BACKGROUND
the male specific lethal  ribonucleoprotein complex is required for x chromosome dosage compensation in the fruit fly drosophila melanogaster  <cit> .
the msl complex binds to most actively transcribed x-linked genes in males  <cit>  and is responsible for a two-fold enhancement in gene transcription  <cit> .
while there has been considerable progress in our understanding of the composition of the msl complex  <cit>  and the nature of the high affinity binding sites on the male x chromosome  <cit> , less is known about the mechanism of transcription regulation.
one protein component of the msl complex that could play an important role in transcription enhancement is mof, a member of the myst family of histone acetyltransferase enzymes   <cit> .
in the presence of a nucleosomal substrate, purified msl complex predominately monoacetylates histone h <dig> at lysine  <dig>   <cit> .
the msl complex has considerably less hat activity when mof has a single glycine to glutamic acid change in the acetyl-coa binding motif   <cit> .
in the presence of free histones recombinant mof is less specific, preferentially acetylating the n-terminal tail of histone h <dig> but also acetylating the n-terminal tail of histone h <dig>  the stringent h4k <dig> substrate specificity of the msl complex requires a nucleosomal substrate and integration of mof into the complex.
association of mof with msl <dig> and msl <dig> appears to be particularly important for hat specificity and activity  <cit> .
the carboxyl terminal domain of msl <dig>  which acts as a scaffold for complex assembly, interacts with both mof and msl <dig>  <cit> .
in addition to being a component of the msl complex, mof also associates with proteins that form the non-specific lethal or nsl complex  <cit> .
protein components of the nsl complex include nsl <dig>  nsl <dig>  nsl <dig>  mcrs <dig> and mbd-r <dig>  genome-wide chip-chip studies of male cells found that mof associates predominately with promoter regions of autosomal genes but has a bimodal distribution on male x-linked genes with peaks at both 5' and 3' ends  <cit> .
in contrast, msl <dig> and msl <dig> show little binding to autosomal genes but are highly enriched across active x-linked genes, with a bias towards the 3' end  <cit> .
h4k16ac is strongly enriched at the 5' region of autosomal genes that have high levels of bound mof  <cit> .
kind et al.
therefore suggest that mof has a role in gene expression independent of the msl complex.
however, a subsequent study found little support that mof was important for 5' h4k16ac of genes  <cit> .
early immunostaining studies of polytene chromosomes demonstrated that there is significant enrichment of h4k16ac on the male x chromosome  <cit> .
further, the msl complex co-localized to the hundreds of sites on the x chromosome enriched for h4k16ac  <cit> .
more recent chip-chip experiments found that nearly all actively transcribed x-linked genes are highly enriched for h4k16ac throughout the body of the gene but with a bias towards the middle and 3' end  <cit> .
there is mounting evidence that genes enriched for h4k16ac have an altered chromatin structure resulting in elevated transcription.
in the crystal structure of the nucleosome core particle, several hydrogen bonds and salt bridges were observed between the basic tail of histone h <dig>  and acidic side chains of histones h2a and h2b of a neighbouring nucleosome core particle  <cit> .
acetylation of h <dig> n terminal lysine residues would reduce this association.
indeed, incorporation of h4k16ac into nucleosomal arrays abolished a salt-dependent compaction into  <dig> nm-like fibres  <cit> .
in vivo, the msl complex appears to counteract the effect of factors that promote compaction of the male x chromosome such as iswi and hp <dig>  for example, in homozygous iswi or su2- <dig>  mutant male salivary gland nuclei the x chromosome has a bloated appearance, which required msl complex function  <cit> .
h4k16ac interferes with binding of iswi to nucleosomal substrate in vitro and antagonizes iswi function in vivo  <cit> .
transcription of nucleosomal templates in vitro is enhanced by incorporation of h4k16ac  <cit> .
in yeast, the expression of a reporter gene was strongly stimulated by a gal4-mof fusion protein  <cit> .
the reporter gene had multiple gal <dig> binding sites in the promoter.
in contrast, little transcription enhancement was obtained with a gal4-mof <dig> fusion protein carrying a g691e mutation.
these results were perhaps somewhat surprising as 80% of histone h <dig> is acetylated at k <dig> in yeast  <cit> .
indeed, it was not shown if the reporter was enriched for h4k16ac in the presence of gal4-mof  <cit> .
while these studies support a role for h4k16ac in gene transcription, it has recently been reported that h4k16ac is more strongly associated with dna replication timing than transcription in drosophila cells  <cit> .
here we have targeted mof to reporter gene promoters by fusing to the dna binding domain of the yeast gal <dig> protein.
this would mimic the observed enrichment of mof at gene promoters but not the 3' enrichment seen on x-linked genes in males.
our aim was to determine if tethering mof to a promoter would stimulate transcription.
if so, was transcription enhancement equal in drosophila males and females?
RESULTS
gal4-mof activates the uas-redstinger reporter gene expression more strongly in females than males
the gal4-mof and gal4-mof gene fusions previously used to control reporter gene expression in yeast  <cit>  were cloned into a drosophila p-element transformation vector that contained the constitutive hsp <dig> promoter .
a hemagluttinin  epitope tag is present between the gal <dig> dna binding domain and mof, which facilitates protein detection in cells.
a control gene construct was made that contains only the dna binding domain of gal <dig> .
to confirm that any difference in reporter gene response between gal4-mof and gal4-mof was due to loss of hat activity, we made two additional active site mof mutants, c680a and e714q .
western blot analysis confirmed expression of the gal <dig> fusion proteins .
there was some variability in the level of gal <dig> fusion protein expression between lines.
lines that had very high or very low levels of gal <dig> fusion protein expression were not examined further.
