males absent on the first


Interaction of Mof with RNA

In Drosophila, compensation for the reduced dosage of genes located on the single male X chromosome involves doubling their expression in relation to their counterparts on female X chromosomes. Dosage compensation is an epigenetic process involving the specific acetylation of histone H4 at lysine 16 by the histone acetyltransferase Mof. Although Mof is expressed in both sexes, it only associates with the X chromosome in males. Its absence causes male-specific lethality. Mof is part of a chromosome-associated complex comprising male-specific lethal (MSL) proteins and at least one non-coding roX RNA. How Mof is integrated into the dosage compensation complex is unknown. Association of Mof with the male X chromosome is shown in this study to depend on its interaction with RNA. Mof specifically binds through its chromodomain to roX2 RNA in vivo. In vitro analyses of the Mof and Msl-3 chromodomains indicate that these chromodomains may function as RNA interaction modules. Their interaction with non-coding RNA may target regulators to specific chromosomal sites (Akhtar, 2000b).

The association of (MSL) proteins (Msl-1, Msl-2, Msl-3, Mof, Mle) and roX RNA with the male X chromosome has been visualized by immunofluorescence analysis of larval polytene chromosomes. Drosophila SL-2 cells can act as a model system for male features, because functional dosage compensation complex (DCC) has been purified from them, and they have also been used to study the post-transcriptional regulation of dosage compensation. The nuclear territory of the X chromosome in SL-2 cells can be visualized by immunostaining with antisera against Msl-1, Mof, the histone H4 isoform acetylated at Lys 16 (K16) and Mle. Msl-1 remains localized to the X chromosome after permeabilization of the cells and RNase treatment. In contrast, the bulk of Mof staining disappears from the X chromosome after RNase treatment. Mle, which interacts with larval polytene chromosomes in an RNase-sensitive manner, dissociates from the chromosome upon permeablization of the cells ; therefore, the RNase sensitivity of Mle's chromosomal association in this system could not be confirmed. Loss of Mof correlates with a reduction of the H4 acetyl-K16 histone isoform at the X chromosome, suggesting a high turnover of the modification under these conditions. These results suggest that the stable integration of Mof into chromosome-bound DCC involves an RNase-sensitive structure, and that the continued association of Mof with DCC is independent of Mle (Akhtar, 2000b).

Candidate RNAs that may contribute to the assembly and chromosomal association of the DCC are the roX1 and roX2 RNAs that are stably expressed in male but not female flies, and that colocalize with MSL proteins on the male X chromosome. The two RNAs have no effect on the integrity of DCC, because deletion of a single roX RNA has no phenotype, but mutation of both genes abolishes the interaction of MSL proteins with the chromosome. SL-2 cells express only roX2 RNA, which forms a complex with MSL proteins and is therefore an excellent candidate for an RNA involved on the targeting of Mof. To test whether Mof interacts with roX2 RNA in vivo, nuclear extracts were prepared from SL-2 cells and the soluble DCC was isolated by immunoprecipitation with antibodies specific for either Mof or Mle. The RNase sensitivity of interactions was tested by RNase treatment of extracts before immunoprecipitation. The immunoprecipitated complexes were characterized by western blot analysis. Under these conditions, Mof, Mle, Msl-3 and Msl-2 co-immunoprecipitate independently of RNase treatment, indicating that the DCC is held together by protein-protein interactions and/or that bridging RNA is protected from the nuclease attack. In the absence of RNase, roX2 RNA is readily detected in the immunopurified complex by reverse transcription of RNA followed by polymerase chain reaction with roX2 specific primers, whether the immunoprecipitation has been performed with antibodies specific for Mof or Mle (Akhtar, 2000b).

These experiments confirm earlier reports that roX2 RNA can be part of soluble DCC, but they raise the issues of how soluble DCC may differ from the chromosome-associated complex and whether Mof interacts with roX2 RNA directly or indirectly (for example, through Mle). To clarify the latter issue, use was made of the observation that the association of Mle with DCC is sensitive to elevated ionic strength. Under these stringent immunoprecipitation conditions, Mle is no longer associated with Mof, although Msl-2 and Msl-3 are still detectable. In the absence of Mle, a significant amount of roX2 RNA remains associated with Mof (Akhtar, 2000b).

Although these experiments rule out that Mle, to date the best candidate for an RNA interacting factor, bridges between Mof and roX2, the involvement of Msl-3 or other unknown protein(s) remain possible. To determine whether Mof interacts with RNA directly, the recombinant enzyme was studied in vitro. Electrophoretic mobility shift assay (EMSA) with fragments derived from roX1 reveals a nonspecific interaction of Mof with RNA. Competition experiments show that Mof interacts with RNA with high preference over DNA. Mof is unable to interact with DNA efficiently. A lack of specificity of the RNA interaction may be due to misfolding of the in vitro transcribed RNAs in the absence of chaperones or, most likely, to the use of arbitrary fragments of roX RNA, which lack optimum binding sites (Akhtar, 2000b).

The specific interaction of Mof with RNA should be distinguished from insignificant 'sticking', by determining whether Mof has a specific domain for RNA interaction. The RNA interaction domain was mapped by creating a series of recombinant Mof derivatives that are all active in chromatin binding and histone acetylation, and analysing their potential to interact with RNA. A Mof protein truncated at its amino terminus (N352) still interacts with RNA, and further deletion of the chromodomain (N518) abolishes this interaction. Since this result suggests that the chromodomain is involved in RNA binding, hydrophobic residues that are conserved in chromodomains from various origins were mutated. Mof derivatives with single amino-acid exchanges (W426G and Y416D) are unable to interact with RNA, whereas a point mutation in the acetyl CoA-binding site, G691E4, does not affect RNA binding. The W426G and Y416D mutations affect the structure of the chromodomain only locally because the mutant enzymes are still active histone acetyltransferases (HATs). Confirmatory results were obtained with an alternative binding assay involving roX1 RNA immobilized on streptavidin-sepharose beads (Akhtar, 2000b).

To establish the physiological relevance of the chromodomain for RNA interaction in vivo Mof derivatives were transiently expressed from a metallothioneine promoter in SL-2 cells. The addition of an N-terminal haemagglutinin (HA) epitope allowed the distinguishing of ectopic enzymes from endogenous Mof. Whole-cell extracts were prepared before or after induction of transgene expression with copper. Under stringent immunoprecipitation conditions with antibody directed against HA, roX2 RNA associates with HA-Mof. When expressed to the same level as intact Mof, both chromodomain mutants W426G and Y416D show an impaired interaction with roX2 RNA. Almost no roX2 RNA could be detected after immunoprecipitation of Y416D, whereas the effect of the W426G mutation was less severe, but still clear (Akhtar, 2000b).

To establish whether RNA interaction is a specific feature of the Mof chromodomain or whether it is a more general property of chromodomains, Msl-3, another dosage compensation protein containing two chromodomains was tested for interaction with RNA. Recombinant Msl-3 interacts efficiently with RNA, forming several complexes in the EMSA assay at higher concentrations. Competition experiments have established that Msl-3 interacts far better with RNA than with DNA. Even the carboxy-terminal chromodomain of Msl-3 (CD2) when fused to glutathione-S-transferase (GST) is able to interact with RNA but not DNA, whereas the GST moiety alone is inactive (Akhtar, 2000b).

These results suggest that the Mof chromodomain interacts with roX RNA in vivo, which may contribute to the integration of Mof into DCC at the male X chromosome. The association of Mof with DCC is a rather late step in the assembly of the complex and does not occur in the absence of Mle. The earlier incorporation of roX2 RNA also depends on Mle. The stable association of any one subunit with DCC may rely on multiple interactions with protein and/or RNA subunits, and an additional direct contact between Mof and Mle remains possible. Whether RNA interaction is a general property of chromodomains or restricted to a subfamily of the chromodomain superfamily remains to be seen. Chromodomains are important for the function of a number of chromatin regulators, but their modes of action have remained enigmatic. Although the related 'chromo shadow domain' of heterochromatin protein 1 (HP1) mediates interactions with several proteins and an interacting peptide has been identified, chromodomains have so far not been shown to contact proteins or peptides. Mutations of the clr protein analogous to the ones made in Mof abolish its silencing capacity (Ivanova, 1998). Small deletions of the polycomb protein containing these residues lead to its delocalization in SL-2 cells. Non-coding RNAs may be more commonly involved in organizing regulatory complexes than has been appreciated to date (Eddy, 1999). Identification of the RNA structure motif that determines the specific interaction with the chromodomain remains a challenge for the future. Interestingly, dosage compensation in mammals also involves a non-coding RNA, Xist, coating the inactive X. It is tempting to speculate that roX and Xist RNAs may target regulatory proteins to the X chromosomes of Drosophila and humans (Akhtar, 2000b).

MOF acetylates MSL-3 in the dosage compensation complex

Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). RNA interference of individual DCC components has been used to define the order of complex assembly in Schneider cells. Interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. Localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. The acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, RPD3 interacts with MSL-3 and MSL-3 can be deacetylated by the RPD3 complex. It is proposed that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome (Buscaino, 2003).

As in male flies, MSL-2 is central to the assembly of the dosage compensation complex in SL-2 cells, since its depletion by RNAi leads to disassembly of the complex. MOF protein requires prior assembly of MSL-1, MSL-2, and MSL-3, while the MSL-3 protein requires prior assembly of at least MSL-1 and MSL-2 proteins. Since in MOF dsRNA-treated cells approximately 10% of MOF protein can still be detected by Western blot analysis, it remains possible that small amount of MOF enzyme may be sufficient for MSL-3 localization to the X chromosome. In MOF mutant flies, MSL-3 protein localization on polytene chromosomes is restricted only to chromatin entry sites, as detected by immunostaining of the polytene chromosomes, suggesting that incomplete knockdown of MOF may be a plausible explanation for the apparent unaffected MSL-3 localization in these cells. However, it is also important to note that due to the limited size of SL-2 cells, it is difficult to resolve entry sites in SL-2 cells in comparison to the polytene chromosomes. Alternatively, MSL-3 localization to the X chromosome in MOF dsRNA-treated cells could also be a feature specific to SL-2 cells. Interestingly, depletion of MSL-2, MSL-3, or MOF led to dissociation of MLE from the X chromosome. It is therefore suggested that MLE localization is sensitive to the assembly of the rest of the complex in SL-2 cells (Buscaino, 2003).

MOF protein is associated with the X chromosome in an RNase-sensitive manner. This observation has been extended and it has been show that MSL-3 protein is also tethered to the X chromosome via RNA. MSL-3 interacts with rox2 in immunoprecipitation experiments and not with another nuclear RNA, suggesting that roX2 is a likely candidate for mediating this interaction in SL-2 cells. However, it remains plausible that an as yet unidentified RNA (or protein) may act as a bridge between MSL-3 and the X chromosome in vivo (Buscaino, 2003).

Surprisingly, it was found that association of MSL-3 with the X chromosome is not only sensitive to RNase treatment but also to TSA treatment. These results suggest that the acetylation status of MSL-3 protein is likely to be of a dynamic nature and that slight imbalance in cellular acetylation levels leads to dramatic consequences for the MSL-3 protein in vivo. This result is intriguing, since MOF, which is also capable of autoacetylation, did not show the same phenotype under these conditions. Whether autoacetylation of MOF leads to other effects remains possible (Buscaino, 2003).

In the case where the MSL-3 protein levels are reduced by MSL-3 dsRNA treatment, the localization of MOF protein to the X chromosome is severely compromised. This result at first sight appears contradictory to the observation that MOF localization to the X chromosome is unaffected upon TSA treatment for 30 min. However, it is important to note that incubation of SL-2 cells with TSA for periods longer than 4 hr also affects localization of the rest of the complex, including MOF, suggesting that the DCC as a whole is sensitive to overall acetylation levels within the cells. Furthermore, it remains possible that additional modifications of DCC factors contribute to the stability and dynamics of the complex as a whole. However, the findings strongly suggest that MSL-3 is particularly sensitive to acetylation changes and therefore follows a more rapid dissociation than the rest of the complex members in the conditions tested (Buscaino, 2003).

