The data reported in this work demonstrate that in vitro mH2A is an efficient repressor of p300- and Gal4-VP16-dependent Pol II-activated transcription. We found that this property of mH2A resides mainly in its NHR domain. Indeed, our experiments show that mH2A and the fusion H2A-NHR were able to impede both Gal4-VP16-dependent Pol II-activated transcription and histone acetylation as well as nucleosome remodeling by SWI/SNF and ACF. Since the presence of mH2A was found to affect weakly the efficiency of Gal4-VP16 binding to the mH2A nucleosomes, the contribution of this effect to the repression of transcription is expected to be small. Bearing in mind that Gal4-VP16 is responsible for the recruitment of p300 to the promoter (2
) and that Gal4-VP16 is able to invade mH2A nucleosomes, one could hypothesize that the NHR domain of mH2A is involved in the impediment of histone tail acetylation by p300. The mechanism of this impediment is presently unknown. NHR does not exhibit histone deacetylase activity (1
), suggesting that the involvement of NHR in the impediment of histone acetylation is rather steric.
Interestingly, the H2A-like domain of mH2A does not affect p300- and Gal4-VP16-dependent Pol II-activated transcription (this work) but interferes with SWI/SNF nucleosome mobilization (4
). Thus, mH2A exhibits some redundancy in function with respect to nucleosome remodeling since each individual domain of mH2A (either H2A-like or NHR, when fused to H2A) was able to impair nucleosome remodeling.
We speculate that in vivo mH2A could contribute to the repression of transcription by affecting at least two different pathways: histone acetylation and chromatin remodeling. Since these two events, i.e., histone acetylation and nucleosome remodeling, are essential for the activation of transcription, it appears that mH2A could be viewed as a major stopper of transcriptional activation. Interestingly, the efficiencies of Pol II passage through conventional H2A, mH2A, and fusion H2A-NHR nucleosomes were essentially the same for the three types of particles. This suggests that the presence of a positioned single mH2A nucleosome on the promoter of specific genes could be sufficient to impede transcription activation by repressing the initiation of transcription.
Our data suggest that the interference of the NHR domain with histone acetylation through steric hindrance would be one of the reasons for this repression. In addition, as shown in a recent report, NHR specifically interacts with HDAC1,2 (11
). Consequently, the NHR domain could interfere with the ability of HAT to acetylate the histones of the promoter associated with the macroH2A nucleosome, and in addition, it could recruit histone deacetylase, which further abrogates the possibility of histone acetylation.
One cannot exclude, however, that the presence of several mH2A nucleosomes, some of which reside on the gene coding region, would affect transcription more efficiently. Indeed, the structure of chromatin domains which contain mH2A could be distinct from the 30-nm fiber canonical structure, which in turn might be more refractive to transcription.
Our finding that mH2A behaves as a major stopper of Pol II activation of transcription in vitro raises several questions, since to fulfill such function in vivo, mH2A should be localized specifically on the promoter of transcriptionally inactive genes. The presence of mH2A on such genes would repress transcription. For the transcriptional activation of these genes, the repressive function of mH2A should be eliminated. This could be achieved by the specific removal of mH2A from the promoter and its replacement by conventional H2A by an mH2A-specific histone chaperone as recently described for the histone variant H2A.Z (28
). The identification of genes for which expression is controlled by mH2A as well as the understanding of the mechanism of specific deposition and removal of mH2A from these genes remains a challenge for future studies.