- Email: [email protected]
Dosage compensation: an intertwined world of RNA and chromatin remodelling
161
Dosage compensation: an intertwined world of RNA and chromatin remodelling Asifa Akhtar Dosage compensation mechanisms in flies and mammals provide an exquisite example of chromatin associated RNAs in chromosome-wide transcription regulation. Recent progress shows that chromatin modifications are also closely linked to these processes. Concerted action of the RNA/chromatinmodifying enzymes may play a crucial role in determining transcriptional output. Furthermore, non-coding RNAs appear to play a dual role, being targeting modules as well as encoding for target sites for complex recognition. Addresses European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:161–169 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by John Tamkun and David Stillman 0959-437X/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(03)00016-9
Abbreviations DCC dosage compensation complex ES embryonic stem HAT histone acetyltransferase MLE maleless MOF males absent on the first MSL male specific lethal roX RNA on the X chromosome Xic X inactivation centre Xist X inactive specific transcript
females [3] (Figure 1). Themes common to these different processes is the presence of factors that specifically decorate the compensated X chromosome and the recent findings that chromatin modifications may play an important role [4,5,6,7]. In this review, I focus on what is known about Drosophila dosage compensation and discuss the interesting parallels with mammalian X inactivation.
Drosophila: XX = XY In D. melanogaster, the single male X is hyper-transcribed approximately twofold in comparison to females. Several genetic screens, scoring for male-specific lethality, have identified proteins required for dosage compensation [8,9]. Male specific lethal (MSL) proteins include MSL-1, MSL-2 and MSL-3, maleless (MLE) and males absent on the first (MOF) [10–15]. In addition to these proteins, two non-coding RNAs, roX1 and roX2 (‘RNA on the X chromosome’) have also been identified as members of the dosage compensation complex (DCC) [16–18]. An essential histone H3 kinase, JIL-1, also associates with the dosage compensation members [19] (Figure 2; Table 1). The dosage-compensated X chromosome has a more decondensed chromatin structure relative to the autosomes that correlates with hyperacetylation of histone H4 at lysine 16 residue (H4K16) along the length of the X chromosome by MOF histone acetyltransferase (HAT) [4,15,20,21]. The DCC preferentially associates at hundreds of sites on the male X but fails to assemble in females as a result of a translation block of the MSL-2 subunit by a protein called Sexlethal [22]. In the absence of MSL-2, MSL proteins and the roX RNAs, except MOF, are expressed in very low amounts in females [10,11].
Chromatin entry sites on the X chromosome Introduction In diploid species, the dosage-compensation mechanisms have evolved to equalise for the difference in gene dosage occurring caused by unequal number of sex chromosomes between males and females. Failure to compensate leads to lethality early in embryo development. Dosage compensation has been studied in mammals, fruit flies and nematode worms, and, interestingly, each of these species tackles the problem differently. In mammals, it is achieved by inactivation of one of the two female X chromosomes in comparison to the XY males [1]. In Caenorhabditis elegans hermaphrodites, transcription from the two X chromosomes is repressed by half to equalise for the expression from the XO males [2]. By contrast, in Drosophila melanogaster, transcription is upregulated twofold in XY males to equalise for the expression from XX www.current-opinion.com
An intact RNA/protein DCC is required for hyper-transcription of genes on the X chromosome. However, in backgrounds where MOF, MSL-3 or MLE have been mutated, a partial complex containing MSL-1 and MSL-2 can be still detected on 35 ‘chromatin entry sites’ along the X chromosome. Chromatin entry sites act as initial docking sites for the complex assembly from where the complex spreads in cis and paints the rest of the X chromosome [23,24]. To date, two of these presumed entry sites have been identified and, remarkably, they include the genes encoding roX1 and roX2 [23,24,25]. The detailed characterisation shows that the roX1 cDNA is sufficient for attraction of the complex on an autosome. Furthermore, active transcription from the roX1 gene is not a prerequisite Current Opinion in Genetics & Development 2003, 13:161–169
162 Chromosomes and expression mechanisms
Figure 1
Mammals
XY
XX
Dosage compensation C. elegans
XO
X-Inactivation
Drosophila
XX
XY
Repression 2×
Hyperactivation 2×
XX
X
X
X
X
X X
X
XX
Current Opinion in Genetics & Development
Mechanisms of dosage compensation. X-linked gene expression is equalised in males and females by either transcriptional silencing of one of the female X chromosomes relative to males (mammals); by transcription repression so that transcription from both X chromosome of an hermaphrodite is repressed by half relative to males (C. elegans); or by transcriptional activation of the single male X chromosome twofold in comparison to females (Drosophila). The size of the X chromosome symbols represents transcription activity.