The results demonstrate that the MSL-3 protein is regulated by acetylation and that MOF acetylates a single lysine residue in MSL-3 in vitro. This finding underscores the stringent substrate specificity of MOF, consistent with the fact that MOF also acetylates only a single lysine in a nucleosomal substrate. A striking consequence of MSL-3 acetylation is the loss of its interaction with RNA and its failure to localize to the X chromosome. The sensitivity of acetylated MSL-3 protein seems to be specific for roX2 in vitro since neither nonspecific DNA nor nonspecific RNA binding was affected. The MSL-3 protein contains two chromodomains, and MSL-3 has been shown to bind RNA in vitro via its chromodomain; remarkably, the acetylation occurs next to one of its chromodomains (Buscaino, 2003).

Acetylation of the X chromosome seems to have a high turnover, and the dosage compensation complex members, particularly MSL-3, appear to be sensitive to changes in endogenous acetylation levels. It is therefore proposed that regulated acetylation of MSL-3 may cause a conformational change that leads to temporary loss of interaction with RNA from one of the chromodomains. A cycle of deacetylation may follow that will allow MSL-3 to contact RNA again on a nearby affinity site. By continuous cycle of acetylation and deacetylation, MSL-3, along with the rest of the complex, may be able to spread from a chromatin entry site. These findings also provide further insight into the essential nature of MOF histone acetyl transferase, which is required not only for acetylation of the X chromosome but also for regulation of other members of the complex. Association of RPD3 with the dosage compensation members provides strong supporting evidence for this hypothesis. Interestingly, RPD3 hypomorphic mutants show a reduced male to female ratio. Furthermore, the S. cerevisiae homolog of MSL-3, EAF3, part of the NuA4 complex that contains ESA1, copurifyies with yeast RPD3 using the tandem affinity purification (TAP) procedure. It therefore appears likely that this property of MSL-3/EAF3 is conserved and that MSL-3/EAF3 may act as a bridge between two different histone-modifying activities. The transient interaction of dosage compensation complex members with a histone deacetylase may be required for fine tuning the effects of hyperacetylation by MOF protein to achieve the proper level of dosage compensation (Buscaino, 2003).

The activation potential of MOF is constrained for dosage compensation

The H4K16 acetyltransferase MOF plays a crucial role in dosage compensation in Drosophila but has additional, global functions. This study compared the molecular context and effect of MOF in male and female flies, combining chromosome-wide mapping and transcriptome studies with analyses of defined reporter loci in transgenic flies. MOF distributes dynamically between two complexes, the dosage compensation complex and a complex containing MBD-R2, a global facilitator of transcription. These different targeting principles define the distribution of MOF between the X chromosome and autosomes and at transcription units with 5' or 3' enrichment. The male X chromosome differs from all other chromosomes in that H4K16 acetylation levels do not correlate with transcription output. The reconstitution of this phenomenon at a model locus revealed that the activation potential of MOF is constrained in male cells in the context of the DCC to arrive at the 2-fold activation of transcription characteristic of dosage compensation (Prestel, 2010).

The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset

The MOF (males absent on the first)-containing NSL (non-specific lethal) complex binds to a subset of active promoters in Drosophila and is thought to contribute to proper gene expression. The determinants that target NSL to specific promoters and the circumstances in which the complex engages in regulating transcription are currently unknown. This study shows that the NSL complex primarily targets active promoters and in particular housekeeping genes, at which it colocalizes with the chromatin remodeler NURF (nucleosome remodeling factor) and the histone methyltransferase Trithorax. However, only a subset of housekeeping genes associated with NSL are actually activated by it. The analyses reveal that these NSL-activated promoters are depleted of certain insulator binding proteins and are enriched for the core promoter motif 'Ohler 5'. Based on these results, it is possible to predict whether the NSL complex is likely to regulate a particular promoter. It is concluded that the regulatory capacity of the NSL complex is highly context-dependent. Activation by the NSL complex requires a particular promoter architecture defined by combinations of chromatin regulators and core promoter motifs (Feller, 2012).

MOF is best known for its key role in the Drosophila dosage compensation process. It is a subunit of the dosage compensation complex [DCC, also known as male-specific lethal (MSL) complex], which brings about the 2-fold transcriptional activation of genes on the single male X chromosome to equalize expression with the corresponding genes transcribed from the two female X chromosomes. The DCC is constituted only in male flies and the five protein components, MSL1, MSL2, MSL3, maleless (MLE) and MOF, as well as the non-coding roX RNAs are essential for male viability. According to the current model, the DCC recruits MOF to the transcribed regions of X-chromosomal genes. Subsequent acetylation of H4K16 renders chromatin more accessible and potentially facilitates transcriptional elongation) (Feller, 2012).

With the exception of MSL2, all DCC protein subunits are also expressed in female flies, and therefore also serve more general, yet barely understood functions. For example, the acetyltransferase MOF appears to be involved in more global transcription regulation as it has recently been found in an alternative complex together with MCRS2, the WD40-repeat protein WDS (will-die-slowly), NSL1, NSL2, NSL3 and the plant homeo domain (PHD) protein MBD-R2 (Mendjan, 2006; Raja, 2010; Prestel, 2010). With reference to the dosage compensation 'MSL complex', this alternative MOF-containing assembly was termed 'NSL complex' (for 'non-specific lethal'), as its subunits are essential in both sexes (Mendjan, 2006). The incorporation of MOF into either the DCC or the NSL complex is determined by association of MOF with the PEHE domains of the respective MSL1 or NSL1 subunits (Mendjan, 2006). Genome-wide mapping by chromatin immunoprecipitation (ChIP) coupled to DNA microarrays (ChIP-chip) identified MOF binding sites at many, but not all active promoters in male and female cells. Subsequent studies revealed that MBD-R2 colocalizes with MOF at many active promoters in both sexes, suggesting that the NSL complex recruits MOF to these sites (Prestel, 2010). This is compatible with a recent ChIP-Seq study (ChIP DNA analyzed by massive parallel sequencing), which found MCRS2 and NSL1 peaks at promoters in mixed-sex 3rd instar larval salivary glands (Feller, 2012).

In male cells the association of MOF with NSL subunits is in competition with its incorporation into the DCC, which redirects it to the transcribed regions of X chromosomal genes. However, key aspects of MOF's targeting in the context of the NSL complex are unclear. What determines the binding of the NSL complex to only a subset of the active promoters? The available data also are ambiguous when it comes to the role of the NSL complex; does it activate or repress target genes, or perhaps both? Ablating the NSL subunit MBD-R2 in male embryonic cells resulted in a reduced expression of many MBD-R2 target genes. In contrast, a similar fraction of genes was found up- and downregulated when MBD-R2 and NSL3 were depleted in 3rd instar salivary glands (Feller, 2012).

This study created novel data sets and analyzed existing ones to compare functional interactions of NSL subunits in different developmental tissues to better define the targets of the NSL complex. The common properties of the NSL target genes were systematically explored, searching for colocalizing chromatin factors and prevalent sequence motifs in target promoters. The NSL complex was traced through monitoring the NSL1 subunit, and it was found it preferentially bind to promoters of housekeeping genes, which are also approached by the chromatin remodeler NURF and the methyltransferase Trithorax. There, NSL1 binding correlates best with the core promoter element DNA replication-related element (DRE). However, only a defined fraction of NSL1-bound genes are actually regulated by the complex. Those promoters are depleted for insulator proteins and are enriched for an E-box-derived promoter motif. This analysis provides a functional classification of housekeeping genes according to their NSL coregulator requirements (Feller, 2012).

The NSL complex regulates housekeeping genes in Drosophila

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. This study reports a comprehensive ChIP-seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. An increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac was found in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP-seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, it was demonstrated that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication-related Element (DRE). Taken together, these findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription (Lam, 2012).

This study has revealed that the majority of the NSL-complex-bound targets are housekeeping genes in Drosophila. While chromatin-modifying complexes that regulate tissue-specific genes, such as SAGA, polycomb and trithorax complexes, have been studied extensively, global regulators of housekeeping genes are poorly understood. The NSL complex is the first identified major regulator of housekeeping genes (Lam, 2012).

The promoters of NSL target genes exhibit prominent enrichment of certain histone modifications (H4K16ac, H3K9ac, H3K4me2, H3K4me3) as well as specific core promoter elements (such as DRE, E-box and motif 1). Furthermore, these genes display distinct nucleosome occupancy and dispersed promoter configuration characterized by multiple transcription start sites. The correlation between these promoter characteristics (well-defined chromatin marks, TATA-less DNA sequences and broad initiation patterns) was previously identified for housekeeping genes in mammals and flies, but how these promoter features are translated into gene transcription had remained elusive. This study now conclusively demonstrates that the NSL complex modulates transcription at the level of transcription initiation by facilitating pre-initiation complex loading onto promoters. Therefore, it is proposed that the NSL complex is a key trans-acting factor that bridges the promoter architecture, defined by the DNA sequence, histone marks and higher chromatin structures with transcription regulation of constitutive genes in Drosophila (see Summary model: NSL-dependent Pol II recruitment to promoters of housekeeping genes) (Lam, 2012).

The enrichment of DNA motifs on NSL target gene promoters in combination with the genome-wide Pol II binding data has established functional links between the motifs enriched on housekeeping genes and the NSL-dependent Pol II binding to promoters. The abundance of DRE motifs, for example, was found to be positively associated with the magnitude of Pol II loss upon NSL knockdowns. The DRE binding factor (DREF) interacts tightly with TRF2 to modulate the transcription of DRE-containing promoters in a TATA-box-independent fashion (Hochheimer, 2002). It is tempting to speculate that the NSL complex might also cooperate with the TRF2 complex to facilitate transcription in a specific manner, rendering DRE-containing promoters more sensitive to NSL depletions. As the NSL-bound promoters are associated with a large variety of transcription factors, it will be of great interest to study whether the NSL complex communicates with different transcription regulators, perhaps making use of distinct mechanisms (Lam, 2012).

In contrast to DRE, motif 1 (YGGTCCACTR) showed an opposing effect on Pol II recruitment to NSL-complex-bound genes as the presence of strong motif 1 sequences was associated with decreased Pol II loss upon NSL depletion. The mechanistic reasons for this remain unclear. However, one can envisage several possible scenarios. It is possible that motif 1 may recruit another transcription factor, which can also function to recruit the transcription machinery. Alternatively, the turnover of the transcription machinery might be slower on promoters containing strong motif 1 sequences. There is precedent for the transcription machinery having various turnover rates on different promoters. For example, in yeast, it has been shown that TBP turnover is faster on TATA-containing than on TATA-less promoters. It is therefore possible that certain levels of the initiation complexes may still be maintained on motif-1-containing promoters, even though the recruitment of the transcription machinery will be compromised in the absence of NSL complex. Further work is required to understand the importance of sequence determinants for NSL complex recruitment and this analysis sets the grounds for targeted experiments in the future (Lam, 2012).

Taking MOF-mediated H4K16 acetylation into consideration, a putative role of the NSL complex might be to coordinate the opening of promoter architecture by histone acetylation and the assembly of the pre-initiation complex (PIC). Coupling of histone acetylation and PIC formation has been described before. For example, TAF1, a component of TFIID, is a histone aceyltransferase. The SAGA complex, which contains Gcn5 and can acetylate H3K9, is reported to interact with TBP and other PIC components to regulate tissue-specific gene and the recruitment of P300 to the promoter and H3 acetylation have been shown to proceed binding of TFIID in a coordinated manner. H4K16ac is also well-known for its role in transcription regulation of the male X chromosome, yet how H4K16 acetylation and PIC assembly are coordinated remains elusive. Interestingly, absence of the NSL complex does not severely abolish H4K16ac from target genes. Since the turnover of H4K16ac on target promoter is unknown, it remains possible that H4K16ac could remain for some time at the promoter after the NSL complex is depleted. Further studies will be crucial in unraveling the functional relevance of H4K16 acetylation and NSL complex function on housekeeping genes (Lam, 2012).

Sequence signatures involved in targeting the Male-Specific Lethal complex to X-chromosomal genes in Drosophila melanogaster

In Drosophila melanogaster, the dosage-compensation system that equalizes X-linked gene expression between males and females, thereby assuring that an appropriate balance is maintained between the expression of genes on the X chromosome(s) and the autosomes, is at least partially mediated by the Male-Specific Lethal (MSL) complex. This complex binds to genes with a preference for exons on the male X chromosome with a 3' bias, and it targets most expressed genes on the X chromosome. However, a number of genes are expressed but not targeted by the complex. High affinity sites seem to be responsible for initial recruitment of the complex to the X chromosome, but the targeting to and within individual genes is poorly understood. This study has extensively examined X chromosome sequence variation within five types of gene features (promoters, 5' UTRs, coding sequences, introns, 3' UTRs) and intergenic sequences, and assessed its potential involvement in dosage compensation. Presented results show that: (1) the X chromosome has a distinct sequence composition within its gene features, (2) some of the detected variation correlates with genes targeted by the MSL-complex, (3) the insulator protein BEAF-32 preferentially binds upstream of MSL-bound genes, (4) BEAF-32 and MOF co-localize in promoters, and (5) that bound genes have a distinct sequence composition that shows a 3' bias within coding sequence. Although, many strongly bound genes are close to a high affinity site neither the promoter motif nor the coding sequence signatures show any correlation to high affinity binding sites (HAS). Based on the results presented in this study, it is believed that there are sequences in the promoters and coding sequences of targeted genes that have the potential to direct the secondary spreading of the MSL-complex to nearby genes (Philip, 2012).

