In the study reported in Epigenetics and Chromatin, John Gurdon and colleagues describe the investigation of reprogramming that is independent of DNA replication, by transferring mammalian somatic cell nuclei into Xenopus oocytes, which are mitotically arrested, and following the resulting chromatin changes [4]. They focused on the incorporation of the histone variant H3.3, which is a hallmark of sites of high nucleosome turnover, and is associated with active genes and their regulatory elements [5,6]. When they microinjected mRNA encoding epitope-tagged H3.3 into the oocytes prior to nuclear transfer, they observed early incorporation of H3.3 into the pluripotency gene Oct4 coincident with the onset of transcription of the gene. To check the requirement for H3.3, they injected into the oocyte polyclonal antibodies against HIRA, the chaperone responsible for the incorporation of histone H3.3 into chromatin, and were able to show that this abrogates transcriptional reprogramming. They also showed, by the use of the polymerase II inhibitor alpha-amanitin, that H3.3 incorporation depends on transcriptional activity as well as HIRA. Impaired reprogramming in the absence of HIRA and H3.3 deposition could not be compensated for by the increased deposition of histone variant H3.2. These results imply that some specific function is attributable to the H3.3 deposition pathway in promoting reprogramming, and raises the question of what that function might be.

Reprogramming is far less efficient than differentiation and this may reflect the need for reprogramming factors to overcome changes to the chromatin environment that occur with differentiation. Embryonic stem cells are characterized by a highly dynamic chromatin state compared with that of more differentiated cell types [7], and pluripotency genes in particular have been shown to gain repressive chromatin marks during differentiation. It is, however, at these silenced sites that the reprogramming factors must bind to elicit their effects. Whilst c-Myc binding occurs early in the reprogramming process, Oct4, Sox2 and to a lesser extent Klf4, which co-occupy a large number of promoters, bind only later during reprogramming. Delayed binding of Oct4, Sox2 and Klf4, presumably because of the repressive chromatin environment at their binding sites, is thought to be a major roadblock in the reprogramming process.

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 ​(Figure1a1a).

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.