at least three lines for each construct were selected for assays with a uas-red stinger reporter .
this reporter encodes the fast maturing dsred.t <dig> red fluorescent protein with a nuclear localization sequence  that is under the control of a minimal hsp <dig> promoter with upstream binding sites for gal <dig>   <cit> .
in the presence of gal4-mof, strong activation of dsred.t4-nls expression was observed in female larvae .
much less dsred.t4-nls expression was observed in male larvae.
similarly, female adults showed significantly higher levels of red fluorescence than adult males.
dsred.t4-nls expression was particularly strong in the abdomen but expression was also detected in head and thorax.
subsequent experiments were performed using third instar larvae, as low levels of dsred.t4-nls expression could be detected since background fluorescence was low at this stage of development.
the level of reporter gene activation correlated with the level of gal4-mof protein expression.
this was most easily seen in male larvae, which have a lower level of reporter gene activation than female larvae.
for example, more red fluorescence was observed with line s <dig>  which makes moderate to high levels of gal4-mof, than either of the low expression lines s <dig> and s <dig> .
we noticed lateral clusters of cells that showed strong red fluorescence with all gal4-mof drivers, including lines that express low levels of protein.
the location of the cell clusters and variable number of cells per cluster suggests that these are larval oenocytes  <cit> .
one of the reporter lines, uas-redstinger <dig> , responded more strongly to gal4-mof than the other available autosomal line uas-redstinger <dig> ) .
hence, for most experiments we used the uas-redstinger <dig> line.
an x-linked reporter line, uas-redstinger <dig> , also responded more strongly to gal4-mof in females than males .
hat activity is required for robust transcription enhancement by mof
we next asked if histone acetyltransferase  activity was essential for transcription activation by gal4-mof.
we made the c680a and e714q mof point mutations based on the results of mechanistic studies identifying the homologous residues c <dig> and e <dig> as essential for hat activity in yeast esa <dig>  <cit> .
esa <dig> is a myst hat that is highly similar to mof .
the recent deposition of the protein coordinates of the crystal structure of human mof  allowed us to compare the hmof and esa <dig> three dimensional structures .
overall, hmof and esa <dig> share a high degree of structural similarity.
in particular the active site glutamate and cysteine residues occupy almost identical positions.
the hmof hat domain structure is also highly similar to the three dimensional structures of other human myst family hats, htip <dig> and moz .
hence, we predicted that the mof mutants containing c680a or e714q point mutations should have greatly reduced hat activity.
to test this prediction, hat assays were performed with purified recombinant mof wild type and mutant proteins and hela core histones.
consistent with previously published studies  <cit> , we found that recombinant mof containing the chromo and myst hat domains  catalysed acetylation of histone substrate but mof was at least ten times less active .
mof and mof were about  <dig> to  <dig> times less active than mof .
this supports the prediction that the active site glutamate and cysteine residues would be important for catalysis.
in the presence of gal4-mof protein, dsred.t4-nls expression was higher in female than male larvae .
the level of red fluorescence in male larvae was similar to the gal <dig> control.
in female larvae, red fluorescence was particularly noticeable in larval oenocytes and salivary glands.
however, dsred.t4-nls expression in salivary gland was also detected with the gal <dig> control, as previously reported  <cit> .
that is, since the dna binding domain of gal <dig> is sufficient to activate expression in salivary glands, all gal4-mof active site mutant fusion proteins showed dsred expression in salivary gland.
the results shown are typical for the three gal4-mof lines that were examined.
similarly, low but readily detectable levels of dsred.t4-nls expression were observed in female larvae with either gal4-mof  or gal4-mof proteins .
in male larvae, dsred.t4-nls expression was similar to the gal <dig> control.
the level of red fluorescence in female larvae that express a gal4-mof active site mutant was much less than female larvae that express gal4-mof .
as similar results were obtained with all three hat mutants we conclude that mof hat activity is required for robust reporter gene transcription activation by gal4-mof.
however, hat activity does not appear to be essential for transcription activation by mof.
to more accurately quantify reporter gene expression levels, we performed real-time quantitative pcr with rna isolated from larvae that contain the uas-red stinger reporter and express a gal-mof protein or gal <dig> control.
samples from three independent experiments were analyzed in triplicate.
pka rna levels were used for normalization of dsred rna levels.
the results are shown in figure  <dig>  the increase in dsred rna is shown relative to the rna level measured in the gal <dig> control.
the results are consistent with the fluorescence microscopy observations.
a large increase in dsred rna is seen in female larvae that express gal4-mof.
gal4-mof males make about  <dig> times less dsred rna.
female larvae that express one of the active site mutants  contain significant levels of dsred rna, comparable to males that express gal4-mof.
in contrast, much less dsred rna was detected in male larvae that express gal4-mof ,  or .
the level of induction of dsred rna in female larvae by the mof active site mutants suggests that histone acetylase activity is not essential for transcription activation.
however, it is possible that the transcription activation is due to residual acetyltransferase activity .
alternatively, transcription activation could be due to an nsl protein that associates with the gal4-mof mutant proteins and is thus recruited to the promoter of the reporter gene .
gal4-mof is incorporated into the msl complex
we considered several possible explanations for why gal4-mof more strongly activated the uas-redstinger reporter in female larvae.
one possibility was that if gal4-mof was incorporated into the msl complex in males, then less gal4-mof would be available to bind to the reporter gene.
to evaluate this possibility we examined polytene chromosomes of third instar larvae salivary glands that express gal4-mof and carry a uas-lacz gene.
in female nuclei, gal4-mof bound to a few sites on the x chromosomes and autosomes .
in general binding was at a low level, although strong binding was consistently seen at the site of the uas-lacz transgene .
