One obvious possibility is that the high turnover of H3.3-containing nucleosomes reflects some intrinsic instability. However, H2A/H3.3-containing nucleosomes seem to be just as stable in vitro
as H2A/H3.1-containing nucleosomes [8
]. Another possibility is that histone H3.3 accumulates at these high turnover sites as a gap-filling mechanism after the eviction of nucleosomes following transcription or the activity of chromatin remodelers. The observations of Gurdon and colleagues, however, indicate failure of histone variant H3.2 to compensate for the lack of H3.3 deposition, suggesting that there is more to H3.3 deposition in the reprogramming process than simple gap filling.
If there is nothing intrinsic to the amino acid sequence of H3.3 that favors a permissive chromatin state, then perhaps the deposition process itself is key. Perhaps, therefore, a specific post-translational modification of H3.3 might be associated with HIRA-dependent deposition, or an additional chromatin remodeling step at the point of H3.3 deposition might promote an active chromatin conformation and accessibility to the underlying DNA sequence. iPSC generation has been suggested to be a stochastic process [9
], which at a molecular level may reflect the stochastic nature of binding by the reprogramming factors to their target sequences due to transient exposure of these sites in the repressive chromatin environment [10
]. Increases in the deposition of H3.3 may therefore facilitate reprogramming by increasing the frequency of exposure of these binding sites (Figure ).
Figure 1 Opposing roles of histone H3.3 in reprogramming. (a) In somatic cells, pluripotency genes are in a repressive chromatin environment. Transient binding by a reprogramming factor to its binding site results in histone H3.3 incorporation, which in turn results (more ...)
A key question for future studies is whether the overexpression of HIRA and/or histone H3.3 may accelerate reprogramming and increase the efficiency of iPSC generation by allowing the remodeling of transcription factor binding sites. A second, related question is whether such overexpression might provide the necessary replication-independent chromatin remodeling, thereby reducing the requirement for c-Myc as a catalyst. This would be desirable because c-Myc, which is thought to aid reprogramming by increasing the rate of cell proliferation and thereby genome-wide chromatin remodeling, is a proto-oncogene; there are thus naturally concerns about its induced expression in the generation of iPSCs as a therapeutic agent.
There is, however, a potential pitfall in the overexpression of HIRA/H3.3 in reprogramming. Whilst it may allow the remodeling of repressive chromatin to promote gene activation, histone H3.3 has also been shown in an earlier study by Gurdon and colleagues [11
] to potentiate transcriptional memory (Figure ). In that study, using reprogramming via somatic cell nuclear transfer into enucleated Xenopus
oocytes, they showed that overexpression of histone H3.3 increased the frequency at which nuclei maintained their original transcriptional program, as determined by MyoD
expression. They have since suggested that the unusually high H3.3 content in eggs is responsible for this transcriptional memory [2
]. This may suggest the need for a delicate balance in the overexpression of HIRA and/or H3.3, in which factor binding will be facilitated through chromatin decondensation, but transcriptional memory will not be evoked. Given the evidence that transcriptional memory in iPSCs may cause some of the observed limitations to the regenerative applications of these cells, clearly the operation of the H3.3 pathway in both reprogramming and memory will need to be taken into account in any manipulation of that pathway for therapeutic purposes. Gurdon's latest work thus forges a connection, through the changes to chromatin required for reprogramming, between the two 2012 Nobel Prize-winning papers published almost a half century apart.