In this work, we document a surprisingly rapid and wide-spread exchange of histones H3/H4 between nucleosomes and unassembled, free histone pools during early G2-phase and show that this exchange likely requires specific acetylation of H4. Histones acetylated at lysines 8 and 16 are preferentially located in the free pool and installation of acetylation mimics at these positions in H4 significantly enhances exchange of exogenous proteins. These results point to a model whereby specific H4 acetylation marks histones for rapid exchange out of nucleosomes as a mechanism for increasing accessibility of specific DNA sequences ().
Figure 5. Model of the acetylation-dependent nucleosome turn over. The model proposes that histone acetylation by HATs within euchromatin induces the displacement of the nucleosome from chromatin, making accessible the genetic information. The unassembled acetylated (more ...)
Like the distribution found in human cell cultures, our experiments show that Physarum
nuclear histones are partitioned into two pools; the vast majority of nuclear histones are assembled into chromatin, whereas a small fraction exists in an unassembled pool ( and A). These results are consistent with FRAP analyses of human cells wherein a subpopulation of H3 (<16%) was found to diffuse freely (41
), and the finding of a small fraction of nuclear core histones is associated with chaperones and not assembled into chromatin (40
). Moreover, we find that despite introduction of trace quantities of exogenous histones, small increases in the amounts introduced into the cell result in the exogenous histones being preferentially partitioned to the unassembled nuclear pool. These results suggest that the amount of histone produced by the cell closely matches the capacity for assembly into chromatin during S-phase. Hence, a fine balance between histone supply and demand is maintained during S-phase (25
). Thus, we are able to control the amount of H3/FH4 in the free pool by modest increases in the amount of proteins applied to the cell during S-phase.
In absence of obvious degradation of the exogenous histones throughout the G2
-phase, we observed that the apparent exchange in early G2
-phase depended on the amounts of the exogenous histone introduced into the cells and their partitioning in the nucleus. Indeed, introduction of the lowest amount of exogenous histones we used, resulted in their near-complete assembly into chromatin during S-phase. In this case, we detected the eviction of chromatin-associated tagged proteins in early G2
-phase. In contrast, when higher amounts of exogenous histones were introduced, a greater fraction was partitioned to the free pool, and the amount of tagged protein associated with chromatin actually increased during G2
under some conditions. These results suggest that the two pools of nuclear histones are in constant flux, and the apparent replication-independent exchange is affected by the amount of tagged histones available in the free pool. We previously observed rapid H2A/H2B dimer exchange associated with actively transcribed regions in Physarum
, whereas H3/H4 exchanged with proteins in the free pool at much slower rate (22
). Thus, our finding that levels of H3/H4 associated with chromatin remain constant when ‘mid’ amounts of histones are introduced () may be because of equilibration of tagged exogenous protein between the two nuclear histone pools. This steady state may also be reached after extended periods when the constant flux of histone leads to the equilibrium of target histone within the two pools (, T0
). Alternatively exchange may be a more active and prevalent feature in early G2
(). In this model, nucleosomes early G2
still retain deposition-related acetylation or are present in ‘open’ chromatin regions, more exposed to exchange machinery (46
), whereas mature chromatin in late G2
exhibits much lower levels of exchange ().
Importantly, inspection of the acetylation state of nuclear proteins indicated that the vast excess of acetylated H4 was present within the Triton-extractable, free pool. As the pan-acetyl H4 antibody reacts most strongly with hyper-acetylated protein, we tested acetylation at individual sites with specific antibodies. Interestingly, we find that while acetylation at lysines 5 or 12 is associated with H4 stably assembled into chromatin, acetylation of lysines 8 and 16 is preferentially represented in the free histone pool (A). These results are consistent with a role in chromatin assembly for H4 acetylated at lysines 5 and 12 (38
). Interestingly, in yeast substitution of H4 lysine 16 with arginine indicates a critical function in acetylation at this position in transcription of chromatin (48
). This H4 lysine 16 acetylation plays an important role in Drosophila
dosage compensation, as this specific acetylation mark is concomitant with the RNA polymerase II recruitment (49
). In these experiments, the H4 acetylation has been examined by ChIP, which provides information on the location of the modification within the genome, but did not examine the relative abundance of the acetylation mark within the assembled and free pools of histones. Our analyses revealed that the lysine 16 acetylation of H4 is mainly found in the free pool, suggesting that this modification plays a key role in eviction of core histones. Consistently, it has shown that H4 acetylation at lysine 8 and 16 facilitates H2A/H2B dimer exchange, although in these experiments, H3/H4 tetramer displacement was not investigated and could also be exchanged (50
). Moreover, we find that installation of glutamine substitutions as mimics of acetylated lysine at all four positions of the H4-tail domain greatly stimulated the rate and extent of histone exchange compared with the native proteins, whereas H4-containing mimics of acetylation at K5 and K12 induced only low levels of exchange (B). These results indicate that the stimulation in histone exchange observed with the tetraacetyl mimic is because of additional acetylation on K8 and/or K16 (40
). Together these results indicate that specific acetylation at K8/K16 within the H4 tail marks nucleosomes for rapid exchange. This model predicts that incorporation of a mutant H4 in which the four acetylable lysines are substituted for arginine would result in a protein that undergoes much less frequent exchange. Unfortunately, this hypothesis cannot be tested in our system, as H3/FH4 K5,8,12,16Q tetramers are not efficiently transported into Physarum
nuclei and assembled into chromatin, likely because of the role of H4 K5,12 acetylation in these processes (33
It is well-established that transcription activity coincides with histone acetylation. Interestingly, our results showed that epigenetic marks of transcription, such as the histone variant H3.3 and acetylated H4, are preferentially recovered within the unassembled pool of histones (A and A). Moreover, HATs and HDACs are preferentially co-localized at active regions within yeast and human genomes (9
). Thus, we propose that a recycling mechanism occurs, wherein specific acetylation induces histone displacement, whereas rapid deacetylation occurs after re-deposition of histones into chromatin from the free pool (). Recent genome-wide analyses in yeast and Drosophila
revealed that histone acetylation promotes nucleosome turn over, suggesting that this model is likely conserved throughout eukaryotes (49
). It will be interesting to determine whether other reversible histone modifications within the nucleosome influence rates of nucleosome eviction and histone deposition.