in male nuclei, gal4-mof bound to hundreds of sites on the male x chromosome and to many sites on the autosomes.
binding was generally stronger than seen in female nuclei.
msl <dig> co-localized with gal4-mof to sites on the x chromosome and autosomes in male nuclei.
in contrast, msl <dig> did not co-localize with gal4-mof to the site of the uas-lacz transgene in female nuclei.
in wild type male nuclei there is little binding of msl <dig> to the autosomes  <cit> .
this suggests that gal4-mof has been incorporated into the msl complex but has somehow altered the normal binding profile of the complex.
possibly the fusion of the dna binding domain of gal <dig> has altered the normal function of mof.
if so, gal4-mof may not be able to functionally replace mof in the msl complex.
thus, we next asked if gal4-mof could rescue mof <dig> mutant males, which die at third instar or prepupal stage  <cit> .
we found that expression of gal4-mof  did not rescue males from the lethal effects of the mof <dig> mutation .
similar results were obtained with a different gal4-mof line .
thus it appears that, although the fusion of the dna binding domain of gal <dig> to the amino end of mof has not disrupted hat activity  or incorporation into the msl complex, the msl complex containing gal4-mof is not fully functional.
in their recent study, becker and colleagues reported that mof <dig> males were rescued by expression of a gal4-mof fusion protein driven by the endogenous mof promoter  <cit> .
thus an alternative explanation for our results is that the hsp <dig> promoter does not provide the required expression profile of mof fusion protein to rescue mof <dig> males.
afull genotype of cross: w cv mof1; fm <dig>  y31d sc wa b females crossed with w <dig> y1; s <dig> {w+, h83-gal4-mof}/tm <dig>  ser y+ males.
for clarity, the number of flies that inherited the tm <dig>  ser balancer chromosome are not shown.
the msl complex does not assemble in females because translation of msl <dig> rna is repressed by sxl  <cit> .
however, constitutive expression of msl <dig> from a transgene causes the msl complex to assemble in females  <cit> .
if the difference in the chromosome-binding pattern between male and female nuclei is because gal4-mof is incorporated into the msl complex in male nuclei, then constitutive expression of msl <dig> in females should lead to an altered distribution of gal4-mof.
as predicted, in female nuclei that carry the hsp83-msl <dig>  hsp83-gal4-mof and uas-lacz transgenes, we observed strong binding of gal4-mof to many sites on the x chromosomes and autosomes .
the binding of gal4-mof was stronger than seen in female nuclei that don't express msl <dig> .
further, msl <dig> co-localized with gal4-mof, confirming incorporation of gal4-mof into the msl complex.
the enrichment of the msl complex on x chromosomes was less than typically observed in female nuclei that express msl <dig>  <cit> , which is consistent with the interpretation that gal4-mof has interfered with the normal chromatin binding pattern of the msl complex.
we next examined dsred fluorescence in female larvae that constitutively express msl <dig> and gal4-mof and carry the uas-redstinger reporter gene.
we observed a dramatic decrease in dsred.t <dig> fluorescence in female larvae that express msl <dig> .
the strongest fluorescence was seen in oenocytes, as also seen in male larvae that express gal4-mof.
interestingly, there was also a small but consistent decrease in dsred.t <dig> fluorescence in male larvae that express msl <dig> compared to normal males.
these results suggest that if more gal4-mof protein is driven into the msl complex then there is less activation of the uas-redstinger reporter gene.
if low dsred.t <dig> expression in males is because gal4-mof is incorporated into the msl complex then reporter gene expression should increase in msl <dig> mutant males.
alternatively, since mof requires msl <dig> for robust histone acetyltransferase activity in vitro  <cit> , reporter gene expression driven by gal4-mof could decrease in msl <dig> mutant males and females.
homozygous and heterozygous msl1l <dig> larvae were distinguished using a constitutively expressed gfp marker for the second chromosome that had a normal copy of the msl <dig> gene.
for these experiments it was necessary to use the third chromosome-linked uas-redstinger <dig> reporter, which shows a significant response to gal4-mof but the response is less strong than the uas-redstinger <dig> line used previously.
we observed higher dsred.t <dig> fluorescence in homozygous msl1l <dig> male larvae compared to their heterozygous brothers .
in female larvae, dsred.t <dig> expression was similar in homozygous and heterozygous msl1l <dig> mutants.
these results show that the lower reporter gene response in males is because gal4-mof is incorporated into the msl complex.
further, it does not appear that msl <dig> is essential for transcription activation by gal4-mof.
gal4-mof elevates expression of a uas-arm-lacz reporter gene
the uas-redstinger reporter gene is an artificial construct consisting of five gal <dig> dna binding sites and a minimal promoter from the autosomal hsp <dig> gene.
in the absence of activator, background dsred.t <dig> expression is negligible.
to examine the effect of gal4-mof on a promoter from an x-linked gene, we inserted twelve copies of the gal <dig> binding site upstream of the armadillo promoter in the arm-lacz reporter.
we called this reporter  <dig> × uas-arm-lacz.
we previously used the arm-lacz reporter to examine the effect of msl complex binding on gene expression  <cit> .
we found that insertion of a strong msl complex binding site upstream of the arm promoter led to a two-fold increase in lacz expression in males  <cit> .
in contrast to the robust transcription activation of the uas-redstinger reporter, there was only a modest increase in lacz expression in hemisected adults in the presence of gal4-mof .
further, the increase in lacz expression was similar in males and females, in contrast to the female-biased redstinger expression seen in larvae.
however, the increase in lacz expression was significant in both sexes .
we also measured β-galactosidase activities in larvae and found only a modest increase in  <dig> × uas-arm-lacz expression in males  and females  in the presence of gal4-mof.