This study has thoroughly investigated X chromosome sequence variation in D. melanogaster and related this variation to the targeting of the dosage compensation complex, using frequencies of two to six base pair sequence 'words' and multivariate statistical analyses. The advantage of this approach is that it is unbiased and focused on finding sequences with predictive value, rather than merely over-represented sequences. First, the genome sequence was divided into intergenic, promoter, 5' UTR, coding, intron and 3' UTR sequences. Interestingly, there is more divergence among these six sequence types or gene features than within the sequence types on different chromosomes. The findings also show that sequences are present in promoters and coding sequence that could be involved in the spreading of the MSL-complex from the high affinity sites on the X chromosome. The coding sequences that were identified share a similar 3' bias with the MSL-complex. Further, the highest scoring promoter sequences form the target motif of the insulator protein BEAF-32, and BEAF-32 mapping data indicate that this protein binds preferentially upstream of genes strongly bound by MSL (Philip, 2012).

Different gene features are known to vary in sequence composition, but their variation is not normally taken into account in attempts to discover new sequence motifs. This study shows the extent of this sequence variation, and that coding sequences have the most distinct sequence composition followed by 5' UTRs, 3' UTRs and promoters. This has important implications for studies of sequence variation and motif discovery; when groups of sequences are compared it is important to take gene features into account (e.g. when using the MEME option of discriminative motif discovery), otherwise the results may reflect differences in gene feature composition rather than biologically relevant sequence variation (Philip, 2012).

The separate analyses of the six gene features clearly show that the sequence composition of those in the X chromosome differs from the composition of corresponding features in all other chromosomes. This distinction of the X chromosome is mainly due to differences in frequencies of various di-nucleotides, many of which have been previously found to be enriched on X . These sequences could, in principle, be involved in recruiting X chromosome-specific factors, such as the MSL-complex. Apart from being dosage-compensated in males, the X-chromosome might also be under selective forces that do not act on the autosomes. Some of the sequence variation of the X-chromosome is likely a result of its evolution as a sex chromosome. The MSL-complex is the only known protein complex involved in dosage compensation in Drosophila with an X chromosome-specific distribution. This study has focused on the sequence variation that could be related to the targeting of this complex. It has been shown that the MSL-complex is initially targeted to X by binding to so-called high affinity sites (HAS) that contain the GA-rich MSL recognition element (MRE)]. The MSL-complex can be recruited to autosomes by inserting MRE-containing high affinity sites, but the mechanism involved in the spreading of MSL to X-chromosomal genes is under debate. This study has investigated whether sequence patterns may be involved in this spreading of the MSL-complex, as discussed below (Philip, 2012).

The genome distribution of the MSL-complex has been mapped in several studies. This study used the data from (Kind, 2009) to select genes that are expressed and strongly MSL-bound, expressed and weakly MSL-bound as well as unexpressed genes. This is the only currently available dataset where mapping of several MSL-complex components and transcription in mutants/knock-downs of MSL-components was done in parallel and in the same cell-type. When merging all strongly MSL-bound expressed genes into one observation and all weakly MSL-bound expressed genes into another, it was found that all six sequence types have sequences that differ between strongly bound and weakly bound genes. It was observed that sequence variation between expressed genes strongly bound and weakly bound by MSL complex is much higher than that between expressed and unexpressed genes on chromosome X. Further, expressed genes that are weakly bound by the MSL complex group more closely to unexpressed genes than to expressed MSL-bound genes in Principal Component Analysis (PCA) score plots. Therefore, the small but significant expression difference detected between the expressed genes that are strongly bound and weakly bound by the MSL complex did not have any major correlation on the sequence variation observed between the two groups. Sequence words extracted from PCA models of intron, 3' UTR and 5' UTR sequences were more GA, CA or adenine rich, in agreement with the previous identification of CA dinucleotide repeats, runs of adenines and GA-rich MRE motif from High Affinity Sites (HAS). It is conclude that there are differences in sequences of all six features between expressed genes that are strongly bound and weakly bound by the MSL-complex. However, these results merely identify sequence words that are overrepresented in groups of genes strongly or weakly bound by MSL. In order to search for predictive sequence patterns for MSL-binding to individual genes, Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA, was applied (Philip, 2012).

Using OPLS-DA, differences were examined between features of individual genes that are strongly MSL-bound and expressed versus weakly MSL-bound and expressed, sequence words with the highest predictive power were extracted, and attempts were made to combine them into more complex motifs using the algorithm described in this study. Interestingly, both coding sequence and promoter models yielded sequence words that could be used to predict the MSL-binding status of genes excluded from the modeling. Neither nucleotide content nor expression level significantly influence these promoter and coding sequence models and the top sequence words that were identified are only weakly overrepresented on the X-chromosome. It is concluded that promoters and coding sequences contain sequence signatures that are potentially involved in the spreading of the MSL-complex from high affinity sites. In principle, there may be motifs in unbound, expressed genes that block the binding of the MSL-complex, but no evidence was obtained for such motifs (Philip, 2012).

From the promoter model a motif was extracted which could be used to predict promoters of genes strongly bound by MSL. This motif proved to correspond to the targeting motif for the insulator protein BEAF-32, which binds to hundreds of sites across the genome, generally located upstream of active genes. Although the molecular mechanisms of BEAF-32 activity are unknown, it seems to be linked with active transcription (Jiang, 2009). In order to test whether the BEAF-32 protein itself is enriched at strongly MSL-bound genes BEAF-32 ChIP-chip mapping data obtained from modENCODE was used, and it was found that BEAF-32 preferentially binds proximal to transcription start sites of genes strongly bound by MSL. This exciting link between BEAF-32 and dosage compensation is supported by the observation that beaf-32 mutants have a male-specific defect in X-chromosome morphology. Further, Laverty (2011) found that reporters inserted on the X chromosome are better able to recruit the MSL-complex if they have binding sites for GAGA and DREF factors. The DREF binding site is very similar to the BEAF-32 binding site and although DREF might be involved in dosage compensation it is possible that increased BEAF-32 recruitment is the true cause of the effects observed by Laverty. However, since DREF has not been mapped genome wide the possibility cannot be excluded that the promoter motif correlate better with DREF. BEAF-32 is associated with active transcription and might facilitate the MSL-complex targeting of active genes. Since MSL-complex bound genes show MOF binding in the promoter and MOF clearly co-localizes with BEAF-32, it is hypothesized that BEAF-32 and MOF interact in promoters of MSL-complex bound genes. BEAF-32 is a DNA-binding protein and might recruit MOF to active genes on the X-chromosome, genes that are then targeted by the MSL-complex. However, further experimental efforts are needed to understand the link uncovered in this study between BEAF-32 and the MSL-complex (Philip, 2012).

The finding of sequence patterns that are predictive of MSL-binding genes within coding sequences is intriguing. Scoring the sequence words only in the transcribed strand or the correct frame did not improve the coding sequence model, suggesting that the relationships are not attributable to (for instance) specific variations in amino acid composition. Neither was any codon usage bias between strongly bound and weakly bound expressed genes found, nor any model correlation with expression and AT-content. However, Orthogonal Partial Least Squares Discriminant Analysis found that bound coding sequences are rich in AG di-nucleotides, which have been previously reported to be abundant in dosage-compensated chromosomes (Philip, 2012).

The MSL-complex binds to genes with a preference for exons. The relatively low binding to introns might suggest that the complex targets spliced RNA transcripts. However, it was recently found that the complex targets chromatin rather than transcribed RNA. The exon specificity could be explained by various chromatin factors, nucleosome density and/or sequence specificity. Variations in nucleosome density may partially explain the exon bias, as it is higher in exons and thus may provide more targets for H4K16 acetylation, a modification that is strongly linked to the MSL-complex. In addition, the MSL-complex binding profile clearly shows that it binds most strongly towards the 3' end of genes. Accordingly, the models predicted the MSL-binding status of genes better from the 3' thirds than from the 5' thirds of the coding sequences. This is in contrast to the lack of 3' bias of the [G(GC)N]4 motif reported in another study. Taken together, these results strongly indicate that the MSL-complex distribution within genes on the X-chromosome is influenced by the primary DNA sequence (Philip, 2012).

The MSL-complex evidently targets a limited number of High Affinity Sites along the X-chromosome. Although, many strongly bound genes are close to a HAS neither the promoter motif nor the coding sequence signatures found in this study show any correlation to HAS. Based on the results presented in this study, it is believed that there are sequences in the promoters and coding sequences of targeted genes that have the potential to direct the secondary spreading of the complex to nearby genes. However, a number of genes are dosage-compensated by MSL-independent mechanisms and expression on the X-chromosome is only reduced to ~80% of wild type levels in males when msl genes are mutated or knocked down using RNAi. Apart from the dosage compensation mediated by the MSL-complex there is evidence for a more general buffering system that targets haploid regions in the genome. So other, as yet unknown, factors are likely involved in compensating the X chromosome and these factors could potentially act on a number of levels, such as transcription regulation, mRNA export, mRNA stability and translation. The observed optimal codon usage on the X-chromosome likely represents compensation on the translational level. However, even if additional factors involved in dosage compensation remain to be discovered, this study shows that there are plenty of sequences within all types of gene features that could act as X-targeting elements (Philip, 2012).

Protein Interactions

Msl1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila

In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X-linked genes are transcribed at a higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage compensation and male viability. Here it has been shown that one of these proteins, Msl1, interacts with three others, Msl2, MSL3 and Mof. The latter is a putative histone acetyl transferase. Overexpression of either the N- or C-terminal domain of Msl1 has dominant-negative effects, i.e. causes male-specific lethality. The lethality due to expression of the N-terminal domain is reduced if msl2 is co-overexpressed. Msl2 co-purifies over a FLAG affinity column with the tagged region of Msl1, and both MSL3 and Mof co-purify with the FLAG-tagged Msl1 C-terminal domain. Furthermore, the Msl1 C-terminal domain binds specifically to a GST-Mof fusion protein and co-immunoprecipitates with HA-tagged MSL3. The Msl1 C-terminal domain shows similarity to a region of mouse CBP, a transcription co-activator. It is concluded that a main role of Msl1 is to serve as the backbone for assembly of the MSL complex (Scott, 2000).

In general, the amino acid sequences of the MSLs suggest regions or domains within the proteins that could be important for function in vivo. Indeed, this has been confirmed by mapping loss-of-function mutations to the domain, such as the helicase domain of MLE, the putative acetylase domain of Mof and the RING finger region of Msl2. The amino acid sequence of Msl1 is the least informative, containing no recognizable domains, although regions rich in acidic amino acids and possible PEST sequences have been identified. To identify regions within Msl1 that are important for function in vivo, it was determined which regions have dominant-negative effects when overexpressed. Two regions of Msl1, one near the N-terminus and the other at the C-terminus, are likely to be important for assembly of the MSL complex in vivo, because overexpression of either region causes male-specific lethality. Genetic evidence, decreased male viability of msl2 heterozygotes and increased male viability by co-overexpression of Msl2, suggests that the region of Msl1 at the N-terminus interacts with Msl2. This has been confirmed by co-purification of Msl2 with FLAG-tagged versions of Msl1 over FLAG affinity columns. Similarly, the C-terminal region of Msl1 interacts with both Mof and MSL3. Furthermore, expression of the C-terminal domain results in significant loss of Mof from the male X chromosome (Scott, 2000).