for the complex assembly. Within the roX1 locus, a 217 bp region is sufficient for attracting the complex. What seems remarkable is that, despite being entry sites, roX1 and roX2 DNAs bear no homology except for a 30 bp sequence at the 30 end of the DNA [26]. The significance of this 30 bp region at the DNA or RNA level still remains unclear; it may contain a protein-binding site for either one of the complex members or for an unidentified protein. Analysis of the chromatin entry site using MSL mutants shows that, unlike other entry sites, roX1 and roX2 entry sites are sensitive to the absence of MLE because a partial DCC complex is unable to form in these mutants [24,27]. These results suggest that different entry sites may have different component requirements and therefore may play specialised roles in the context of global male X chromosome regulation.
Assembly and spreading of the dosage compensation complex The Drosophila dosage compensation appears to be a twostep process (Figure 3a). The first step includes assembly of the complex on chromatin entry sites mediated by MSL-1 and MSL-2 [23]. Whether MSL-1 and MSL-2 recognise some specific feature of the DNA sequence or a special chromatin structure around an entry site in preference to other X chromosomal regions remains unknown. Characterisation of additional entry sites will be an important step in identifying these features. Current Opinion in Genetics & Development 2003, 13:161–169
Once correctly assembled, the DCC proteins seem to occupy many additional distinct regions along the chromosome whereas other regions seem devoid of the complex [27]. It is therefore an enigma how the DCC gets targeted to entry sites, spreads from these relatively well defined sites and occupies distinct X-chromosomal regions. Whether there are unidentified boundary elements dispersed along the X chromosome that prevent the access of DCC from certain X chromosomal regions remains unclear. A similar problem is faced in the mammalian system where, despite downregulation of most X linked genes, some X chromosomal regions escape inactivation. The potential mammalian complex is thus allowed access to most but not all of the X chromosome. In either MOF or MLE mutant males — where the catalytic sites of these enzymes have been mutated — partial complexes containing MSL-1, MSL-2 and MSL-3 are found restricted to chromatin entry sites, suggesting these enzymes affect the second step namely ‘spreading’ along the X chromosome [15,28]. It is interesting to note that, MLE but not MOF protein affects stability of roX RNAs, which implies that even though MLE and MOF work in concert to help the complex spread, they are clearly required for distinct steps during this process [26]. Whether MOF’s participation in the spreading phenomenon is merely to create a more fluid chromatin environment by hyper-acetylation or in addition, MOF can also post-translationally modify other DCC members to modulate their activity remains an interesting issue to be explored.