anumber of independent experiments
brelative to β-galactosidase activity of control  <dig> × uas-arm-lacz hemisected adults
cmean +/- standard deviation.
activity in  <dig> mod min- <dig> mg protein
in hemisected adults, the β-galactosidase activity in the uas-arm-lacz strain was well above the background activity in the parental y w strain , as anticipated from previous studies  <cit> .
there was no significant elevation in lacz expression with either gal <dig> or full length gal <dig> .
the lack of stimulation by full length gal <dig> was perhaps not surprising as the binding sites are greater than  <dig> bp upstream from the first transcription start site of the arm gene.
in yeast  <cit>  and drosophila  <cit>  the level of transcription stimulation by gal <dig> decreases with increased distance between binding sites and the promoter.
surprisingly, in the presence of either gal4-mof, gal4-mof or gal4-mof, lacz expression was also significantly greater than the respective arm-lacz controls .
there was, however, some line-to-line variation in the degree of elevation of lacz expression  lines s <dig> and s65).
however, the elevation in lacz expression with any of the mof mutants was consistently less than observed with gal4-mof.
in males the elevation in lacz expression by gal4-mof was significantly higher than the increase in lacz expression with any of the gal4-mof mutants .
we conclude that recruitment of gal4-mof to the arm-lacz reporter leads to a small but significant increase in gene expression, which at least in part, is due to mof hat activity.
in the  <dig> × uas-arm-lacz reporter, the gal <dig> binding sites are about  <dig>  kb upstream of the lacz gene.
we next asked if binding of gal4-mof led to an increase in h4k16ac over the body of the lacz gene.
chromatin immunoprecipitation was performed using an antibody that recognises h4k16ac.
we found that there was an enrichment for h4k16ac across the lacz gene in nuclei from third instar larvae of mixed sex .
there was no enrichment of h4k16ac in the lacz gene in larvae that express the gal <dig> control protein.
thus binding of gal4-mof upstream of the promoter does lead to an increase in h4k16ac levels across the  <dig> × uas-arm-lacz reporter gene.
we also examined larvae that express gal4-mof and have a lacz reporter with a minimal promoter and gal <dig> binding sites immediately upstream of the promoter .
as the gal4-mof binding sites are very close to the start of transcription we predicted that the lacz gene would be enriched for h4k16ac.
indeed, we found an enrichment for h4k16ac across the lacz gene in larvae of mixed sex .
similar enrichments in h4k16ac were observed in independent chip experiments with the lacz reporter genes .
discussion
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  <cit> .
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  <cit> .
we found that the human mof and yeast esa <dig> 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 esa <dig> catalysis, c <dig> acts to nucleophilically displace the acetyl group from acetylcoa  <cit> .
then e <dig> deprotonates the substrate lysine amino group to nucleophilically attack the acetyl-c <dig> covalent adduct, generating acetyl-lysine as the final product.
the esa <dig> mutants e338q and c304a had essentially no hat activity in vitro.
while another in vitro study suggested that c <dig> did not play an essential role in the esa <dig> mechanism  <cit> , an in vivo study supports the earlier conclusion that c <dig> and e <dig> are essential for hat activity in esa <dig>  <cit> .
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  <cit> , 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 msl <dig> 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 msl <dig> in female nuclei that constitutively express msl <dig>  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 msl <dig> 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 msl <dig>  <cit> .
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  <cit> .
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  <cit> .
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.
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  <cit> .
the authors provide several lines of evidence that the nsl complex members and mof act synergistically to regulate gene expression.
for example, a gal4-nsl <dig> 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  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 nsl <dig> 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 gal <dig> 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 h <dig> at lysines  <dig> and  <dig> in addition to lysine  <dig>  <cit> .
during activation of transcription of the human ifnβ gene, acetylation of histone h <dig> at k <dig> leads to recruitment of the swi/snf chromatin remodelling complex  <cit> .
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 h4k <dig> 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  <dig> × 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 , which has a minimal promoter.
it has recently been reported that knockdown of mof expression in drosophila s <dig> had a greater effect on those autosomal genes that were expressed at low levels  <cit> .
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  <dig> × 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  <cit> .
thus, if the nsl complex is bound to the arm promoter in the  <dig> × 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  <dig> × uas-arm-lacz but may have little effect on transcription as the gal <dig> binding sites are further upstream from the transcription start site than for the uas-redstinger reporter.
in this regard, we observed that full length gal <dig> did not stimulate  <dig> × uas-arm-lacz expression but gal <dig> is a potent activator of uas-lacz.
surprisingly, expression of the  <dig> × 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 .
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.
CONCLUSIONS
in this study we have shown that targeting the histone acetyltransferase mof to reporter genes via the dna binding domain of gal <dig> led to transcription enhancement and acetylation of histone h <dig> at lysine  <dig>  highly conserved active site residues cys <dig> and glu <dig> were important for mof catalytic activity in vitro and for uas-reporter gene activation in drosophila.
gal4-mof strongly induced expression of a uas-dsred reporter gene, particularly in females.
in males, gal4-mof was incorporated into the male specific lethal  complex.
the lower uas-dsred response in males could be because there is less gal4-mof protein available to bind to the reporter gene or the msl proteins inhibit the transcription activation by mof.
alternatively, it could be that the reporter gene response is higher in females as more gal4-mof protein is available to recruit the non-specific lethal  complex proteins to the promoter of the reporter gene.
in contrast, gal4-mof only modestly increased expression of a  <dig> × uas-arm-lacz reporter driven by a constitutive promoter.
this could be because of a higher basal activity of the reporter, greater distance between the gal <dig> binding sites and the transcription start site or because the arm promoter can independently recruit the nsl complex.

