In this study we asked if targeting MOF to a reporter gene would lead to an enrichment of H4K16ac across the gene and therefore result in transcription enhancement in Drosophila. We found that H4K16ac was increased over a reporter gene driven either by a minimal promoter with low basal activity or by a more generally active promoter from the X-linked armadillo gene. The increase in H4K16ac was significant because robust transcription enhancement by MOF required full HAT activity. However, the answer to the question of whether or not targeting MOF to a gene would enhance transcription was more complex than initially supposed. The degree of transcription enhancement depended upon the sex, tissue, reporter gene location and promoter. Interestingly, targeting MOF to the arm-lacZ reporter gene led to an approximately two-fold increase in gene expression. We had previously observed a two-fold increase in lacZ expression in males that carried the arm-lacZ reporter with an upstream MSL complex high affinity binding site . Our results are consistent with the suggestion that the increase in H4K16ac over X-linked genes in males plays an important role in doubling gene expression .
We found that the human MOF and yeast Esa1 three dimensional structures were very similar. Importantly, the active site glutamate and cysteine residues occupied almost identical positions. In the proposed ping-pong mechanism for Esa1 catalysis, C304 acts to nucleophilically displace the acetyl group from acetylCoA [30, 31]. Then E338 deprotonates the substrate lysine amino group to nucleophilically attack the acetyl-C304 covalent adduct, generating acetyl-lysine as the final product. The Esa1 mutants E338Q and C304A had essentially no HAT activity in vitro. While another in vitro study suggested that C304 did not play an essential role in the Esa1 mechanism , an in vivo study supports the earlier conclusion that C304 and E338 are essential for HAT activity in Esa1 . We found that the highly conserved active site cysteine and glutamate amino acids were important for MOF catalytic activity in vitro and for UAS reporter gene activation in Drosophila.
We observed a particularly strong reporter gene response to Gal4-MOF in larval oenocytes. This was most easily seen in male larvae that otherwise responded weakly to the activator and in female larvae that expressed an active site mutant form of MOF. Presumably there is a tissue-specific factor in these cells that enhances the transcription stimulation by MOF. Oenocytes are highly specialized cells that regulate lipid metabolism in Drosophila , so it is not unlikely that such tissue-specific factors may exist.
Gal4-MOF more strongly activated expression of the UAS-RedStinger reporter gene in females than males. There are several lines of evidence that this is because Gal4-MOF is incorporated into the MSL complex in males. Firstly, Gal4-MOF co-localized with MSL1 in male salivary gland polytene chromosomes to sites on the X chromosome and autosomes. In female nuclei the strongest binding site corresponded to the location of the UAS-lacZ reporter gene. Gal4-MOF co-localized with MSL1 in female nuclei that constitutively express MSL2, and Gal4-MOF only weakly activated the UAS-RedStinger reporter in these females. Lastly, UAS-RedStinger was activated more strongly by Gal4-MOF in males that were unable to assemble the MSL complex due to a mutation in the msl1 gene.
