- Research article
- Open Access
Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila
© Schiemann et al; licensee BioMed Central Ltd. 2010
Received: 30 April 2010
Accepted: 9 November 2010
Published: 9 November 2010
In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). 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 Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) 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 MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) 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 H4 at lysine 16. 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.
The male specific lethal (MSL) ribonucleoprotein complex is required for X chromosome dosage compensation in the fruit fly Drosophila melanogaster [1–3]. The MSL complex binds to most actively transcribed X-linked genes in males [4–6] and is responsible for a two-fold enhancement in gene transcription [7, 8]. While there has been considerable progress in our understanding of the composition of the MSL complex [1, 2] and the nature of the high affinity binding sites on the male X chromosome [9, 10], 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 (HATs) . In the presence of a nucleosomal substrate, purified MSL complex predominately monoacetylates histone H4 at lysine 16 (H4K16ac) [12, 13]. The MSL complex has considerably less HAT activity when MOF has a single glycine to glutamic acid change in the acetyl-CoA binding motif (G691E, the mof1 allele) . In the presence of free histones recombinant MOF is less specific, preferentially acetylating the N-terminal tail of histone H4 but also acetylating the N-terminal tail of histone H3. The stringent H4K16 substrate specificity of the MSL complex requires a nucleosomal substrate and integration of MOF into the complex. Association of MOF with MSL1 and MSL3 appears to be particularly important for HAT specificity and activity . The carboxyl terminal domain of MSL1, which acts as a scaffold for complex assembly, interacts with both MOF and MSL3 [14, 15].
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 NSL1, NSL2, NSL3, MCRS2 and MBD-R2. 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, MSL1 and MSL3 show little binding to autosomal genes but are highly enriched across active X-linked genes, with a bias towards the 3' end [4, 5, 17]. 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 H4 (K16 to N25) and acidic side chains of histones H2A and H2B of a neighbouring nucleosome core particle . Acetylation of H4 N terminal lysine residues would reduce this association. Indeed, incorporation of H4K16ac into nucleosomal arrays abolished a salt-dependent compaction into 30 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 HP1. For example, in homozygous ISWI or Su(var)2-5 (the gene encoding HP1) mutant male salivary gland nuclei the X chromosome has a bloated appearance, which required MSL complex function [23, 24]. 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 [12, 25]. In yeast, the expression of a reporter gene was strongly stimulated by a Gal4-MOF fusion protein . The reporter gene had multiple Gal4 binding sites in the promoter. In contrast, little transcription enhancement was obtained with a Gal4-MOF1 fusion protein carrying a G691E mutation. These results were perhaps somewhat surprising as 80% of histone H4 is acetylated at K16 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 Gal4 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
At least three lines for each construct were selected for assays with a UAS-Red Stinger reporter (Figure 1D). This reporter encodes the fast maturing DsRed.T4 red fluorescent protein with a nuclear localization sequence (NLS) that is under the control of a minimal hsp70 promoter with upstream binding sites for Gal4 (UAS) . In the presence of Gal4-MOF, strong activation of DsRed.T4-NLS expression was observed in female larvae (Figure 1E). 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 S41, which makes moderate to high levels of Gal4-MOF, than either of the low expression lines S38 and S44 (Figure 1E). 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-RedStinger4 [chromosome 2, FlyBase ID FBst0008546], responded more strongly to Gal4-MOF than the other available autosomal line UAS-RedStinger6 [chromosome 3, FlyBase ID FBst0008547]) (Figure 1E). Hence, for most experiments we used the UAS-RedStinger4 line. An X-linked reporter line, UAS-RedStinger3 [FlyBase ID FBst0008545], also responded more strongly to Gal4-MOF in females than males (Additional file 1, Figure S1).
HAT activity is required for robust transcription enhancement by MOF
Gal4-MOF is incorporated into the MSL complex
Gal4-MOF does not rescue males from the lethal effects of the mof 1 mutationa
mof1/Y; S49/+ male
FM7/Y; S49/+ male
mof1/+; S49/+ female
FM7/+; S49/+ female
The MSL complex does not assemble in females because translation of msl2 RNA is repressed by SXL . However, constitutive expression of MSL2 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 MSL2 in females should lead to an altered distribution of Gal4-MOF. As predicted, in female nuclei that carry the hsp83-msl2, hsp83-Gal4-MOF and UAS-lacZ transgenes, we observed strong binding of Gal4-MOF to many sites on the X chromosomes and autosomes (Figure 7B). The binding of Gal4-MOF was stronger than seen in female nuclei that don't express MSL2 (Figure 7A). Further, MSL1 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 MSL2 , which is consistent with the interpretation that Gal4-MOF has interfered with the normal chromatin binding pattern of the MSL complex.