 the male specific lethal ribonucleoprotein complex is required for x chromosome dosage compensation in the fruit fly drosophila melanogaster. the msl complex binds to most actively transcribed x-linked genes in males and is responsible for a two-fold enhancement in gene transcription. while there has been considerable progress in our understanding of the composition of the msl complex and the nature of the high affinity binding sites on the male x chromosome, less is known about the mechanism of transcription regulation. one protein component of the msl complex that could play an important role in transcription enhancement is mof, a member of the myst family of histone acetyltransferase enzymes. in the presence of a nucleosomal substrate, purified msl complex predominately monoacetylates histone h at lysine. the msl complex has considerably less hat activity when mof has a single glycine to glutamic acid change in the acetyl-coa binding motif. in the presence of free histones recombinant mof is less specific, preferentially acetylating the n-terminal tail of histone h but also acetylating the n-terminal tail of histone h the stringent h4k substrate specificity of the msl complex requires a nucleosomal substrate and integration of mof into the complex. association of mof with msl and msl appears to be particularly important for hat specificity and activity. the carboxyl terminal domain of msl which acts as a scaffold for complex assembly, interacts with both mof and msl. in addition to being a component of the msl complex, mof also associates with proteins that form the non-specific lethal or nsl complex. protein components of the nsl complex include nsl nsl nsl mcrs and mbd-r genome-wide chip-chip studies of male cells found that mof associates predominately with promoter regions of autosomal genes but has a bimodal distribution on male x-linked genes with peaks at both 5' and 3' ends. in contrast, msl and msl show little binding to autosomal genes but are highly enriched across active x-linked genes, with a bias towards the 3' end. h4k16ac is strongly enriched at the 5' region of autosomal genes that have high levels of bound mof. kind et al. therefore suggest that mof has a role in gene expression independent of the msl complex. however, a subsequent study found little support that mof was important for 5' h4k16ac of genes. early immunostaining studies of polytene chromosomes demonstrated that there is significant enrichment of h4k16ac on the male x chromosome. further, the msl complex co-localized to the hundreds of sites on the x chromosome enriched for h4k16ac. more recent chip-chip experiments found that nearly all actively transcribed x-linked genes are highly enriched for h4k16ac throughout the body of the gene but with a bias towards the middle and 3' end. there is mounting evidence that genes enriched for h4k16ac have an altered chromatin structure resulting in elevated transcription. in the crystal structure of the nucleosome core particle, several hydrogen bonds and salt bridges were observed between the basic tail of histone h and acidic side chains of histones h2a and h2b of a neighbouring nucleosome core particle. acetylation of h n terminal lysine residues would reduce this association. indeed, incorporation of h4k16ac into nucleosomal arrays abolished a salt-dependent compaction into nm-like fibres. in vivo, the msl complex appears to counteract the effect of factors that promote compaction of the male x chromosome such as iswi and hp for example, in homozygous iswi or su2- mutant male salivary gland nuclei the x chromosome has a bloated appearance, which required msl complex function. h4k16ac interferes with binding of iswi to nucleosomal substrate in vitro and antagonizes iswi function in vivo. transcription of nucleosomal templates in vitro is enhanced by incorporation of h4k16ac. in yeast, the expression of a reporter gene was strongly stimulated by a gal4-mof fusion protein. the reporter gene had multiple gal binding sites in the promoter. in contrast, little transcription enhancement was obtained with a gal4-mof fusion protein carrying a g691e mutation. these results were perhaps somewhat surprising as 80% of histone h is acetylated at k in yeast. indeed, it was not shown if the reporter was enriched for h4k16ac in the presence of gal4-mof. while these studies support a role for h4k16ac in gene transcription, it has recently been reported that h4k16ac is more strongly associated with dna replication timing than transcription in drosophila cells. here we have targeted mof to reporter gene promoters by fusing to the dna binding domain of the yeast gal protein. this would mimic the observed enrichment of mof at gene promoters but not the 3' enrichment seen on x-linked genes in males. our aim was to determine if tethering mof to a promoter would stimulate transcription. if so, was transcription enhancement equal in drosophila males and females?  gal4-mof activates the uas-redstinger reporter gene expression more strongly in females than males the gal4-mof and gal4-mof gene fusions previously used to control reporter gene expression in yeast were cloned into a drosophila p-element transformation vector that contained the constitutive hsp promoter. a hemagluttinin epitope tag is present between the gal dna binding domain and mof, which facilitates protein detection in cells. a control gene construct was made that contains only the dna binding domain of gal. to confirm that any difference in reporter gene response between gal4-mof and gal4-mof was due to loss of hat activity, we made two additional active site mof mutants, c680a and e714q. western blot analysis confirmed expression of the gal fusion proteins. there was some variability in the level of gal fusion protein expression between lines. lines that had very high or very low levels of gal fusion protein expression were not examined further. at least three lines for each construct were selected for assays with a uas-red stinger reporter. this reporter encodes the fast maturing dsred.t red fluorescent protein with a nuclear localization sequence that is under the control of a minimal hsp promoter with upstream binding sites for gal. in the presence of gal4-mof, strong activation of dsred.t4-nls expression was observed in female larvae. much less dsred.t4-nls expression was observed in male larvae. similarly, female adults showed significantly higher levels of red fluorescence than adult males. dsred.t4-nls expression was particularly strong in the abdomen but expression was also detected in head and thorax. subsequent experiments were performed using third instar larvae, as low levels of dsred.t4-nls expression could be detected since background fluorescence was low at this stage of development. the level of reporter gene activation correlated with the level of gal4-mof protein expression. this was most easily seen in male larvae, which have a lower level of reporter gene activation than female larvae. for example, more red fluorescence was observed with line s which makes moderate to high levels of gal4-mof, than either of the low expression lines s and s. we noticed lateral clusters