Targeting or being targeted by roX RNAs At least for some of the DCC members, roX RNAs play a role in X-chromosome-specific targeting. MLE helicase was the first protein within the DCC to have RNA sensitive association to the X chromosome [29]. It was later shown that MLE is also important for stability of roX RNAs [28]. A clue as to how specific targeting may be achieved in the case of MOF came from the finding that MOF is also an RNA binding protein, it binds RNA via its chromodomain, and that it is also tethered to the X chromosome via RNA [30]. In accord with this, deletion of roX1 and roX2 show mis-localisation of the MSL proteins, in particular MSL-2 and H4K16 acetylation to the autosomes [31]. Furthermore, in MOF mutant females overexpressing MSL-2, the roX RNAs are dispersed around the nucleoplasm, suggesting that the MOF/RNA complex is important for correct targeting to the X chromosome [28]. Hence, there seems to be an
(Figure 2 Legend) Linearised depiction of the domain structure of dosage compensation associated factors in (a) Drosophila and (b) mammals. The different domains are indicated as cylindrical shapes. The domain size and location are drawn to indicate their relative position within the proteins. The total length of the proteins and RNAs is indicated: aa, amino acid; kb, kilobase. www.current-opinion.com
Dosage compensation Akhtar 163
Figure 2
(a)
MSL-1: 1039 aa
S/TP rich potential phosphorylation sites
Acidic region (25% D or E)
MSL-2: 773 aa
RING finger
Coiled coil domain
Metallothionein-like domain PHD
MSL-3: 512 aa
2 Chromodomains
MLE: 1293 aa
Helicase domain
Double strand RNA binding domain
9 G-rich repeats
MOF: 827 aa
Chromodomain Zn finger HAT domain
JIL-1: 1207 aa Kinase domain 1
roX1RNA: 3.7 kb
(b)
Kinsae domain 2
roX2 RNA: 0.6 kb
Macro H2A: 372 aa
H2A domain
Eed: 535 aa
WD40
Enx1: 747 aa
SANT
SANT
SET
BRCA1: 1812 aa
RING finger
BRCT
Xist RNA: 17 kb
Current Opinion in Genetics & Development
www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:161–169
164 Chromosomes and expression mechanisms
Table 1 Functional regions within the DCC proteins identified by interaction studies. Functional region
References
MSL-1 Residues 1–84; important for X chromosomal localisation. Residues 85–186 important for in vitro interaction with MSL-2. Residues 759–1039 important for interaction with MOF and MSL-3.
[57,58]
MSL-2 Residues 1–190, containing ring finger (37–87), are important for X chromosomal localisation and interaction with MSL-1.
[12,27,58]
MSL-3 MSL-3 directly interacts with MSL-1. Residues 437–516 containing chromodomain interact with RNA in vitro.
[57] [30]
MOF Residues 518–827 containing zinc finger and HAT domain (MYST domain) are sufficient for HAT activity. Point mutation in the HAT domain (G691E ) abolishes HAT activity. C2HC zinc finger within the MYST domain is important for interaction with chromatin especially histone H4 tail. Point mutations C557G, Y572G, L578/Y580 abolish HAT activity and chromatin interaction. MOF specifically interacts with roX2 RNA in vivo. Chromodomain in MOF interacts with RNA in vitro. Point mutations W426G, Y416D abolish RNA interaction both in vitro and in vivo. MLE Point mutations in helicase domain GKT to GET (ATPase A motif) or DEIH-DQIH. (ATPase B motif) reduce ATPase/Helicase activity, and affect male viability. Residues 940–1293 containing GGY domain: important for targeting MLE to the male X via RNA. JIL-1 Kinase domain 1 and 2: catalytic domains; important for kinase activity. Both kinase domains also interact with MSL-1 and MSL-3.
[59] [15,21] [39] [30]
[29]
[19]
Summary of functional regions identified by protein–protein and protein–RNA interaction studies in Drosophila proteins associated with dosage compensation. Point mutations are indicated by the amino acid of interest, its position within the protein followed by the replaced amino acid.