The N-terminal FN region of Msl1 that binds to Msl2 was chosen originally for expression in flies because it was predicted that almost half of FN (amino acids 96-172) would form a two-stranded, alpha-helical, coiled-coil structure. Coiled-coil structures are comprised of a heptad repeat (abcdefg)n where hydrophobic residues occupy positions a and d on the same side of the alpha-helix. The coiled-coil motif of GCN4 mediates dimerization. If a similar structure mediates the formation of the Msl1-Msl2 heterodimer, then part of the region of Msl2 that interacts with Msl1 should form a coiled-coil structure. The Ring finger domain region of Msl2 interacts with Msl1. It is predicted that the region immediately preceding the RING finger could form a coiled-coil structure. It is particularly significant that several of the mutations that disrupt the interaction with Msl1 in yeast introduce amino acid changes that either significantly disrupt the alpha-helix (leucine to proline) or introduce a charged amino acid into the predicted hydrophobic face of the alpha-helix. The RING domain is found in a number of proteins, including the V(D)J recombination-activating protein RAG1. The crystal structure of the RAG1 dimerization domain, which includes the RING finger, reveals that dimerization is stabilized by interaction between alpha-helices that form a hydrophobic core. The RING finger is thought to form the structural scaffold upon which the dimer interface is formed. It is tempting to speculate, by analogy with RAG1, that the association of Msl1 and Msl2 involves the interaction of amphipathic alpha-helices that depend on the RING finger domain. This could best be addressed by determining the crystal structure of the Msl1-Msl2 complex (Scott, 2000).

In vitro translated Msl1 C-terminal domain co-immunoprecipitates with in vitro translated HA·MSL3 but not HA·Mof. Thus, C interacts directly with MSL3 but the interaction with Mof requires either another factor present in fly extracts or post-translational modification of Msl1 or Mof. While the possibility of a nucleic acid component of the FC-Mof complex cannot be ruled out, the possiblity (post-translational modification of Msl1 or Mof) is favored since a silver stain of FLAG affinity-purified FC-Mof complex separated by SDS-PAGE shows only two main bands corresponding to the sizes expected for FC and Mof. The C-terminal domain of Msl1 is rich in serine and threonine residues, and contains several potential phosphorylation sites and a predicted PEST sequence. PEST sequences have been suggested to contribute to the instability of the Msl1 protein. However, the role of these sequences in Msl1 has not been determined. Indeed, an alternative function for the PEST sequences is suggested by the observations that the PEST domains of PU.1 and IB are required for their respective interactions with Pip and c-Rel. In both cases, phosphorylation of a serine residue within the PEST sequence is required for the respective protein-protein interactions. The recent finding that a serine/threonine kinase is associated preferentially with the male X chromosome raises the possibility that Msl1 or another MSL is phosphorylated by this enzyme (Scott, 2000).

In the sequential model for assembly of the MSL complex, the first step involves the binding of the Msl1-Msl2 complex to several 'high affinity' sites on the male X chromosome. Since the localization of both Mof and MSL3 to the X chromosome requires mle+ function, this suggests that the association of Mof and MSL3 with the Msl1-Msl2 complex is Mle dependent. Mle could either bind directly to Mof and/or MSL3, or somehow stabilize the MSL complex together with roX-1 RNA. In support of the latter model, Mof and MSL3 bind directly to the C-terminal domain of Msl1. Furthermore, Mle does not co-purify with an FC-Mof-MSL3 complex over an affinity column. However, the affinity chromatography experiments were designed to maximize the likelihood of detecting protein-protein association and are not quantitative. It is possible that Mof and MSL3 may have a higher affinity for the C-terminal domain of Msl1 than full-length Msl1. Thus, one possible mechanism is that in vivo the C-terminal domain of Msl1 is not freely available to bind to Mof and/or MSL3, and that the binding of Mle to the Msl1-Msl2 complex causes a conformational change in Msl1, such that the C-terminal domain becomes more accessible (Scott, 2000).

Previous searches of the protein sequence database with the complete Msl1 sequence have failed to identify any significant similarities. However, when a search is carried out with just the C-terminal domain sequence, some similarity is found to a 254 amino acid region of mouse CBP. Although the similarity is not high, given that the similarity extends across almost the entire C-terminal domain of Msl1, and that both CBP and the Msl1 C-terminal domain bind to histone acetyl transferases (or putative histone acetyl transferases), it is thought that this homology may be significant. If this similarity reflects a conserved function, then it would be predicted that the Msl1-similar region of CBP, which has no known function, would associate with either an Mof-like histone acetyl transferase or an MSL3-like protein in mammalian cells (Scott, 2000).

It is not known how the MSL complex binds to the male X chromosome. None of the MSLs contain a recognizable DNA-binding motif. The F84 version of Msl1, lacking the first 84 amino acids, binds to Msl2, MSL3 and Mof but does not bind preferentially to the male X chromosome. This suggests that the male lethality that results from overexpression of F84 is due to this protein being able to bind to three MSLs, but not being able to bind to the X chromosome because the first 84 amino acids of Msl1 are required for recognition of the X chromosome. Alternatively, the lack of binding of F84 to the male X chromosome could be because the beginning of Msl1 is required for assembly of the MSL complex in vivo. However, if so, then it would be expected that F84 would have bound to the 'high affinity' sites since F84 does bind to Msl2. Assuming that Msl1 and Msl2 are the only components of the high affinity complex, it would then appear more likely that the first 84 amino acids of Msl1 are required for X chromosome binding rather than complex formation. However, there are several lines of evidence that suggest that the roX RNAs are part of the MSL complex, which raises the possibility that one or both of the roX RNAs could be part of the high affinity complex. Thus it will be of interest to determine if the MSL complex containing the F84 protein binds to roX RNA with a lower affinity than the complex containing full-length Msl1 (Scott, 2000).

Activation of transcription through histone H4 acetylation by Mof, an acetyltransferase essential for dosage compensation in Drosophila

Analysis of the Mof amino acid sequence reveals a putative acetyl-coenzyme A binding pocket close to the C terminus as well as zinc finger and chromodomain motifs in the central part of the protein. In the mof-1 allele, which gives rise to the male-specific lethality and leads to loss of acetylation of H4 at lysine 16 on male X chromosomes, glycine 691 is replaced by a glutamate in the presumed acetyl-coenzyme A binding pocket. In order to analyze the activity of Mof, the full-length protein was expressed in E. coli and purified to homogeneity. The mof-1 allele (hereafter referred to as Mof-G691E) was also produced as a control. To analyze the functional status of the recombinant enzymes, histone acetyltransferase assays were performed using recombinant histones and derivatives lacking the N- and C-terminal domains containing the physiological acetylation targets. Efficient incorporation of 3H-labeled acetyl groups into protein has identified full-length Mof as a robust histone acetyltransferase, whereas Mof-G691E is about ten times less active. Histones lacking their N-terminal 'tails' are only poor substrates. Autoacetylation of Mof may contribute to the residual incorporation of [3H]acetyl into protein in reactions containing truncated histones (Akhtar, 2000a).

Mof exhibits a preference for free histones, and shows a clear substrate preference for histone H4 but also acetylates H3 to some extent. No acetylation of histone H2A and H2B has been observed using the intact protein. However, an N-terminal deletion mutant of Mof that lacks the first 352 amino acids (Mof-N352) is able to acetylate histone H2A, suggesting that the N terminus of Mof may be involved in substrate selectivity. On Triton-Acid-Urea (TAU) gels, the acetylated histone H4 resolves into a mixture of isoforms with a preponderance of the monoacetylated isoform over di- and tri-acetylated species (Akhtar, 2000a).

In order to test Mof activity on a more physiological substrate, nucleosomal arrays were assembled on linear DNA coupled to paramagnetic beads using NAP-1 as a histone deposition vehicle, and this was used as a nucleosomal substrate for acetylation. The analysis of the acetylated histones on the TAU gel demonstrates that nucleosomal arrays serve as substrates for Mof and reveals an increased substrate specificity. When compared to the acetylation of free histones, predominantly monoacetylated histone H4 is obtained. To test directly whether Mof can acetylate lysine 16 (K16) of H4, the products of the acetylation reactions were subjected to Western blot analysis and probed with antibodies against specific histone isoforms. This analysis reveals a clear substrate preference of H4 lysine 16 over lysine 12, when either free histones or nucleosomal substrates are acetylated. The faint signal obtained using the H4K12 antibody is not due to the poor sensitivity of the antibody since the antibody easily detects acetylation of lysine 12 by HAT1. Taken together, these experiments demonstrate that recombinant Mof is able to monoacetylate histone H4 within a nucleosomal array, preferentially at lysine 16. and that the mof-1 mutation largely abolishes this activity. It is therefore very likely that Mof is directly responsible for the acetylation pattern observed on the male X chromosome (Akhtar, 2000a).

To determine whether the poor acetyltransferase activity of Mof-G691E is solely due to a defect in the catalytic mechanism or whether the interaction with the substrate is also affected, a series of substrate binding experiments were carried out. For this purpose, chromatin was assembled on linear DNA attached to paramagnetic beads using the Drosophila embryo extract system. The washed chromatin beads were then incubated with Mof or Mof-G691E; unbound enzyme was washed out, and bound protein was subjected to Western blot analysis. As a control, binding of the proteins to free DNA was assayed. Interaction of wild-type Mof with free DNA is barely detectable, while a small but significant amount of DNA-bound Mof-G691E is observed. In contrast, both wild-type and Mof-G691E are retained on the chromatin beads. Significantly, the interaction of Mof-G691E with chromatin is not impaired; rather, the mutant enzyme interacts consistently better with the chromatin substrate. The Mof-DeltaN352 deletion derivative is also able to interact with nucleosomes (Akhtar, 2000a).

In order to ensure that the Mof-nucleosome interaction is direct and not mediated by other factors endogenous to the chromatin assembly extract, an interaction of Mof with mononucleosomes, reconstituted by salt gradient dialysis from free histones, was sought. As before, an interaction of Mof with the nucleosome, but not with free DNA, was detected. Similar results were obtained when nucleosomal arrays were assembled from pure histones using NAP-1 as chaperone. These results demonstrate a direct interaction of Mof with the substrate and suggest that the phenotype of the mof-1 mutation is due to an impaired catalysis rather than a failure of interaction with the substrate (Akhtar, 2000a).

In vivo, acetylation of histone H4 at lysine 16 correlates with an increased transcriptional activity. The ability to acetylate reconstituted chromatin with recombinant Mof allowed for a direct test of whether acetylation of histone H4 is cause or consequence of transcriptional activity. A cell-free transcription system derived from Drosophila embryos has been shown to responded to the acetylation status of histones. However, in these experiments, histone H4 is tetra-acetylated and other histones are also modified. Nucleosome arrays were assembled on the immobilized template, and the extent of the array was monitored by micrococcal nuclease digestion. The immobilized array was washed in buffer and used as a substrate for acetylation by recombinant wild-type or mutant Mof. Following acetylation, the template was incubated with transcription extract and the resulting transcripts were monitored by primer extension. Assembly of nucleosomes leads to tight repression of an hsp26 template. Acetylation of lysine 16 of H4 with wild-type Mof leads to a remarkable derepression, whereas the acetylation-deficient enzyme is unable to facilitate transcription. If histones are acetylated prior to assembly and then used to reconstitute nucleosomes, the resulting chromatin is also active. Reconstitution of transcriptionally permissive nucleosomal arrays requires acetyl-CoA, in addition to a functional acetyltransferase. The bulk of the effect of Mof is apparently due to acetylation of histone H4 rather than modification of the transcription apparatus since transcription of naked templates is only marginaly affected by the acetylation reaction (Akhtar, 2000a).

The biochemical analysis suggests that Mof may function as a chromatin-specific activator in vivo. To directly test this hypothesis, Mof was targeted to a responsive promoter in yeast by fusing the enzyme to a heterologous Gal4 DNA-binding domain. A similar strategy has previously been used to target general transcription factors or transcription activators to promoters bearing Gal4 binding sites in vivo, making convenient use of the matchmaker system of plasmids and yeast strains (Clontech). In short, the cDNA encoding full-length Mof or Mof-G691E was fused in frame to the 3' end of the yeast Gal4 DNA-binding domain (amino acids 1-147) in an expression vector. These constructs were then transformed into a yeast strain in which the only his3 gene had been engineered to be under the control of the Gal1 promoter. Deletion of the endogenous Gal4 activator renders this promoter dependent on exogenous activators that contain a Gal4 DNA-binding domain. Under these circumstances, survival of yeast under conditions of histidine starvation can be directly correlated to the extent by which the his3 promoter is activated by factors interacting with the Gal4 binding site. A more quantitative assessment of his3 transcription can be obtained from experiments in which yeast are grown in varying amounts of aminotriazole, a histidine analog. Survival of yeast in medium lacking histidine but containing 25 mM aminotriazole and containing 60 mM aminotriazole is supported by the Gal4(1-147)-Mof fusion protein. In contrast, neither the Gal4(1-147)-G691E fusion nor the Gal4-DBD alone are able to activate the Gal1 promoter. These results indicate that Mof can serve as a transcription activator in yeast and that this function relies on an intact acetyl-CoA binding site. The data also suggest that targeted acetylation of histone H4 at lysine 16 suffices to reverse the inhibitory effects of native yeast chromatin (Akhtar, 2000a).

