattern of acetylation, especially histone H4, which is acetylated on lysines 5 and 12 (Sobel et al., 1995; Annunziato and Hansen, 2000). Interestingly, we found that lacking the H4 tail domain of H3/H4 impedes the nuclear recovery of the complex. We assumed that the absence of accumulation of the complex within the nucleus might be caused by the loss of replication-dependent acetylable residues within the H3/H4n complex. To verify this hypothesis, we generated histone mutants by substituting K5 and K12 for glutamine (FH4-Q5/12) and for arginine (FH4-R5/12), which are commonly used for mimicking acetylated lysine and unacetylated lysine, respectively (Figure 3A) (Megee et al., 1990; Blackwell et al., 2007; Wang and Hayes, 2008). Histone mutant complexes were then purified and incorporated into Physarum at the beginning of S-phase for 1 h (Figure 3B). Importantly, incorporation analyses revealed that mimicking replication-dependent acetylation at positions 5 and 12 of H4 (FH4-Q5/12) improved nuclear import of H3/H4 complex compared with the wild type. In contrast, preventing acetylation of H4 at lysines 5 and 12 (FH4-R5/12) inhibited nuclear import of the histone complex. Furthermore, the failure to recover the exogenous FH4-R5/12 by Western blot following incorporation and by anti-FLAG IP of the cytoplasmic and nuclear soluble fractions suggested that the FH4-R5/12–containing complex was degraded (Supplemental Figure S2). Chromatin assembly was then examined to determine whether acetylation at positions 5 and 12 of H4 affects histone deposition into chromatin (Figure 3C). The results showed that assembly of histone mutants reflects the efficiency of nuclear import of these histone complexes. These results strongly suggested that acetylation at positions 5 and 12 facilitates nuclear import of histones and chromatin assembly in vivo.FIGURE 3.

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