Gal4-MOF elevates expression of a UAS-arm-lacZ reporter gene
Transcription regulation of a 3 × UAS-arm-lacZ reporter by Gal4-MOF fusion proteins in hemisected adults
Mean male relative β-galactosidase activitiesb
Mean female relative β-galactosidase activitiesb
1.04 ± 0.06c
1.1 ± 0.0003
1.057 ± 0.041
0.96 ± 0.031
0.95 ± 0.06
0.95 ± 0.03
2.17 ± 0.12
1.67 ± 0.08
2.47 ± 0.3
2.05 ± 0.21
1.78 ± 0.11
1.54 ± 0.05
1.56 ± 0.09
1.30 ± 0.03
1.70 ± 0.03
1.49 ± 0.12
1.32 ± 0.27
1.04 ± 0.01
1.45 ± 0.22
1.60 ± 0.15
1.32 ± 0.13
1.27 ± 0.11
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 (not shown), as anticipated from previous studies [8, 38]. There was no significant elevation in lacZ expression with either Gal4[DB] or full length Gal4 (Table 2). The lack of stimulation by full length Gal4 was perhaps not surprising as the binding sites are greater than 650 bp upstream from the first transcription start site of the arm gene. In yeast  and Drosophila  the level of transcription stimulation by Gal4 decreases with increased distance between binding sites and the promoter. Surprisingly, in the presence of either Gal4-MOF(G691E), Gal4-MOF(C680A) or Gal4-MOF(E714Q), lacZ expression was also significantly greater than the respective arm-lacZ controls (ANOVA, P < 0.01 for both sexes). There was, however, some line-to-line variation in the degree of elevation of lacZ expression (e.g hsp83-Gal4-MOF(E714Q) lines S64 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 (ANOVA, P < 0.05). 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 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.
In this study we have shown that targeting the histone acetyltransferase MOF to reporter genes via the DNA binding domain of Gal4 led to transcription enhancement and acetylation of histone H4 at lysine 16. Highly conserved active site residues Cys680 and Glu714 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 (MSL) 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 (NSL) complex proteins to the promoter of the reporter gene. In contrast, Gal4-MOF only modestly increased expression of a 3 × 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 Gal4 binding sites and the transcription start site or because the arm promoter can independently recruit the NSL complex.
Construction of plasmids
pAS2-MOF and pAS2-MOF G691E plasmids  were used as a template for PCR. The Gal4-MOF and Gal4-MOF G691E constructs were made by PCR using primers AS7 5'-TTCGGTACCGAAGCAAGCCTCCTG-3' and AS8 5'-TTCGGTACCCCCGGGCTAGCCGGAATTACCCGG-3". The PCR products were digested with Asp 718 and cloned into pCaSpeR-h83. The Gal4-MOF plasmid served as a template to make the Gal4-MOF[E714Q] and Gal4-MOF[C680A] constructs by PCR using primers carrying the point mutations. The Gal4 DB construct was made by PCR using AS7 5'-TTCGGTACCGAAGCAAGCCTCCTG-3' and AS9 5'-ATAAAGAATGCGGCCGCCTACGGCGATACAGTCAAC-3". The PCR product was digested with Kpn I and Not I and cloned into pCaSpeR-h83. The UAS Gal4 binding site was excised from plasmid pBS-2N17mer by digestion with Not I and inserted into pRHO7  to create p3 × UAS-arm-lacZ. For expression of MOF in E. coli, a fragment of the Drosophila mof open reading frame encoding amino acids 370 to 827 was inserted into the GST expression vector pGEX-6P3. This fragment contains the intact chromodomain and the MYST HAT domain. Active site mutants were made with the Stratagene quick change kit and verified by DNA sequencing. Primer sequences are available upon request.
Recombinant MOF purification and HAT assays
E. coli cell pellets were resuspended in cleavage buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA), protease inhibitors were added and then treated with 1 mg/mL hen egg white lysozyme (Sigma), 2.5 mg of DNaseI (10 mg/mL), and 50 U Benzonase (Invitrogen) per liter of cells at 4°C for 25 min. GST-fusion proteins were purified using Glutathione-Sepharose (GS) affinity chromatography with an ÄKTA FPLC and a HR 16/5 column (GE Life Science) packed with 10 - 15 mL of Glutathione SepharoseTM 4B (GE Life Science). Eluted protein was concentrated using an Ultracel regenerated cellulose Amicon® Ultra-15 centrifugal filter device. HAT assays were performed in triplicate with each sample with [3H]acetyl-CoA and HeLa cell core histones .