The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation

A cDNA fragment containing the putative Mof catalytic domain (aa 518 to 827) was expressed and it was determined that the recombinant peptide can acetylate Drosophila histones with a preference for histone H4. This pattern is similar to that for a related yeast protein, Esa1p. Active full-length Mof could not be expressed, Mof was isolated as a component of a partially purified MSL complex. Tissue culture cells were used for the initial characterization of the MSL complex. S2 cells are male, based on the following criteria: they do not express the Sxl (Sex-lethal) gene product, which is necessary for female differentiation, and they express Msl2, a limiting component of the dosage compensation machinery whose synthesis is prevented by Sxl. S2 cells can be stably transfected, allowing the use of commercially available antibodies recognizing epitope tags. Transient transfection of S2 cells with Msl2 tagged at its carboxy terminus with the HA epitope reveals that the localization of the HA epitope is coincident with the location of endogenous Mof. After selection with hygromycin, most cells exhibit HA staining on the male X chromosome, the location of which is revealed by antibodies to H4Ac16 (Smith, 2000).

Immunoprecipitation of nuclear extracts from Msl2-HA cells with the 12CA5 (anti-HA) antiserum results in the same proteins as those obtained from S2 cells with an Msl1 antiserum. In salivary gland nuclei, Mle is released from the male X chromosome with RNase treatment. Furthermore, the roX1 and roX2 RNAs are found along the X chromosome with a distribution that mimics that of the MSL complex. Therefore, attempts were made to obtain a partially purified complex containing Mle and a roX RNA and to see whether the presence of either of these components depended on the other. 'RNA-friendly' conditions were developed to increase the chances of purifying Mle and roX RNA-containing complex. The method involved a cell line expressing Flag-tagged MSL3 and sonication under low-salt conditions, immunoprecipitation with Flag antibodies followed by peptide elution, and a second immunoprecipitation with either an MSL antibody or with the corresponding preimmune serum. By using this two-step procedure, a faint band was detected by silver staining that corresponds to Mle protein. Clear enrichment of Mle was seen in the Msl1 immunoprecipitate relative to the preimmune serum. However, following a brief treatment with 0.4 M NaCl, the Mle levels were significantly reduced (Smith, 2000).

To determine if roX RNAs are expressed in S2 cells, Northern blot analysis was performed and it was observed that roX2, but not roX1, is expressed in these cells, consistent with the observation that roX1 is dispensable in flies. The size of the major roX2 transcript observed by Northern analysis was ~ 600 nucleotides. To test if roX2 RNA is present in the Mle-containing immunoprecipitates, RNA was extracted from the immunoprecipitation pellets and a RT-PCR was performed with roX2-specific primers in the linear range. The results show a clear enrichment of roX2 RNA in the immune over the preimmune serum precipitates (Smith, 2000).

The MSL complex specifically acetylates lysine 16 of histone H4. When MSL-containing immunoprecipitates were incubated with nucleosomal substrates, significant acetyltransferase activity toward histone H4 was detected. Msl1 immunoprecipitates from S2 nuclear extracts and 12CA5 immunoprecipitates from Msl2-HA nuclear extracts contain H4-specific acetyltransferase activity, while control immunoglobulin G or 12CA5 immunoprecipitates from S2 cells do not. To demonstrate that the acetyltransferase activity of the MSL complex is ascribable to Mof, complexes were purified containing either wild-type Mof or a protein produced by the mutant allele mof1. This allele is a point mutation resulting in a glycine-to-glutamic acid replacement at the most highly conserved residue of the acetyl-CoA binding domain (G691E). Wild-type Mof-HA or G691E Mof-HA were overexpressed in S2 cells and immunoprecipitated with anti-HA antibodies to obtain complexes with only transfected Mof fusion proteins. Immunoprecipitates from G691E cells have markedly reduced acetylation, consistent with the conclusion that Mof is the sole acetyltransferase in the MSL complex (Smith, 2000).

Given the specificity of the MSL complex toward H4, it was of intereset to determine which particular lysines were acetylated. When acetylated histones were separated by acid-urea gel electrophoresis, predominantly monoacetylated H4 was detected. A similar acid-urea gel was blotted to PVDF, and the mono-acetylated band was subjected to microsequencing. Counts were found at lysine 16, while other potential acetylation sites (at position 5, 8, or 12) were unlabeled. This result provides a causative link between the presence of histone H4 acetylated at lysine 16 and the MSL complex on the X chromosome in Drosophila males (Smith, 2000).

Histone acetylation and gene expression analysis of Sex lethal mutants in Drosophila

In this report, an examination was carried out of how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in SxlM mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases relatively little effect is found on X chromosomal gene expression (Bhadra, 2000).

In SxlM males, in which Sxl is ectopically expressed, the slow accumulation of the Sxl protein during development eventually prevents significant Msl-2 expression and hence reduces the Msl complex association with the X chromosome. This results in X and autosomal gene expression quite similar to that found in the mle mutant, i.e., little response of the X-linked genes, but an overall increase in autosomal expression. Similarly, the association of the Msl proteins in ~50% of the cells of heteroallelic Sxl females causes sequestration of MOF to the two X chromosomes. This sequestration reduces the H4Ac16 on the autosomal loci, resulting in a lowered expression. There is a concomitant increase of acetylation on the X chromosome, but little overall response of the X-linked genes (Bhadra, 2000).

In the SxlM and Sxlf;mle genotypes a low level of Msl-1/Msl-2 shows chromosomal binding to some degree, although binding takes present in distinct patterns in the two cases. This low level of binding, however, is insufficient to sequester all the available Males absent on the first (Mof) present in the cell. Previous and present data suggest that in the absence of a functional Msl complex, Mof still associates with the chromosomes and is active in modifying H4. The reduced amount of Msl-1/Msl-2 appears saturated with Mof, allowing the remainder to be uniformly distributed across the genome, which modulates gene expression (Bhadra, 2000).

The general trends of X and autosomal gene expression in SxlM and Sxlf mutants match the published autoradiographic data when the latter is considered as absolute levels rather than relative X to autosomal ratios. Autoradiographic grain counts over the X chromosome were changed very little in SxlM larvae compared to normal, but the counts over the autosomes were increased. Conversely, in Sxlf females, the autosomal counts were lower than in normal females with little change over the X. Because the data reported here are anchored to rRNA levels, which in turn do not vary per unit of DNA, the 'per cell' expression trends can be determined on an absolute rather than a relative comparison basis; they indicate greater changes of the autosomes compared to the X chromosome (Bhadra, 2000 and references therein).

The loss of individual components of the Msl complex in the msl mutant males releases the Mof acetylase from the X and a uniform H4 acetylation distribution results. Accordingly, autosomal gene expression is generally increased, reflecting an inverse effect of the X on the autosomes, because the normally sequestered acetylase is now dispersed: this results in higher acetylation levels on the autosomes. An increase or decrease of acetylation level on the X is not reflected in major changes in gene expression, suggesting that some member of the Msl complex insulates genes on the X from responding to the much increased acetylation level (Bhadra, 2000 and references therein).

The collective data indicate that models that posit an association of the Msl complex with a gene for dosage compensation to occur are not supported. First of all, genetic destruction of the complex does not eliminate dosage compensation of most X-linked genes. However, one could perhaps argue that this action eliminates compensation of the regulatory genes on the X, which, because they are now dosage dependent, will compensate most of the 'housekeeping' genes that were assayed. This alternative is not favored for three reasons. (1) Ectopic expression of MSL2 in females as a transgene or in Sxlf mutants (present study) have the Msl complex present on their Xs but gene expression in general is not increased as predicted if the MSL complex alone conditions hyperactivation. (2) Dosage compensation also occurs in metafemales (3X chromosomes with diploid autosomes), where there is no complete complex and this compensation is related to that occurring in males. (3) Autosomal insertions of many X-derived genes still exhibit some degree of compensation despite the fact that these genes have no association with the Msl complex. Thus, there are several circumstances known in which compensation occurs without the Msl complex. The function of the Msl complex on the X chromosome appears to be to inhibit the response of most X-linked genes to high levels of histone acetylation (Bhadra, 2000 and references therein).

When these data are taken together along with previous studies on mle, a consistent model is supported, indicating that the effect of the Sex lethal gene is mediated through its control of the presence or absence of Msl-2. When Msl-2 protein is expressed, the sequestration of the MSL complex occurs with a resultant increase of H4Lys16 acetylation on the X at the expense of acetylation on the autosomes. In the absence of MSL-2, there is a uniform genomewide distribution of Mof and H4Lys16 acetylation. In general, gene expression on the autosomes responds positively to the level of acetylation, but the X is refractory to it in the presence of the Msl complex. In this way, the twofold inverse-dosage effect of the X is used to achieve a proper level of dosage compensation, but the effect on the autosomes is diminished. Thus, as the heteromorphic sex chromosomes have evolved, both the X and the autosomes have maintained nearly equal expression between the sexes (Bhadra, 2000).

Targeting the chromatin-remodeling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities

Dosage compensation in Drosophila is mediated by a multiprotein, RNA-containing complex that associates with the X chromosome at multiple sites. Investigated were the roles that the enzymatic activities of two complex components, the histone acetyltransferase activity of Mof and the ATPase activity of Mle, may have in the targeting and association of the complex with the X chromosome. Mle and Mof activities are necessary for complexes to access the various X chromosome sites. The role that histone H4 acetylation plays in this process is supported by the observations that Mof overexpression leads to the ectopic association of the complex with autosomal sites (Gu, 2000).

The normal association of the MSL complex at hundreds of sites along the X chromosome appears to be a process with at least three major steps. The first is the formation of functional complexes at the two entry sites where the roX RNAs are transcribed. It should be noted that although the Msl1 and Msl2 proteins are able to access the X chromosome at the entry sites and to recruit Mle, further complex assembly can only occur in the presence of the roX RNAs. This contention is supported by the observation that, in the absence of the two roX genes, no complex is seen to form in embryonic stages where it is normally evident. A caveat is that removal of roX2 was accomplished by using a deletion of such size that other roX-like genes or other unidentified components of the complex or genes whose product is required for complex stability, closely linked to roX2, may have been deleted as well. In any event, since the roX RNAs are unstable unless they are associated with the complex, the process of assembly can proceed only at the entry sites containing the roX genes. Once complexes are formed, they access the X chromosome through all of the entry sites, presumably via the affinity of their Msl1/Msl2 components for these sites. Finally, the complexes spread from the entry sites to the many other sites along the X chromosome where they are normally found. This last step requires the histone acetyltransferase activity of Mof. It is suggested that the spreading process involves the acetylation of neighboring nucleosomes, thereby altering the conformation of adjacent chromatin and rendering it more accessible to the entry of additional MSL complexes. The latter may require the presence of acetylated histone H4 tails in order to stabilize their chromatin association. This conclusion is consistent with the observation that, in S2 cells overexpressing Mof, the resulting abnormal ectopic acetylation of histone H4 at Lys16 leads to the association of the MSL complex along autosomal chromatin. This may mirror the normal situation in vivo where complexes, initially attracted to the entry sites, acetylate histone H4 at Lys16 and thereby make adjacent chromatin regions accessible to more complexes. The affinity of the MSL complex for histone H4 tails implied in this model is reminiscent of a similar role played by histone tails in the spreading of complexes containing SIR2, 3 and 4 during the silencing of mating type loci and telomeric heterochromatin formation in yeast. Although critical to the spreading process, the role played by the ATPase function of Mle, either directly or in conjunction with roX RNA, is not sufficiently understood to be incorporated in the model (Gu, 2000).

