Our results support a model where nucleosome unwrapping during elongation exposes the histones so that they dissociate from the core octamer unless they interact with another segment of DNA. We propose that because Pol II sharply bends the DNA, it positions the exposed histones very close to the DNA immediately upstream of the polymerase, thus mediating histone transfer to the same DNA molecule via looping. This positioning hypothesis has been proposed before18,19
and explains both the small size of the loops that allow histone transfer by bridging upstream and downstream DNA, and the small upstream shift in the position of transcribed nucleosomes. Note that our observation that only a minority of nucleosomes change position after transcription may be influenced by our use of a strong NPS that could bias the histones to transfer and rewrap at the same location as before transcription. The total percentage of shifted nucleosomal particles (hexamer and octamers together) decrease slightly at the NTP concentration is lowered (Supplementary Table 2
). Presumably, the slower transcription is more likely to allow the histones to equilibrate on their original position during rewrapping.
In this model of nucleosomal transcription, faster transcription leads to faster overall nucleosome unwrapping, favouring histone dissociation. However, other factors affecting the rewrapping of the histones could influence the outcome of the competition. For example, a trailing polymerase blocking access of the unwrapped histones to upstream DNA15,35
and histone mutations that destabilize histone-DNA wrapping36
were both shown to inhibit histone transfer and to promote histone dissociation in vitro
, as expected according to our model.
This competition model also explains why faster polymerases produce a mix of octamers and bare DNA but yield little or no hexamers upon transcription. For instance, in vitro,
the majority of Pol III complexes complete transcription through a nucleosome in approximately 30 seconds12
(, vertical black line), so we predict that octamer transfer is likely – approximately 40%, while bare DNA production should be about 50%. To obtain hexasomes, on the other hand, two slow processes have to occur before Pol III can finish transcription: dimer dissociation and histone transfer, making the probability of hexamer transfer very unlikely, approximately 10%, under these fast transcription conditions. Note that here we only consider histone transfer within the same DNA molecule and do not include the probability of histone rebinding to other DNA molecules after complete dissociation. These predictions match previous experimental studies with Pol III reporting ~50% octamers and ~50% bare DNA12
in the presence of competitor DNA, when only transfer in cis is measured.
Moreover, in vitro
transcription by the even faster SP6 polymerase also leads to the formation of octamers and bare DNA, without hexamer formation, with the percentage of bare DNA increasing as the speed of elongation is increased11
. Since we estimate that SP6 RNAP is faster than 5bp s−1
, our model predicts that the outcome of transcription should be dominated by bare DNA. This prediction might seem contradictory at first with the experimental results, where much lower levels of bare DNA were observed, especially in the absence of competitor DNA11
. However, our model only considers transfer of the histones in cis (within the same DNA molecule). While for Pol II this is the prevalent scenario, we believe that for faster polymerases (such as SP6) a lot of the histone transfer happens in trans. Since Pol II moves slower, the histones have time to equilibrate with the DNA upstream (which is at a higher local concentration than other pieces of DNA). In contrast, for faster polymerases, the histone octamer detaches quickly, and since it’s floating free in solution, it is now just as likely to bind to any piece of DNA (in cis or trans). This interpretation is supported by the observation that for the SP6 polymerase, addition of competitor DNA to the reaction increases the amount of bare transcribed DNA11
. Moreover, it appears that transfer in trans is seen at higher NTPs concentrations, while at lower NTPs, transfer in cis dominates, in agreement with our model. However, a quantitative comparison with data obtained for the SP6 RNAP is difficult, since there are multiple – and unknown – transcription rounds for each DNA molecule, leading to a higher probability of complete histone dissociation than described by our model. In addition, since we propose that the geometry of the elongation complex influences histone transfer, we expect that polymerases of significantly different sizes and structures could lead to different positions distributions and transfer probabilities of the transcribed nucleosomes; the importance of these effects remains to be tested.
Gene regulation in vivo
may result from the modification of any one of the competing rates involved in elongation on a nucleosomal template. While we use a DNA sequence with a higher affinity for the nucleosome than other naturally occurring sequences, we predict that transcription through a weaker nucleosome is faster (an increased kue
rate, because of higher probability of finding the nucleosome locally unwrapped17
) and the transfer probability decreases (because of lowered rewrapping rates of histones to the upstream DNA). Both these effects would result in higher percentage of bare DNA and hexasomes formations after transcription of weaker positioning sequences. More importantly, elongation factors that increase the net transcription rate of Pol II through the nucleosome would result in an increased probability of complete histone removal from DNA. Alternatively, dimer dissociation from the partially unwrapped octamer could be faster for certain histone variants of H2A37
or in the presence of histone chaperones that bind the dimer, increasing the probability of hexasome formation, as has been shown in vitro38
. Such transcription-induced alterations in chromatin structure may affect gene expression in vivo
by reducing or eliminating nucleosomal barriers for future transcription elongation events in a similar manner to results obtained in vitro15,18
, or by altering the accessibility of transcription factor binding sites39
. Finally, we point out that the findings communicated here might also be relevant to other processes that involve advancement of molecular motors through DNA wrapped in nucleosomes, such as processive DNA replication and chromatin remodeling40