There are three possible explanations for why incorporation of Gal4-MOF into the MSL complex in males would reduce activation of the UAS-RedStinger reporter gene. Firstly, because Gal4-MOF is sequestered into the MSL complex in males, there is less free protein available to bind to the autosomal UAS-reporter gene and enhance transcription. A similar model was proposed by Birchler and colleagues to explain a decrease in expression of autosomal mini-white and yellow transgenes in females that constitutively express MSL2 . A second explanation is that the MSL complex somehow largely represses the transcription enhancement of the UAS-RedStinger reporter gene that would be expected from the increased histone acetylation by MOF. This is similar to the proposed role of the MSL complex in the "inverse dosage effect" model for X chromosome dosage compensation . In this model the MSL complex sequesters MOF to the X chromosome reducing the inverse dosage effect on the autosomes by decreasing histone acetylation. Further the MSL complex is proposed to inhibit any transcription elevation of genes on the X chromosome due to increased level of H4K16ac. After submission of this manuscript, Becker and colleagues published their study on transcription regulation of reporter genes by a Gal4-MOF fusion protein . Consistent with our findings, they found that expression of a UAS-lacZ reporter gene was higher in females than males and that the gene was enriched for H4K16ac. The authors favored a model that the MSL complex inhibits the transcription elevation due to increased H4K16ac such that the net effect is a two-fold increase in reporter gene expression. In contrast to our study, Prestel et al. (2010) did not test if catalytically inactive versions of MOF increased reporter gene expression, so it is unclear what fraction of transcription elevation by Gal4-MOF was due to MOF histone acetyltransferase activity. A third explanation for why reporter gene expression was higher in females is that more of the Gal4-MOF fusion protein is available to be incorporated into the NSL complex. In a recent study, Akhtar and colleagues found that the NSL complex is a potent regulator of gene expression, upregulating the expression of most target genes . The authors provide several lines of evidence that the NSL complex members and MOF act synergistically to regulate gene expression. For example, a Gal4-NSL3 fusion protein is a potent activator of expression of a UAS-luciferase reporter gene but full activation required MOF and other NSL components. Thus if more NSL complex is assembled at the promoter of the UAS-RedStinger reporter gene in females than males, this could explain the higher expression observed in females. Indeed, Prestel et al (2010) showed that Gal4-MOF is incorporated into the NSL complex in female cells. They also found that the NSL complex regulates the expression of many genes in Drosophila. The recruitment of NSL proteins such as NSL3 by Gal4-MOF active site mutant proteins to the promoter region of the UAS-RedStinger reporter gene could explain why we observed a significant increase in DsRed expression in females. Presumably, in males the incorporation of Gal4-MOF mutant protein into the MSL complex and reduced HAT activity led to a very small increase in reporter gene expression compared to the Gal4[DB] control.
Interestingly, a recent study found that in the human NSL complex, MOF has relaxed substrate specificity relative to the MSL complex and acetylates histone H4 at lysines 5 and 8 in addition to lysine 16 . During activation of transcription of the human IFNβ gene, acetylation of histone H4 at K8 leads to recruitment of the SWI/SNF chromatin remodelling complex . It will be interesting to determine if the broader specificity of MOF when incorporated into the NSL complex in part explains why the NSL complex is a potent transcription regulator. Further, the "repressive" effect of the MSL complex on transcription activation by MOF may simply reflect the high specificity of MOF for H4K16 when part of the MSL complex.
In contrast to the sex-bias observed with the UAS-RedStinger reporter gene, both males and females showed only a very modest increase in 3 × UAS-arm-lacZ expression. One difference between the reporter gene constructs is that the baseline expression of arm-LacZ driven by the active armadillo promoter is much higher than the UAS-RedStinger reporter (or UAS-lacZ), which has a minimal promoter. It has recently been reported that knockdown of mof expression in Drosophila S2 had a greater effect on those autosomal genes that were expressed at low levels . Further, reducing mof RNA levels had only a modest effect on autosomal gene expression. This might explain why only a small transcription enhancement was observed with the 3 × UAS-arm-lacZ reporter. Presumably, the NSL complex is recruited by Gal4-MOF to both of the reporter genes. However, the NSL complex is bound to the promoter region of the arm gene at its normal location on the X chromosome [34, 46]. Thus, if the NSL complex is bound to the arm promoter in the 3 × UAS-arm-lacZ autosomal transgene, recruitment of additional NSL complex by Gal4-MOF may have little stimulatory effect on lacZ expression. Alternatively, NSL complex may be recruited to the 3 × UAS-arm-lacZ but may have little effect on transcription as the Gal4 binding sites are further upstream from the transcription start site than for the UAS-RedStinger reporter. In this regard, we observed that full length Gal4 did not stimulate 3 × UAS-arm-lacZ expression but Gal4 is a potent activator of UAS-lacZ. Surprisingly, expression of the 3 × UAS-arm-lacZ reporter gene was increased significantly by all three Gal4-MOF active site mutant proteins. This could be due to residual HAT activity of the mutant proteins (Figure 4). Alternatively, as discussed above, the transcription elevation of the reporter gene could be due, in part, to recruitment of other proteins to the arm promoter by the Gal4-MOF mutant proteins.