It is thought that the process just described can provide the following explanations for the gaps in Msl binding that occur along the X chromosome, or at ectopic autosomal sites where the complex has been caused to form at the site of a roX transgene. It is possible that the spread of H4 acetylation and complex association may be stopped by some insulator or some as yet uncharacterized boundary elements. This would not necessarily require that the entry sites be entirely responsible for the pattern seen along the X chromosome. The interphase chromosome is believed to consist of a series of rosettes formed by loops of the chromatid fiber anchored to a central core by dispersed regions that have affinity for one another. In such an arrangement, a cluster of complexes that have been stopped by some boundary element could acetylate the nucleosomes on a neighboring loop, initiating a spreading process on the other side of a gap (Gu, 2000).

The above considerations raise a number of questions that remain to be resolved. Is the pattern of complex association on the X chromosome tissue specific? Is it dependent on a tissue-specific distribution of the entry sites (other than those containing the roX loci, which must remain invariant in all tissues)? Is the tissue-specific distribution established when the complex first forms in early embryogenesis and is the pattern perpetuated through the mitotic divisions that give rise to a particular tissue? To answer these questions will require a thorough melding of cytological and biochemical approaches (Gu, 2000).

The MRG domain mediates the functional integration of MSL3 into the dosage compensation complex

The male-specific-lethal (MSL) proteins in Drosophila melanogaster serve to adjust gene expression levels in male flies containing a single X chromosome to equal those in females with a double dose of X-linked genes. Together with noncoding roX RNA, MSL proteins form the 'dosage compensation complex' (DCC), which interacts selectively with the X chromosome to restrict the transcription-activating histone H4 acetyltransferase MOF (Males-absent-on-the-first) to that chromosome. MSL3 is essential for the activation of MOF's nucleosomal histone acetyltransferase activity within an MSL1-MOF complex. By characterizing the MSL3 domain structure and its associated functions, it has been found that the nucleic acid binding determinants reside in the N terminus of MSL3, well separable from the C-terminal MRG signatures that form an integrated domain required for MSL1 interaction. Interaction with MSL1 mediates the activation of MOF in vitro and the targeting of MSL3 to the X-chromosomal territory in vivo. An N-terminal truncation that lacks the chromo-related domain and all nucleic acid binding activity is able to trigger de novo assembly of the DCC and establish an acetylated X-chromosome territory (Morales, 2005).

The MSL1 interaction surface maps to the C-terminal half of MSL3. This part of MSL3 is characterized by similarities to the MRG domain that subsumes MRG15, MSL3, and related proteins in multiple species into the so-called MRG family. The msl3 gene is related to the Drosophila mrg15 gene, suggesting an early gene duplication event. Accordingly, MRG sequences in MSL3 are highly conserved between D. melanogaster and Drosophila virilis. The MRG domain consists of three blocks of strong sequence similarity separated by short amino acid stretches of lesser conservation. Interestingly, these 'linker' regions harbor rather long insertions in MSL3 of flies and humans. The C terminus of MSL3 may thus be organized by folding of MRG signature sequences, which are disconnected in the primary sequence, into a compact unit from which the MSL3-specific structures 'loop out.' Consistent with this idea, it was found that every deletion in the C terminus of MSL3 compromises interaction with MSL1. Most of these deletions affect at least one of the blocks of MRG sequence similarity, most likely leading to global misfolding. However, one deletion that abolishes MSL1 binding (Delta328-433) selectively removed MSL3-specific sequences between two MRG blocks. There is considerable conservation of these sequences in the Drosophila species for which sequence information has recently become available, suggesting a conserved function, but whether this sequence contains a dedicated MSL1 interface remains to be explored. In any case, this analysis suggests that the MRG sequence similarity reflects a functional domain. The MRG-MSL1 contact is essential for targeting MSL3 to the X-chromosomal territory, confirming the functional importance of the interactions defined in vitro. It is suggested that MRG modules in other MRG family members may also constitute protein-protein interaction units (Morales, 2005).

In vitro analysis showed that MSL3 interacts better with single-stranded nucleic acids than with dsDNA. The significance of ssDNA interaction, if any, is unclear at the moment. In contrast, there is evidence that MSL3 interacts with roX RNA in vivo and in vitro, but the domain involved in RNA binding had not been defined. Biochemical analysis demonstrates that the nucleic acid binding structures reside in the N-terminal half of MSL3, which also contains the CRD. Previously, it has been suggested that RNA interaction of MSL3 is affected by its acetylation at lysine 116, close to the CRD. In the current studies, a fragment comprising the first 140 amino acids (and hence the CRD as well as K116) was not sufficient for nucleic acid binding, but sequences up to amino acid 259 contributed significantly. To what extent the CRD of MSL3 contributes to RNA binding needs to be established. The CRDs of MSL3 and MOF appear more related to each other than to canonical chromodomains. They lack the alpha-helix supporting the ß-sheet bundle and aromatic residues that may be involved in recognition of methylated histone N termini. The CRD of MOF also appears not to be sufficient for RNA binding. A further interesting similarity between MOF and MSL3 is that nucleic acid interactions are not the primary targeting determinant for either MOF (Morales, 2004) or MSL3. Although impairment of the CRDs leads to somewhat increased binding of the corresponding GFP fusion protein to autosomes, their concentration on the X-chromosomal territory is still obvious. However, the CRDs and noncoding RNA may have functions that are not assayed for in simple recruitment experiments. It is also possible that the CRDs of MOF and MSL3 provide partially redundant functions for DCC assembly. In contrast, mutations in MOF or MSL3 that abrogate their interaction with the C terminus of MSL1 prevent faithful recruitment to the X chromosome. Obviously, the recruitment assay employed may just reveal the strongest binary interaction that MSL3 or MOF are involved in. However, the fact that overexpression of an MSL3 lacking all nucleic acid binding capacity was able to complement an MSL3 deficiency and to trigger the accumulation of MOF and H4K16 acetylation on the X-chromosomal territory emphasizes the importance of the MSL protein interactions for the assembly of a functional DCC (Morales, 2005).

MSL complexes can be formed in vitro in the absence of RNA. A deficiency of roX RNA in vivo can be partially overcome by overexpression of the 'platform' proteins MSL1 and MSL2. It is possible that transient overexpression of MSL3 overcomes the RNA requirement and that under normal conditions of limiting MSL protein concentrations RNA is required for faithful DCC assembly (Morales, 2005).

The remarkable stimulation of MOF's HAT activity upon association of MSL3 with an MSL1-MOF complex was not due to enhanced binding of MSL3 to nucleic acids but rather required contact of MSL3 with the MSL1 scaffold. MOF and MSL3 are brought into proximity by interaction with adjacent structures in the C terminus of MSL1 (Morales, 2004). It is possible that the MSL1 scaffold stabilizes an otherwise transient and therefore nonproductive direct contact between MSL3 and MOF (Morales, 2004). The existence of such a contact has been inferred from the fact that MSL3 can be acetylated by MOF. However, when it comes to acetylation, MSL1 is a much better substrate for MOF than MSL3 (Morales, 2004). The new data reinforce a previous model of an acetylation 'checkpoint' built into DCC assembly. Accordingly, the regulatory potential of H4K16 acetylation would only be fully realized upon binding of MOF with MSL1 and the completion of the complex by association of MSL3 (Morales, 2004). Such a checkpoint would render full activation of MOF dependent on proper DCC assembly and hence 'maleness' and serve to restrict the critical epigenetic mark to the X chromosome (Morales, 2005).

Targeting determinants of dosage compensation in Drosophila

The dosage compensation complex (DCC) in Drosophila melanogasteris responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. Chromatin immunoprecipitation was used to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and it has been demonstrated that these fragments possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, evidence is provided that this range of recruitment potential is due to differences in affinity of the complex to these sites. It was also established that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, a number of short sequence motifs have been uncovered, that in combination may contribute to DCC recruitment. These findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high- and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs (Dahlsveen, 2006).

Using a ChIP strategy, several new DCC binding fragments have been identified and it has been demonstrated that they possess a wide range of potential to recruit the DCC. Because the majority of the isolated candidate fragments co-map with endogenous DCC binding sites at the resolution afforded by staining of polytene chromosomes, it is believed that the ChIP selection procedure is appropriate. By tuning DCC levels in vivo, it was concluded that the difference in recruitment ability is due to different affinity of the DCC for these fragments. At limiting concentrations of complex, only the sites of highest affinity are occupied. Conversely, at non-physiologically high concentrations of DCC, even 'cryptic' binding sites on autosomes are recognized by the complex. This suggests, in accord with previous observations, that selective interaction of the DCC with the X chromosome is a function of tightly controlled levels of complex components that are adjusted to assure interaction with binding sites of varying affinity clustered on the X, but insufficient to occupy cryptic sequences on autosomes. These data are also in broad agreement with observations that numerous sites on the X chromosomes contain DCC binding determinants. These determinants are not all equal, but represent a diverse set of DCC targets that differ by a wide range of affinities for the complex, as expected from a sequence determinant that during evolution became gradually enriched on the X chromosome (Dahlsveen, 2006).

The use of the term 'chromatin entry sites' for the subset of DCC binding sites that are still occupied by partial complexes in the absence of MSL3, implies that these sites were somehow qualitatively and perhaps functionally distinct from the remaining sites that only attract the intact complex. Although it is possible that not all DCC binding sites are functionally equivalent, the characterization of several new examples of both types of DCC binding sites suggests support for the 'affinities model'. According to this model, 'chromatin entry sites' are not qualitatively different from other sites, but only represent those sites with the highest affinity for the complex. A prediction from this model that is further substantiated by the results is that non-functional complexes that lack MSL3 or the acetyltransferase activity of MOF have lower affinity for target sites. Only those determinants with highest affinity for the DCC are able to recruit partial complexes in the absence of MSL3. Sites with slightly lower affinity are still able to recruit the complex in the mof1 mutant. Because the interaction of the DCC with the X chromosome is thought to be largely mediated by MSL1 and MSL2, it remains to be explored whether MSL3 and the acetylase activity of MOF affect the active concentration of MSL1 and MSL2 or lead instead to the adoption of a high-affinity conformation of the complex. Conversely, it remains to be seen if over-expression of MSL1 and MSL2 in the msl-31 and mof1 mutants would allow partial complexes to bind additional sites. In this respect it is intriguing that the mutation of both roX RNAs, which is presumed to lead to incomplete and non-functional complexes, can be partially rescued by the over-expression of MSL1 and MSL2 (Dahlsveen, 2006).

During analysis of DCC recruitment to high-affinity sites inserted into autosomes of wild-type males, an additional band of DCC binding was observed close to the insertion site in three independent cases (one insert each of DBF9, DBF5, and DBF7). Such minimal and rare 'spreading' has previously been observed for ectopic insertions of the 18D high-affinity site and from roX transgenes in the wild-type male background. This study now reveals that these additional DCC binding sites are not a result of random spreading, but are most likely due to interaction of the DCC with one of the low-affinity sites on autosomes that happened to reside close to the insertion site. These sites are usually observed only when the DCC concentrations are globally increased by over-expression of MSL1 and MSL2. Accordingly, it is suggested that the autosomal insertion of a high-affinity DCC binding site leads to a local rise in complex concentration, which allows these low-affinity sites to be recognized by the DCC even in wild-type males. However, additional requirements must clearly be met to allow low-affinity sites to profit from local increases in complex concentration, since not all ectopic high-affinity sites support the phenomenon. Permissive conditions may include active transcription or the presence of specific epigenetic marks (Dahlsveen, 2006).

It is envisioned that the clustering of DCC binding determinants of high and intermediate affinity on the X chromosome (combined with the transcription of the roX RNAs) elevates the concentration of the DCC within the X chromosomal territory and ensures the occupancy of lower-affinity sites in a context-dependent manner. This may explain the observation that autosomally derived transgenes often acquire dosage compensation. The transgenes may contain cryptic DCC binding determinants and may thus acquire binding if placed in the context of the X chromosomal territory. Conversely, an X chromosomal fragment that harbors only low-affinity sites may not be recognized if translocated to an autosomal context, and the fragment DBF3 may be an example for such a scenario. The presence of a large number of low-affinity sites may also contribute significantly to restricting the binding of the DCC to the X chromosome (Dahlsveen, 2006).