intimate connection between MOF and roX RNAs. However, the detailed mechanism is still poorly understood. Apart from MLE and MOF, MSL-3 is the third RNAbinding component within the complex, underscoring the importance of RNAs in integrity and specificity of the DCC [30]. Future experiments will tell us whether the specificity for RNA comes from folding of these RNAs into a distinct structure, which act as a scaffold for protein assembly and subsequent targeting, or whether RNAs act as flags signalling for the X chromosome. Alternatively, the DCC proteins themselves may remodel the RNA structure by interaction and act instead as chaperones for the RNA to be transported to and along the X chromosome. Interesting links between the RNA interference machinery and heterochromatic silencing in Schizosaccharomyces pombe and the involvement of RNA in regulation of pericentric heterochromatin have been made recently [32–34]. These findings further reinforce the versatile nature of non-coding RNAs in various regulatory processes. Detailed functional studies will be instrumental in deciphering the true mechanisms operating behind regulation using non-coding RNAs in the chromatin context. Current Opinion in Genetics & Development 2003, 13:161–169
Association with additional chromatin remodelling factors Chromatin remodelling factors come in two flavours: either enzymes that utilise ATP to mobilise nucleosomes or enzymes that modify chromatin structure by posttranslational modification of histones [35]. Recent work on the chromatin remodelling enzyme ISWI ATPase [36] has shed light into new possible interactions between the DCC and ISWI. In the fly, ISWI is essential
(Figure 3 Legend) Dosage compensation in (a) Drosophila and (b) mammals. (a) Dosage compensation proteins assemble on 35 distinct ‘chromatin entry sites’ on the male X chromosome. The two identified entry sites include roX1 and roX2 RNA encoding genes. The assembly of a functional complex leads to hyperacetylation of the surrounding chromatin at histone H4 lysine 16 residue followed by spreading of the complex along the X chromosome at numerous additional sites. Dosage compensation in Drosophila leads to transcription enhancement of most X-linked genes. (b) In mammals, X inactivation initiates at a single site, the X inactivation centre (Xic), which includes the Xist RNA encoding gene. An as yet unidentified RNA/protein complex spreads bidirectionally from Xic. The onset of X inactivation proceeds with hypoacetylation of chromatin and hypermethylation of histone H3 lysine 9 residue. During the maintenance phase of X inactivation, macro H2A, a histone variant, is incorporated into the silent chromatin. X inactivation in mammals leads to transcriptional repression of most X-linked genes. www.current-opinion.com
Dosage compensation Akhtar 165
Figure 3
(a)
K16
K16
6
K1
K16 K16
K1
? K1 6
6
K16
K16
6
K1
K16
K16
K16
K16
Entry site
MLE
MOF
MSL-3
MSL-1/MSL-2
roX1/roX2
(b) ? ? ?
?
? ?
?
K9 K9
K1
?
?
6
K9
K16
K9
K16
K9
6
K1
K16
K9
K9 K16
K9
?
?
K1 K9
6
K9
K9
K9
Xic
K9
K9
K9
HAT
K9
K9
HDAC
K9
K9
K9
HMTase
K9
BRCA1/BARD1
K9
macroH2A
Xist Current Opinion in Genetics & Development
www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:161–169
166 Chromosomes and expression mechanisms
for organism viability. In the mutant, the dying male larvae display a dramatically altered X chromosome [37]. This phenotype probably occurs due to an imbalance caused by hyperacetylation of the male X chromosome by MOF that works towards a more open chromatin structure and ISWI that may lead to a more condensed chromatin state. In the absence of ISWI, this opposing activity is lost and therefore the X chromosome appears extremely decondensed. In agreement with this, the ATPase activity of ISWI on histone H4 substrate, which is also a substrate for MOF interaction on nucleosomes, is inversely correlated with its acetylation status [38,39]. Taken together with the observation that ISWI staining does not coincide with RNA polymerase II staining and that yeast ISW2 functions to repress genes induced early in meiosis [40,41], it will be interesting to find out whether or not ISWI functions to downregulate gene expression also on the X chromosome. Association of a chromatin-remodelling factor that may function as a putative repressive activity corroborates the model which proposes that the DCC functions to modulate the action of MOF histone acetyl transferase so that the autosomal genes are not over transcribed which may occur due to the presence of a single copy of the X chromosome in males [42,43]. Like ISWI, JIL-1 kinase also plays a more general role in chromosome architecture. JIL-1 has been shown to phosphorylate histone H3 at Serine 10 residue [19]. The connection between the DCC and JIL-1 originates from the following observations. JIL-1 immunostaining shows a twofold enrichment on the X chromosome relative to the autosomes [19]. Hypomorphic JIL-1 mutants show a variety of nuclear defects with alterations in chromosome morphology, the X chromosome seems most severely affected. In addition, these mutants also show a skewed male to female ratio [44,45] (Figure 2; Table 1). The exact role of JIL-1, similar to ISWI, in the context of dosage compensation, remains to be defined as targeting of the DCC members is not affected in JIL-1 mutants [19]. An attractive hypothesis is that JIL-1 may regulate DCC by post-translational modification of the DCC members. H4K16 acetylation on the X chromosome seems to have a high turnover [30] because loss of MOF from the X chromosome leads to a rapid deacetylation of the X chromosome. General histone deacetylase activity in Drosophila may be responsible for this effect, MOF being required on the X chromosome to maintain the hyperacetylated status. Alternatively, a specific histone deacetylase may associate with the complex to regulate dosage compensation. The interplay between this putative histone deacetylase and MOF histone acetyl transferase may have important consequences for the function of the complex. Current Opinion in Genetics & Development 2003, 13:161–169
It appears that, in addition to the well-defined components tailored for male specific functions, the DCC may also interact with additional chromatin-remodelling factors. This larger protein–protein and protein–RNA network might function to fine tune the X chromosomal transcriptional activity.
Function of the dosage compensation complex To date, it has remained a mystery how the dosagecompensation complex functions at the molecular level to activate transcription. It may function at the level of chromatin remodelling, transcription initiation or transcription elongation. Apart from the close correlation between hyper-acetylation and transcriptional upregulation, evidence that dosage compensation may play an active role in transcription regulation comes from several recent observations. Tethering of a chromatin entry site like roX1 or roX2 genes adjacent to a b-galactosidase reporter at an autosomal location increases transcription activation of an heterologous promoter [46]. Interestingly, the exon sequence rather than genomic sequence was more efficient in transcription activation. Furthermore, it was observed that even though a 217 bp region within roX1 is sufficient for complex targeting [26], surrounding sequences within roX1/2 entry sites may have additional function in transcriptional modulation. A similar question was posed by studying the chromatin status along dosage-compensated genes in vivo using chromatin immunoprecipitation experiments [25]. The X-linked dosage compensated genes Phosphogluconate dehydrogenase and Zwischenferment showed higher acetylation levels in the coding region than in the promoter region. These results suggest that the DCC might enhance transcription elongation. It will be interesting to know whether this increased acetylation correlates with increased efficiency of the polymerase read through and whether the dosage-compensation machinery interacts with basal transcription factors.
Mammals: Xx = XY Despite the difference in transcriptional output, the X inactivation process in mammals, similar to flies, requires several steps that include choice of the X chromosome to inactivate followed by initiation and then spreading of inactivation along the X chromosome (Figure 3b). The choice and initiation of inactivation are early steps during embryo development and occur when the totipotent embryonic cells start to differentiate. Unlike in flies, inactivation in mammals is initiated at a single site at the X inactivation centre (Xic), and from there propagates bidirectionally. The Xist gene is located within the Xic, which encodes a non-coding, spliced, polyadenylated RNA. Xist RNA is expressed exclusively from the inactive www.current-opinion.com
Dosage compensation Akhtar 167
X chromosome and coats the X chromosome. Therefore, similar to flies, the ‘targeting and being targeted’ roles also seem valid for Xist. However, Xist RNA only spreads in cis, whereas roX RNAs can be also be targeted in trans. Furthermore, Xist RNA is essential for the onset of inactivation but not for maintainance [47,48–50].
regulation. It is possible that, despite the evolutionary distance, some aspects of the targeting mechanism remain similar between these two organisms. Combination of genomics and proteomics approaches will yield exciting new insights into these chromosome-wide transcription regulatory processes.