of cells that showed strong red fluorescence with all gal4-mof drivers, including lines that express low levels of protein. the location of the cell clusters and variable number of cells per cluster suggests that these are larval oenocytes. one of the reporter lines, uas-redstinger, responded more strongly to gal4-mof than the other available autosomal line uas-redstinger ). hence, for most experiments we used the uas-redstinger line. an x-linked reporter line, uas-redstinger, also responded more strongly to gal4-mof in females than males. hat activity is required for robust transcription enhancement by mof we next asked if histone acetyltransferase activity was essential for transcription activation by gal4-mof. we made the c680a and e714q mof point mutations based on the results of mechanistic studies identifying the homologous residues c and e as essential for hat activity in yeast esa. esa is a myst hat that is highly similar to mof. the recent deposition of the protein coordinates of the crystal structure of human mof allowed us to compare the hmof and esa three dimensional structures. overall, hmof and esa share a high degree of structural similarity. in particular the active site glutamate and cysteine residues occupy almost identical positions. the hmof hat domain structure is also highly similar to the three dimensional structures of other human myst family hats, htip and moz. hence, we predicted that the mof mutants containing c680a or e714q point mutations should have greatly reduced hat activity. to test this prediction, hat assays were performed with purified recombinant mof wild type and mutant proteins and hela core histones. consistent with previously published studies, we found that recombinant mof containing the chromo and myst hat domains catalysed acetylation of histone substrate but mof was at least ten times less active. mof and mof were about to times less active than mof. this supports the prediction that the active site glutamate and cysteine residues would be important for catalysis. in the presence of gal4-mof protein, dsred.t4-nls expression was higher in female than male larvae. the level of red fluorescence in male larvae was similar to the gal control. in female larvae, red fluorescence was particularly noticeable in larval oenocytes and salivary glands. however, dsred.t4-nls expression in salivary gland was also detected with the gal control, as previously reported. that is, since the dna binding domain of gal is sufficient to activate expression in salivary glands, all gal4-mof active site mutant fusion proteins showed dsred expression in salivary gland. the results shown are typical for the three gal4-mof lines that were examined. similarly, low but readily detectable levels of dsred.t4-nls expression were observed in female larvae with either gal4-mof or gal4-mof proteins. in male larvae, dsred.t4-nls expression was similar to the gal control. the level of red fluorescence in female larvae that express a gal4-mof active site mutant was much less than female larvae that express gal4-mof. as similar results were obtained with all three hat mutants we conclude that mof hat activity is required for robust reporter gene transcription activation by gal4-mof. however, hat activity does not appear to be essential for transcription activation by mof. to more accurately quantify reporter gene expression levels, we performed real-time quantitative pcr with rna isolated from larvae that contain the uas-red stinger reporter and express a gal-mof protein or gal control. samples from three independent experiments were analyzed in triplicate. pka rna levels were used for normalization of dsred rna levels. the results are shown in figure the increase in dsred rna is shown relative to the rna level measured in the gal control. the results are consistent with the fluorescence microscopy observations. a large increase in dsred rna is seen in female larvae that express gal4-mof. gal4-mof males make about times less dsred rna. female larvae that express one of the active site mutants contain significant levels of dsred rna, comparable to males that express gal4-mof. in contrast, much less dsred rna was detected in male larvae that express gal4-mof, or. the level of induction of dsred rna in female larvae by the mof active site mutants suggests that histone acetylase activity is not essential for transcription activation. however, it is possible that the transcription activation is due to residual acetyltransferase activity. alternatively, transcription activation could be due to an nsl protein that associates with the gal4-mof mutant proteins and is thus recruited to the promoter of the reporter gene. gal4-mof is incorporated into the msl complex we considered several possible explanations for why gal4-mof more strongly activated the uas-redstinger reporter in female larvae. one possibility was that if gal4-mof was incorporated into the msl complex in males, then less gal4-mof would be available to bind to the reporter gene. to evaluate this possibility we examined polytene chromosomes of third instar larvae salivary glands that express gal4-mof and carry a uas-lacz gene. in female nuclei, gal4-mof bound to a few sites on the x chromosomes and autosomes. in general binding was at a low level, although strong binding was consistently seen at the site of the uas-lacz transgene. in male nuclei, gal4-mof bound to hundreds of sites on the male x chromosome and to many sites on the autosomes. binding was generally stronger than seen in female nuclei. msl co-localized with gal4-mof to sites on the x chromosome and autosomes in male nuclei. in contrast, msl did not co-localize with gal4-mof to the site of the uas-lacz transgene in female nuclei. in wild type male nuclei there is little binding of msl to the autosomes. this suggests that gal4-mof has been incorporated into the msl complex but has somehow altered the normal binding profile of the complex. possibly the fusion of the dna binding domain of gal has altered the normal function of mof. if so, gal4-mof may not be able to functionally replace mof in the msl complex. thus, we next asked if gal4-mof could rescue mof mutant males, which die at third instar or prepupal stage. we found that expression of gal4-mof did not rescue males from the lethal effects of the mof mutation. similar results were obtained with a different gal4-mof line. thus it appears that, although the fusion of the dna binding domain of gal to the amino end of mof has not disrupted hat activity or incorporation into the msl complex, the msl complex containing gal4-mof is not fully functional. in their recent study, becker and colleagues reported that mof males were rescued by expression of a gal4-mof fusion protein driven by the endogenous mof promoter. thus an alternative explanation for our results is that the hsp promoter does not provide the required expression profile of mof fusion protein to rescue mof males. afull genotype of cross: w cv mof1; fm y31d sc wa b females crossed with w y1; s {w+, h83-gal4-mof}/tm ser y+ males. for clarity, the number of flies that inherited the tm ser balancer chromosome are not shown. the