The term 'spreading' has been used to describe the appearance of additional bands of DCC binding around autosomal insertions of roX cDNAs or fragments derived thereof. However, extensive, long-range spreading from roX transgenes, which leads to the appearance of many ectopic DCC bands at greater distances from the insertion sites, occurs only under unusual conditions and depends on the transcription of the roX RNA rather than the DCC binding sites on DNA. Long-range spreading of the complex also does not occur into autosomal chromatin translocated to the X chromosome. It is suggested that large translocations maintain their original chromosomal context (DCC enriched or not), and therefore no redistribution of DCC over the new chromosomal junction is observable at the resolution of the polytene chromosomes. Importantly, this study does not address the higher-resolution distribution of the DCC within a chromosomal band. It is possible that such a band contains many individual binding sites, also of varying affinity. At this resolution, the term 'spreading' may characterize the local diffusion of the DCC from high- to low-affinity sites. This study does not exclude this type of spreading, or indeed any other kind of complex distribution within a chromosomal band. High-resolution ChIP analyses will be necessary to resolve the detailed nature of DCC distribution (Dahlsveen, 2006).

Previously, only three high-affinity binding sites for DCC were known. This study identified nine more fragments, and this encouraged investigation of common features within a larger pool. Interestingly, all new DBFs were found to map to gene-rich regions and either overlap with or lie close to essential genes. Three high-affinity fragments (DBF12, DBF9, and DBF6) reside entirely within genes. It is possible that specific recruitment sites, such as those inferred to reside within the DBFs, have been enriched in and around genes that require dosage compensation during evolution, and consequently, high-affinity sites may represent loci that are particularly dosage sensitive. Previous experiments indicated that the DCC tends to bind to the coding regions of genes, and it was suggested that this was linked to transcriptional activity. Although recent observations suggest that transcriptional activity alone is not sufficient to attract DCC binding, it is possible that transcription influences DCC recruitment to specific sites. For example, high-affinity sites, which show consistent and strong recruitment of the DCC at many chromosomal positions, may not be influenced by transcription. However, sites with lower affinity and variable recruitment ability may profit from transcriptional activity. Developmental differences in transcriptional activity may therefore also explain the lack of DCC recruitment in salivary glands to fragments isolated by ChIP from embryos (Dahlsveen, 2006).

This study has attempted to identify common sequence elements within previously characterized and new high-affinity DCC binding fragments and have uncovered a number of short sequence elements, whose clustering in combinations could contribute to DCC recruitment. Clearly, the importance of these elements remains to be tested experimentally. Previous analysis of the roX DCC binding sites identified a 110 bp sequence containing several blocks of conservation between roX1 and roX2. DCC binding was affected by mutation in several of the conserved blocks, indicating that DCC binding sites may be made up of combinations of shorter elements. Such combinations have be sought by defining pairs of elements found within a 200 bp window in the high-affinity DCC binding fragments. Those pairs that are significantly enriched on the X chromosome compared to other chromosomes are presented. Importantly, these X-enriched pairs often occur in multiple copies in the high-affinity fragments and at higher frequencies compared to the lower-affinity fragments DBF9-A, DBF1, DBF11, DBF13, and DBF3. Nonetheless, there is no obvious correlation between the location of individual pairs on the X and any specific features such as predicted genes. It is hypothesized that the elements that define these pairs (and other such elements that may have escaped attention) correspond to building blocks of DCC binding sites. Accordingly, a DCC binding site of given affinity for the complex would not be determined by a unique DNA sequence, but by clustering of variable combinations of short, degenerate sequence motifs. Individual low-affinity binding sites may not be unique to the X, but their clustering on the X may contribute to high-affinity binding. There are already indications that the DCC binds to several sites in close proximity. The two parts of DBF9, DBF9-A and DBF9-B, are both able to recruit the DCC, albeit with different affinity. The analysis of the 18D high-affinity fragment also suggested that multiple elements over 8.8 kb contribute to the binding of the complex (Dahlsveen, 2006).

The pairs have been ordered according to sequence similarity. Interestingly, a large family of elements contain GAGA-related motifs. Mutation of GAGA or CTCT motifs in the 110 bp roX1/roX2 consensus severely affects DCC recruitment to that sequence, indicating that GAGA motifs are involved in DCC binding. The fact these elements enriched in several independently identified high-affinity fragments demonstrates the appropriateness of the algorithms used to find them. Besides elements with a clear relationship to GAGA motifs, several other element families were identified defined by sequence similarity. In order to visualize the element families, the related words may be aligned such that sequence logos representing degenerate motifs can be derived using the WebLogo software ( It is considered possible that some of these degenerate motifs may contribute to DCC binding sites. Evaluation of the contributions of these novel motifs to the targeting of the complex will require increased resolution analysis and systematic evaluation of candidate sequences in the in vivo recruitment assay (Dahlsveen, 2006).

This study suggests that high-affinity DCC binding sites are composed of variable combinations of clustered, degenerate sequence motifs. The degeneracy of the sequence motifs indicates that many individual elements may have low affinity. Therefore, the interaction of the DCC with each individual site should be in dynamic equilibrium. However, it was recently observed by photobleaching techniques that the DCC components most likely involved in chromatin binding, MSL2 and MSL1, interact with the X chromosomal territory in cultured cells in an unusually stable manner, which is not compatible with binding equilibria involving off-rates that commonly characterize protein-DNA interactions. Several hypotheses can be formulated, whose evaluation may lead to resolution of this apparent contradiction. (1) Formation of higher-order structures involving many DCC components engaged in numerous simultaneous DNA interactions may lead to a trapping of the DCC within the X chromosome territory. (2) An initial sequence-directed targeting event may be followed by a stabilization of the interaction through positive reinforcement involving additional principles, such as epigenetic marks or a topological linkage. (3) It is considered that the arrangement of the interphase genome in polytene chromosomes may differ in a relevant aspect from the more compact chromosomal territories of diploid cultured cells. Ultimately, the identification of the DNA-binding domains of DCC components and analysis of their mode of DNA interaction will be required to solve the targeting issue (Dahlsveen, 2006).

Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila

Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of the male X chromosome, which ensures equal X-linked gene expression in both sexes. This paper reports the purification of enzymatically active MSL complexes from Drosophila embryos, Schneider cells, and human HeLa cells. A stable association of the histone H4 lysine 16-specific acetyltransferase MOF was found with the RNA/protein containing MSL complex as well as with an evolutionary conserved complex. The MSL complex interacts with several components of the nuclear pore, in particular Mtor/TPR and Nup153. Strikingly, knockdown of Mtor or Nup153 results in loss of the typical MSL X-chromosomal staining and dosage compensation in Drosophila male cells but not in female cells. These results reveal an unexpected physical and functional connection between nuclear pore components and chromatin regulation through MSL proteins, highlighting the role of nucleoporins in gene regulation in higher eukaryotes (Mendjan, 2006).

All Drosophila MSL proteins have mammalian orthologs. To address the evolutionary conservation, the human hMOF-containing complexes were purified from a stable HeLa cell line expressing hMOF tagged with one haemagglutinin (HA) and two FLAG epitopes (HA-2xFLAG-hMOF). The characterization of the interacting proteins revealed striking similarities in the complex composition between flies and humans (Mendjan, 2006).

Copurification of mammalian MSL orthologs showed that DCC is an evolutionary conserved protein complex. hMSL1, hMSL2, and hMSL3 were all present in the hMOF complex. Similar to Drosophila DCC, RNA helicase A (the ortholog of MLE) was not present in the complex, which is consistent with previous observations. Furthermore, two isoforms of hMSL3, hMSL3a and hMSL3c, were identified, copurifying with hMOF. The former represents the full-length protein, while the latter is an alternative splice isoform lacking the N-terminal chromobarrel domain (Mendjan, 2006).

In addition to the MSL proteins, most of the other proteins copurifying with TAP-MOF were also found in the hMOF complex. Z4 and Chriz/Chromator (Chr) lack clear mammalian orthologs, which could explain their absence. However, the Mtor ortholog TPR was identified in the HA-2xFLAG-hMOF purification. Human-specific proteins included the transcriptional coactivator HCF-1, O-linked N-acetylglucosaminetransferase OGT, and the forkhead and FHA domain containing transcription factor ILF-1/FOXK2. Interaction of hMSL3, hNSL1, hNSL2, hNSL3, and HCF-1 was further confirmed by Western blot analysis of eluted complex. Similar to the TAP-MOF and MSL-3FLAG complexes, the HA-2xFLAG-hMOF complex specifically acetylated histone H4 at lysine 16 on mononucleosomes (Mendjan, 2006).

Taken together, the data demonstrate that MOF interactions are evolutionary conserved and that the DCC is an evolutionary ancient complex that acetylates histone H4 at lysine 16 (Mendjan, 2006).

The purification of the MSL complex revealed quite an unusual complex composition. One would expect that a complex thought to modulate transcription and/or chromatin structure would contain a significant number of classical transcription factors, some of the numerous components associated with RNA polymerase II, or at least subunits of the ubiquitous chromatin remodeling and modifier complexes. However, none of these components was found. Instead, there seems to be a core MSL complex that interacts substoichiometrically with nucleoporins (Mtor, Nup153, Nup160, Nup98, and Nup154), interband binding proteins (Z4, Chromator/Chriz), and exosome components (Rrp6, Dis3) (Mendjan, 2006).

The results suggest that MOF is a subunit of two independent complexes in mammals and fruit flies. Several lines of evidence support this notion. This includes coimmunoprecipitation experiments and glycerol gradient centrifugation. Furthermore, hMOF was recently found in the MLL1 methyltransferase complex together with HCF-1, MCRS2, WDR5, NSL1, and PHF20, but this complex did not contain hMSL1. Finally, purification of the hMSL3 complex provides further evidence that hMSL3 does not associate with many of the MOF-interacting proteins. Therefore, it is suggested that the NSL complex contains at least MOF, NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS, and in humans also HCF-1 and OGT (Mendjan, 2006).

The results presented here also suggest a molecular mechanism as to how the MOF complexes bifurcate. Both MSL-1 and NSL1 contain a PEHE domain in their C terminus. The NSL1 PEHE domain interacts directly with hMOF in vitro, and Drosophila MSL-1 has been shown to interact directly with MOF through the same domain. Furthermore, MSL-1 is required for full activity of MOF in vitro and for the assembly of the DCC on the male X chromosome. MSL-1 and NSL1 are the only two genes with a PEHE domain in the Drosophila genome, suggesting that it is an evolutionary conserved MOF-interacting domain. It is postulated that MSL1 and NSL1 serve as mutually exclusive bridging factors that assemble two different complexes around MOF, a histone H4 lysine 16-specific acetyltransferase (Mendjan, 2006).

In the current study, focus was placed on the mechanism of DCC function in Drosophila. All three purifications resulted in enzymatically active complexes with consistent copurification of MSL-1, MSL-2, MSL-3, MOF, roX1, and roX2 but not of MLE or JIL-1. The absence of MLE was expected, since its interaction with MSLs has reported to be salt and detergent sensitive. It is likely that JIL-1, like MLE, is sensitive to the purification conditions used in this study (Mendjan, 2006).

To examine the function of the new interacting proteins in dosage compensation, mutant flies were studied and RNAi was used in cell culture. In Z4 mutants or in MBD-R2-depleted SL-2 cells, MSL localization on the X chromosome was not affected. Consequently, these proteins are not required for MSL recruitment, or they have an alternative function with MOF that is independent of its role in dosage compensation (Mendjan, 2006).

However, an unexpected link was discovered between dosage compensation and the nuclear pore. Depletion of either Mtor or Nup153 but not of other nucleoporins or NXF1 delocalized MSL proteins from the X chromosome. The effects observed were not due to a general transport defect, since all the five MSL proteins and roX2 RNA remained nuclear in Mtor- and Nup153-depleted cells, and no accumulation was observe of bulk mRNA in these cells. Consistent with these observations, Mtor and Nup153 are required for proper dosage compensation of several classical MSL-dependent dosage-compensated genes in SL-2 cells. The expression of these genes was not affected in female Kc cells (Mendjan, 2006).

An important question raised from this study is whether the observed effects are due to a soluble fraction of Mtor and Nup153 in the nucleus or due to their function as components of the NPC. The latter is favored: (1) Nup153 staining is exclusively peripheral; (2) depletion of Nup153 delocalizes Mtor from the nuclear periphery and increases the soluble pool of Mtor in the nucleoplasm, but MSL proteins still remained delocalized in Nup153-depleted cells; (3) the fact that several nucleoporins, which exist together only at the nuclear pore, were copurified with the MSL complexes strongly favors the idea that there is an interaction between the DCC and the intact NPC. This interaction is substoichiometric but with clear functional importance for DCC assembly or maintenance on the X chromosome (Mendjan, 2006).