Fly transgenesis, polytene chromosomes and β-galactosidase assays
Maintenance of Drosophila cultures and generation of P transformant lines were done as previously described . Male and female larvae were identified based on the size of the genital disc. arm-Gal4, UAS-lacZ and UAS-RedStinger lines were obtained from the Bloomington Drosophila stock center. Polytene chromosome squashes and immunostaining were carried out as described previously . β-galactosidase assays were performed on hemisected adults as described previously . Assays were performed in triplicate on 3 separate collections. The β-galactosidase activity was standardized by total protein microassays (Bio-Rad). Means and standard deviations of ratios were calculated from the 3 separate collections. Statistical analyses of β-galactosidase activities were performed using the mini-tab and SAS software packages.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation assays using male and female third instar larvae were performed as described previously . Undiluted immunoprecipitated DNA (2 μl) and 100-fold diluted input DNA (2 μl) were assayed with each primer set in triplicate. The primer pairs used were Gpdhr (5'-GTGCCCGACCTGGTTGAG-3") and Gpdhf (5'-CTTGCCTTCAGGTGACGC-3"), armr (5'-TTCCAAGACACAGAGAGGGTG-3") and armf (5'-GCCCTCGACAATCTCCTCC-3"), pgd 10f (5'-GAAGGGCACGGGCAAGTG-3") and pgd 10r (5'-CAATGCCGCCGTAATTAAGTCTC-3"), lacZ-2823F (5'-GCGCGAATTGAATTATGGCCC-3") and lacZ-2950R (5'-GCCATGTGCCTTCTTCCG-3"), lacZ-1540F (5'-GCTGTGCCGAAATGGTCC-3") and lacZ-1670R (5'-CGAAACGCCTGCCAGTATTTAG-3"), lacZ-324F (5'-GGTCAATCCGCCGTTTGTTC-3") and lacZ-493R (5'-TGTCCTGGCCGTAACCG-3").
Fold enrichment was determined by 2^(CP input lacZ or control gene - CP ChIP lacZ or control gene)/2^(CP input Gpdh - CP ChIP Gpdh).
Quantitative real-time PCR was conducted in triplicate using the LightCycler FastStart DNA MasterPLUS SYBR Green I reaction mix (Roche) in a LightCycler Instrument (Roche). An annealing temperature of 55°C and an extension time of 18 s were used.
The crossing point (CP) was automatically determined by the LightCycler software (Roche).
RNA isolation, cDNA synthesis and Quantitative real-time PCR
UAS-DsRed.T4-NLS females were crossed to males with Gal4-MOF, Gal4-MOF mutant or Gal4[DB] transgenes. The 3rd instar larval offspring were collected, sorted by sex and checked for red fluorescence by stereo fluorescence microscopy. Total RNA was extracted with TRIzol™ and treated with turbo DNase (Ambion). cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix (invitrogen). RNA isolation and cDNA synthesis was carried out from three independent experiments. The sequences of DsRed primers are forward primer: 5'-GCGTGATGAACTTCGAGG-3' and reverse primer: 5'-GCCCATAGTCTTCTTCTGC-3". For normalization, we used primers for pka transcripts: forward primer: 5'-TTCTCGGAGCCGCACTCGCGCTTCTAC-3", reverse primer: 5'-CAATCAGCAGATTCTCCGGCT-3". qRT-PCR reactions were performed using Maxima™ SYBR Green/ROX qPCR Master mix (Fermentas), amplifications were run on standard 384-well reaction plate using Applied Biosystems7900HT Fast Real-Time PCR System. PCR efficiencies were determined from the slopes of standard curves from cDNA dilution series (triplicate). DsRed mRNA levels were calculated from the CT values of triplicate samples and three independent experiments using the 2-ΔΔCT method.
We thank Dahlia Nielsen for expert assistance with statistical analysis and Jim Mahaffey and Corey Laverty for comments on the manuscript. We are grateful to Matthias Prestel and Asifa Akhtar for information on the distribution of bound NSL complex around and within the arm gene. Peter Becker, Mitzi Kuroda, Rick Kelley and John Lucchesi generously provided plasmid DNAs, antibodies and fly strains. Work in SAM's laboratory was funded by an operating grant (MOP 79377) from the Canadian Institutes for Health Research. Research in MJS's laboratory was funded by grant MAU204 from the Royal Society of New Zealand Marsden Fund to MJS and a FRST high achiever doctoral scholarship to VMW.
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