msl complex does not assemble in females because translation of msl rna is repressed by sxl. however, constitutive expression of msl from a transgene causes the msl complex to assemble in females. if the difference in the chromosome-binding pattern between male and female nuclei is because gal4-mof is incorporated into the msl complex in male nuclei, then constitutive expression of msl in females should lead to an altered distribution of gal4-mof. as predicted, in female nuclei that carry the hsp83-msl hsp83-gal4-mof and uas-lacz transgenes, we observed strong binding of gal4-mof to many sites on the x chromosomes and autosomes. the binding of gal4-mof was stronger than seen in female nuclei that don't express msl. further, msl co-localized with gal4-mof, confirming incorporation of gal4-mof into the msl complex. the enrichment of the msl complex on x chromosomes was less than typically observed in female nuclei that express msl, which is consistent with the interpretation that gal4-mof has interfered with the normal chromatin binding pattern of the msl complex. we next examined dsred fluorescence in female larvae that constitutively express msl and gal4-mof and carry the uas-redstinger reporter gene. we observed a dramatic decrease in dsred.t fluorescence in female larvae that express msl. the strongest fluorescence was seen in oenocytes, as also seen in male larvae that express gal4-mof. interestingly, there was also a small but consistent decrease in dsred.t fluorescence in male larvae that express msl compared to normal males. these results suggest that if more gal4-mof protein is driven into the msl complex then there is less activation of the uas-redstinger reporter gene. if low dsred.t expression in males is because gal4-mof is incorporated into the msl complex then reporter gene expression should increase in msl mutant males. alternatively, since mof requires msl for robust histone acetyltransferase activity in vitro, reporter gene expression driven by gal4-mof could decrease in msl mutant males and females. homozygous and heterozygous msl1l larvae were distinguished using a constitutively expressed gfp marker for the second chromosome that had a normal copy of the msl gene. for these experiments it was necessary to use the third chromosome-linked uas-redstinger reporter, which shows a significant response to gal4-mof but the response is less strong than the uas-redstinger line used previously. we observed higher dsred.t fluorescence in homozygous msl1l male larvae compared to their heterozygous brothers. in female larvae, dsred.t expression was similar in homozygous and heterozygous msl1l mutants. these results show that the lower reporter gene response in males is because gal4-mof is incorporated into the msl complex. further, it does not appear that msl is essential for transcription activation by gal4-mof. gal4-mof elevates expression of a uas-arm-lacz reporter gene the uas-redstinger reporter gene is an artificial construct consisting of five gal dna binding sites and a minimal promoter from the autosomal hsp gene. in the absence of activator, background dsred.t expression is negligible. to examine the effect of gal4-mof on a promoter from an x-linked gene, we inserted twelve copies of the gal binding site upstream of the armadillo promoter in the arm-lacz reporter. we called this reporter × uas-arm-lacz. we previously used the arm-lacz reporter to examine the effect of msl complex binding on gene expression. we found that insertion of a strong msl complex binding site upstream of the arm promoter led to a two-fold increase in lacz expression in males. in contrast to the robust transcription activation of the uas-redstinger reporter, there was only a modest increase in lacz expression in hemisected adults in the presence of gal4-mof. further, the increase in lacz expression was similar in males and females, in contrast to the female-biased redstinger expression seen in larvae. however, the increase in lacz expression was significant in both sexes. we also measured β-galactosidase activities in larvae and found only a modest increase in × uas-arm-lacz expression in males and females in the presence of gal4-mof. anumber of independent experiments brelative to β-galactosidase activity of control × uas-arm-lacz hemisected adults cmean +/- standard deviation. activity in mod min- mg protein in hemisected adults, the β-galactosidase activity in the uas-arm-lacz strain was well above the background activity in the parental y w strain, as anticipated from previous studies. there was no significant elevation in lacz expression with either gal or full length gal. the lack of stimulation by full length gal was perhaps not surprising as the binding sites are greater than bp upstream from the first transcription start site of the arm gene. in yeast and drosophila the level of transcription stimulation by gal decreases with increased distance between binding sites and the promoter. surprisingly, in the presence of either gal4-mof, gal4-mof or gal4-mof, lacz expression was also significantly greater than the respective arm-lacz controls. there was, however, some line-to-line variation in the degree of elevation of lacz expression lines s and s65). however, the elevation in lacz expression with any of the mof mutants was consistently less than observed with gal4-mof. in males the elevation in lacz expression by gal4-mof was significantly higher than the increase in lacz expression with any of the gal4-mof mutants. we conclude that recruitment of gal4-mof to the arm-lacz reporter leads to a small but significant increase in gene expression, which at least in part, is due to mof hat activity. in the × uas-arm-lacz reporter, the gal binding sites are about kb upstream of the lacz gene. we next asked if binding of gal4-mof led to an increase in h4k16ac over the body of the lacz gene. chromatin immunoprecipitation was performed using an antibody that recognises h4k16ac. we found that there was an enrichment for h4k16ac across the lacz gene in nuclei from third instar larvae of mixed sex. there was no enrichment of h4k16ac in the lacz gene in larvae that express the gal control protein. thus binding of gal4-mof upstream of the promoter does lead to an increase in h4k16ac levels across the × uas-arm-lacz reporter gene. we also examined larvae that express gal4-mof and have a lacz reporter with a minimal promoter and gal binding sites immediately upstream of the promoter. as the gal4-mof binding sites are very close to the start of transcription we predicted that the lacz gene would be enriched for h4k16ac. indeed, we found an enrichment for h4k16ac across the lacz gene in larvae of mixed sex. similar enrichments in h4k16ac were observed in independent chip experiments with the lacz reporter genes. discussion 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 esa 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 esa catalysis, c acts to nucleophilically displace the acetyl group from acetylcoa. then e deprotonates the substrate lysine amino group to nucleophilically attack the acetyl-c covalent adduct, generating acetyl-lysine as the final product. the esa mutants e338q and c304a had essentially no hat activity in vitro. while another in vitro study suggested that c did not play an essential role in the esa mechanism, an in vivo study supports the earlier conclusion that c and e are essential for hat activity in esa. 