A wealth of information has been generated in budding yeast regarding nuclear organization and gene regulation. For instance, yeast telomeres associate with the nuclear periphery and form a transcriptionally silenced chromatin domain. However, a number of recent studies have shown that nuclear periphery is not just a domain of gene inactivation but also of activation. Consistent with these observations, yeast MLP1 and MLP2 (Mtor orthologs in yeast) associate with transcriptionally active genes and are involved in relocalization of active genes to the nuclear periphery. Furthermore, MLPs are involved in chromatin domain formation and pre-mRNA quality control (Mendjan, 2006 and references therein).

Interestingly, in Schneider cells, male embryos, salivary glands, and imaginal discs, the Drosophila male X chromosome appears localized at or near the nuclear periphery and in most cases even follows the nuclear rim curvature. The inactive X in mammals also localizes close to the nuclear periphery as the Barr body. Like the Drosophila male X chromosome, the inactive X has to be globally controlled (inactivated) and is characterized by a special histone modification (trimethylation of lysine 27 of histone H3). Another common feature between mammals and Drosophila is that noncoding RNAs play an essential role. A possible model that can account for these intriguing similarities is that the nuclear periphery is used to generate transcriptional domains that can be transcriptionally active or inactive in order to achieve coregulation of gene expression for a subset of genes. In the case of the Drosophila male X chromosome, hundreds of genes with different basal transcriptional properties need to be coactivated by a factor of two. This kind of a subtle transcriptional coregulation of a whole chromosome may be achieved by partial compartmentalization of the X chromosome mediated by the nucleoporin-MSL interaction, allowing the formation of hyperacetylated chromatin domains with unique transcriptional and/or posttranscriptionalproperties (Mendjan, 2006).

It is important to emphasize that Mtor and Nup153 may be required for general chromatin organization (not just individual chromosomes) through their interaction with chromatin-associated proteins. The DCC might mediate X-chromosomal tethering to the nuclear pore as a mechanism to coregulate a large set of genes by creating chromosomal loops or domains. This could happen by direct or indirect interactions of MSLs with Mtor/Nup153 located at or near high-affinity sites along the X chromosome, which are the binding sites of the DCC. Interactions with nuclear pore components may also be used to 'economize resources' and/or for efficient coupling of transcription to processing of the newly transcribed coregulated messages (Mendjan, 2006).

In summary, the purification of the MSL complex has revealed an unexpected link between dosage compensation and the NPC. In the context of data from other systems, this allows formulation of new hypotheses about the mechanism of dosage compensation that will be exciting to test in the future (Mendjan, 2006).

Genome-wide analysis reveals MOF as a key regulator of dosage compensation and gene expression in Drosophila

Dosage compensation, mediated by the MSL complex, regulates X-chromosomal gene expression in Drosophila. This study reports that the histone H4 lysine 16 (H4K16) specific histone acetyltransferase MOF displays differential binding behavior depending on whether the target gene is located on the X chromosome versus the autosomes. More specifically, on the male X chromosome, where MSL1 and MSL3 are preferentially associated with the 3' end of dosage compensated genes, MOF displays a bimodal distribution binding to promoters and the 3' ends of genes. In contrast, on MSL1/MSL3 independent X-linked genes and autosomal genes in males and females, MOF binds primarily to promoters. Binding of MOF to autosomes is functional; H4K16 acetylation and the transcription levels of a number of genes are affected upon MOF depletion. Therefore, MOF is not only involved in the onset of dosage compensation, but also acts as a regulator of gene expression in the Drosophila genome (Kind, 2008).

Consistent with previous MSL1 and MSL3 profiling studies, MSL1, MSL3, MOF, and H4K16Ac display enrichment to 3' end of genes in SL-2 cells. Surprisingly, MOF displays a bimodal binding pattern on genes residing on the X chromosome, associating with both the 3' ends of dosage-compensated genes as well as with promoter regions (Kind, 2008).

Recent observations on individual X-chromosomal target genes using transgene analysis in vivo have revealed that there are at least two classes of sites; transcription-independent 'high-affinity sites' such as roX2 and transcription-dependent 'low-affinity sites' such as mof or CG3016. Integrating the observations obtained from the genome-wide binding and RNAi-mediated knockdown analysis shown in this study, it appears that MOF plays a central role in targeting the MSL complex to 'low-affinity sites' where recruitment of MSL1 and MSL3 is found to be dependent on the presence of MOF. This is in contrast to the 'high-affinity sites' where partial complexes of MSL1/MSL2 can be recruited independently of MOF, MSL3, and MLE (Kind, 2008).

Interestingly, MOF was found to bind not only to the male X chromosome, but also to autosomes and female chromosomes. Different from the bimodal binding pattern of MOF on the male X chromosome, in Kc cells, MOF is enriched to promoters of all chromosomes similarly to the situation on the male autosomes in SL-2 cells. However, although the binding pattern between the X and the autosomes in Kc cells looks practically identical, the amplitude of promoter binding is significantly higher on the X chromosome than on the autosomes in Kc cells, as is the case in SL-2 cells. It is possible that X-chromosomal genes have as-yet-unidentified sequence elements that contribute toward MOF binding to promoters of X-chromosomal genes in males and females. Alternatively, since reduced amount of MSL1 is expressed in females and MSL1 displays low-level promoter binding on the X chromosome in SL-2 cells, it may contribute to higher amplitude of MOF binding on X chromosomal genes in both SL-2 and Kc cells compared to autosomes. Since the gene density on the X chromosome is similar to that of other chromosomes (except for the fourth chromosome), this does not explain the higher amplitude of MOF binding on the X chromosome. It is therefore possible that MOF, in addition to its role in facilitating transcriptional elongation by acetylating gene loci in an MSL context, is also involved in transcriptional initiation in an MSL-independent manner, perhaps by interaction with additional factors. Another interesting possibility is that the enrichment of MOF to promoters may provide a reservoir of enzyme, held in check by other factors, to be readily used by the MSL proteins or other promoter-bound complexes when needed for modulating transcription levels (Kind, 2008).

Intriguingly, the MSL3 profile across gene loci appeared very similar to that of H4K16Ac, suggesting a role for MSL3 in activation and/or stabilization of H4K16Ac on X-linked genes. In support of this hypothesis, MSL3 has been shown to stimulate MOF's HAT activity in vitro. MSL3 has been shown to bind H3K36 trimethylated (H3K36me3) nucleosomes, and H3K36me3 (which also peaks at 3' end of genes, similar to MSLs) was shown to influence MSL binding. In S. cerevisiae, Eaf3 recognition of H3K36me3 has been shown to direct Rpd3(S) to actively transcribed genes to deacetylate histones in the wake of polymerase II, preventing spurious transcription within genes from cryptic promoters. It has been proposed that the MSL complex on the X chromosome may compete for the Rpd3(S) complex, thereby increasing the overall H4K16Ac levels by reducing the turnover rates of this modification (Kind, 2008).

Since the 3' ends of genes are indispensable for MSL target recognition on the X chromosome (Kind, 2007), it is proposed that MSL1 and MSL2 initially target 3' regions by occasional recognition of degenerative DNA target elements, possibly made accessible by low levels of H4K16Ac brought about by MOF occupancy of the promoter. MSL3 may serve to stabilize the association of MSL1/MSL2 with dosage-compensated genes by binding to H3K36me3, which in turn may lead to the recruitment and stimulation of MOF to the body of the gene. It has also been proposed that local recycling of RNA polymerase II could result in enhanced mRNA production. MOF, with its enrichment to promoter-proximal and 3' regions, is a likely candidate to bridge such a loop formation. Gene structural studies should reveal whether such a gene-loop formation is involved in the process of dosage compensation (Kind, 2008).

This study presents four independent lines of evidence that show that MOF is involved in H4K16Ac of a large number of genes in the male and female genome. 1) MOF binding significantly correlates with H4K16Ac of all chromosomes in both SL-2 and Kc cells. 2) The H4K16Ac profile across genes correlates strongly with the diversified binding of MOF between the X chromosome (peaking toward the 3' end of genes), and autosomes (peaking toward the 5' end of genes. 3) Depletion of MOF results in a marked decrease in H4K16Ac of a number of genes on both the X chromosome and the autosomes. 4) In MOF-depleted SL-2 and Kc cells a more than 50% reduction in total H4K16Ac levels is found by mass specterometery analysis (Kind, 2008).

Several studies have implied a structural role for histone acetylation and H4K16Ac acetylation in particular, in the packaging of DNA into chromatin. Interestingly, H4K16Ac has been shown to cause an increase in the α-helical content of histone H4, and to prevent 30 nm chromatin-fiber formation and crossfiber interactions. H4K16Ac might therefore serve a structural role, imparting a relaxed chromatin state that, in turn, reduces the energy required for RNA polymerase II to affect transcription through a nucleosomal template and thereby enhancing elongation efficiency (Kind, 2008).

Regulation of ubiquitously expressed (housekeeping) genes on the X chromosome by the MSL complex probably necessitates a state of continual association with its target binding sites. Elevated levels of H4K16Ac are reached on the X chromosome presumably by constant activation of MOF by MSL1 and MSL3. On the autosomes, since MOF appears to be present independently of other MSL proteins, it does not associate to the interior of gene loci but is instead promoter bound, similar to its behavior on the X chromosome in the MSL1-depleted condition (Kind, 2008).

Assuming that MOF is involved in general transcription regulation, apart from dosage compensation, it is not surprising that MOF is required for most H4K16 acetylation. Similarly, MOF in mammals has been found to be responsible for most, if not all, H4K16Ac. Interestingly, in line with a possible role for MOF in the G2/M cell-cycle checkpoint in mammals it was found that in both SL-2 and Kc cells, MOF-bound targets are significantly enriched for certain cell-cycle functional categories. It would therefore be very interesting to study gene regulation by MOF in a cell-cycle context in synchronized cells (Kind, 2008).

The role of MOF mediated H4K16Ac on the autosomes remains speculative. One possibility: H4K16Ac modification on autosomes by MOF may create an opportunity for transcription initiation/reinitiation, rather than being an essential mark for transcriptional activity itself. This could also explain why it was observed that, although MOF is generally bound to active genes, approximately 30% of the autosomal bound genes are affected by MOF depletion. MOF's presence on autosomal genes may therefore provide a minimal landscape of H4K16Ac, maintaining a local environment with relatively open chromatin structure, presumably similar to the condition of mating type loci in. Upon transcriptional cues, those genes would be able to rapidly and efficiently respond to meet the cell's requirements, as would be the case for cell cycle-related genes (as discussed above) (Kind, 2008).

Another possibility: MOF may work together with as-yet-uncharacterized proteins, which may allow RNA polymerase II to move efficiently through the chromatin template similar to the situation on the X chromosome. In fully elucidating the molecular mechanisms behind this process, a vital step will be the characterization of additional protein complexes associated with MOF, apart from the MSL complex. It is proposed that such complexes, comprising different trans-activating or repressive factors, may modulate MOF's HAT activity resulting in differential transcriptional outputs. Furthermore, MOF binding to promoters may allow efficient and rapid response to cellular events by recruitment/exclusion of H4K16 binding proteins or, more generally, by unique H4K16Ac-induced conformational changes to the chromatin fiber. Interestingly, one of the evolutionary conserved interacting partners of MOF is WDS, a protein in mammals shown to associate with histone H3 lysine 4 methylation, a histone mark enriched at promoters. It would be interesting to study the potential involvement of WDS or other promoter-bound factors in recruiting MOF to promoters (Kind, 2008).

In summary, it has been shown that the MSL complex members do not conform to a uniform binding behavior on their target genes on the X chromosome: MSL1 and MSL3 are enriched at the 3' end of genes, while MOF shows a bimodal distribution with enrichment at promoter-proximal regions as well as 3' ends. The data reveal that MOF plays a central role in the targeting process on low-affinity sites where recruitment of MSL1 and MSL3 appear to be dependent on the presence of MOF, in contrast to high-affinity sites such as roX2 where targeting of MSL1 appears to be MOF independent. Furthermore, the previously unappreciated binding of MOF to promoter-proximal regions on X-chromosomal as well as autosomal sites provides an opportunity to investigate additional roles of this enzyme in other cellular processes (Kind, 2008).

The nonspecific lethal complex is a transcriptional regulator in Drosophila

The nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, it is concluded that the NSL complex acts as a major transcriptional regulator in Drosophila (Raja, 2010).

Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex. This study reports an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, it was shown that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8 (Cai, 2010).

males absent on the first: Biological Overview | Evolutionary Homologs | Developmental Biology | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.