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 msl 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 msl in female nuclei that constitutively express msl 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 msl 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 msl. 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. 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-nsl 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 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 nsl 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 gal 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 h at lysines and in addition to lysine. during activation of transcription of the human ifnβ gene, acetylation of histone h at k 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 h4k 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 × 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, which has a minimal promoter. it has recently been reported that knockdown of mof expression in drosophila s 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 × 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. thus, if the nsl complex is bound to the arm promoter in the × 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 × uas-arm-lacz but may have little effect on transcription as the gal binding sites are further upstream from the transcription start site than for the uas-redstinger reporter. in this regard, we observed that full length gal did not stimulate × uas-arm-lacz expression but gal is a potent activator of uas-lacz. surprisingly, expression of the × 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. 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.  in this study we have shown that targeting the histone acetyltransferase mof to reporter genes via the dna binding domain of gal led to transcription enhancement and acetylation of histone h at lysine highly conserved active site residues cys and glu were important for mof catalytic activity in vitro and for uas-reporter gene activation in drosophila. gal4-mof strongly induced expression of a uas-dsred reporter gene, particularly in females. in males, gal4-mof was incorporated into the male specific lethal complex. the lower uas-dsred response in males could be because there is less gal4-mof protein available to bind to the reporter gene or the msl proteins inhibit the transcription activation by mof. alternatively, it could be that the reporter gene response is higher in females as more gal4-mof protein is available to recruit the non-specific lethal complex proteins to the promoter of the reporter gene. in contrast, gal4-mof only modestly increased expression of a × uas-arm-lacz reporter driven by a constitutive promoter. this could be because of a higher basal activity of the reporter, greater distance between the gal binding sites and the transcription start site or because the arm promoter can independently recruit the nsl complex. 
we discuss how association of gal4-mof with the msl or nsl proteins could explain our results. mof was fused to the dna binding domain of gal and targeted to the promoter region of uas-reporter genes in drosophila. in the presence of gal4-mof, h4k16ac levels were enriched over uas-lacz and uas-arm-lacz reporter genes. targeting mof to reporter genes led to transcription enhancement and acetylation of histone h at lysine histone acetyltransferase activity was required for the full transcriptional response. the latter utilizes the constitutive promoter from the arm gene to drive lacz expression. immunostaining of polytene chromosomes showed that gal4-mof co-localized with msl to many sites on the x chromosome in male but not female nuclei. however, in female nuclei that express msl gal4-mof co-localized with msl to many sites on polytene chromosomes but dsred expression was reduced. the msl complex is bound within active genes on the male x chromosome with a 3' bias. however, dsred rna levels were about seven times higher in female than male larvae. mof also associates with the non-specific lethal or nsl complex. mutation of conserved active site residues in mof reduced hat activity in vitro and uas-dsred activation in drosophila. in contrast, the nsl complex is enriched at promoter regions of many autosomal and x-linked genes in both sexes. in male drosophila melanogaster, the male specific lethal complex is somehow responsible for a two-fold increase in transcription of most x-linked genes, which are enriched for histone h acetylated at lysine. incorporation of gal4-mof into the msl complex in males led to a lower transcription enhancement of uas-dsred but not uas-arm-lacz genes. in contrast to the strong induction of uas-dsred expression, uas-arm-lacz expression increased by about 2-fold in both sexes.
 in male drosophila melanogaster, the male specific lethal complex is somehow responsible for a two-fold increase in transcription of most x-linked genes, which are enriched for histone h acetylated at lysine. this acetylation requires mof, a histone acetyltransferase that is a component of the msl complex. mof also associates with the non-specific lethal or nsl complex. the msl complex is bound within active genes on the male x chromosome with a 3' bias. in contrast, the nsl complex is enriched at promoter regions of many autosomal and x-linked genes in both sexes. in this study we have investigated the role of mof as a transcriptional activator.  mof was fused to the dna binding domain of gal and targeted to the promoter region of uas-reporter genes in drosophila. we found that expression of a uas-red fluorescent protein reporter gene was strongly induced by gal4-mof. however, dsred rna levels were about seven times higher in female than male larvae. immunostaining of polytene chromosomes showed that gal4-mof co-localized with msl to many sites on the x chromosome in male but not female nuclei. however, in female nuclei that express msl gal4-mof co-localized with msl to many sites on polytene chromosomes but dsred expression was reduced. mutation of conserved active site residues in mof reduced hat activity in vitro and uas-dsred activation in drosophila. in the presence of gal4-mof, h4k16ac levels were enriched over uas-lacz and uas-arm-lacz reporter genes. the latter utilizes the constitutive promoter from the arm gene to drive lacz expression. in contrast to the strong induction of uas-dsred expression, uas-arm-lacz expression increased by about 2-fold in both sexes.  targeting mof to reporter genes led to transcription enhancement and acetylation of histone h at lysine histone acetyltransferase activity was required for the full transcriptional response. incorporation of gal4-mof into the msl complex in males led to a lower transcription enhancement of uas-dsred but not uas-arm-lacz genes. we discuss how association of gal4-mof with the msl or nsl proteins could explain our results. 