Because of the complexity of the mammalian system, an analogous X inactivation complex has not been identified yet, but several recent observations make it a possibility (Figure 2). The inactive X chromosome has long been known to be hypoacetylated, but interestingly it has been reported that methylation at the histone H3 lysine 9 (H3K9) residue constitutes an early mark on the inactive X chromosome [5,6]. At least for imprinted inactivation, the eed/enx1 complex appears to be a good candidate for the histone methyltransferase complex because, in trophectoderm cells, these proteins are localised on the inactive X chromosome, and eed mutants fail to maintain imprinted X inactivation [50,51,52].
Acknowledgements
The inactive X (Xi) chromatin is enriched in a histone variant macro H2A, within a nuclear structure known as the macrochromatin body. However, macro H2A associates with the inactive Xi 5–6 days after the onset of X inactivation and therefore appears to be important in the later step of maintenance of Xi chromatin structure. Macro H2A association also appears to be sensitive to cell-cycle progression [50,53]. Unexpectedly, an exciting new connection between BRCA1 and Xist RNA was reported recently [54]. BRCA1 is a tumour suppressor gene involved in breast and ovarian tumours. BRCA1 seems to colocalise with Xist RNA in female somatic cells and loss of BRCA1 leads to defects in X inactivation, altered Xist RNA appearance, loss of H3K9 methylation as well as loss of Macro H2A association. BRCA1 also appears to interact with a particular region of Xist RNA via its ring finger domain. Whether proteins associated with BRCA1 constitute an analogous DCC in mammals remains to be seen. It is noteworthy that some of the Drosophila dosage compensation members are also conserved in mammals, with as yet undefined function, in particular, chromodomaincontaining proteins MSL-3 and MOF [55,56]. Whether these proteins have evolved into a new and distinct function instead of dosage compensation remains to be determined.
Conclusions Evolutionarily distinct dosage compensation mechanisms in Drosophila and mammals seem to have evolved interesting parallels with noncoding RNAs and chromatin modifications. Recent advances in Drosophila suggest that the dosage compensation complex is a dynamic entity with multiple interactions with additional chromatin machinery, and RNAs play a very integrated role in this www.current-opinion.com
I thank Peter Becker, Elisa Izaurralde, Stefan Kass and Mikko Taipale for critical reading of the manuscript. I am also grateful to Mikko Taipale for help with the artwork.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Lyon M: Gene action in the X-chromosome of the mouse (Mus musculus). Nature 1961, 190:372-373.
2.
Meyer BJ, Casson LP: Caenorhabditis elegans compensates for the difference in X chromosome dosage between the sexes by regulating transcript levels. Cell 1986, 47:871-881.
3.
Mukherjee AS, Beermann W: Synthesis of ribonuleic acid by the X-chromosomes of Drosophila melanogaster and the problem of dosage compensation. Nature 1965, 207:785-786.
4.
Turner BM, Birley AJ, Lavender J: Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 1992, 69:375-384.
5.
Heard E, Rougeulle C, Arnaud D, Avner P, Allis DC, Spector DL: Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation. Cell 2001, 107:727-738. See annotation [6]. 6.
Mermoud JE, Popova B, Peters AH, Jenuwein T, Brockdorff N: Histone H3 lysine 9 methylation occurs rapidly at the onset of random X chromosome inactivation. Curr Biol 2002, 12:247-251. By using specific antibodies raised against methylated peptides in embryonic stem (ES) cells, these studies [5,6] were the first documenting that histone H3 lysine 9 methylation constitutes an early mark on the female inactive X chromosome. 7.
Fong Y, Bender L, Wang W, Strome S: Regulation of the different chromatin states of autosomes and X chromosomes in the germ line of C. elegans. Science 2002, 296:2235-2238. This study showed that in C. elegans eed1/enx1 homologues MES-6 and MES-2 also form a stable complex required for gene silencing and germline development, and this also correlates with histone H3 lysine 9 methylation. 8.
Belote JM, Lucchesi JC: Control of X chromosome transcription by the maleless gene in Drosophila. Nature 1980, 285:573-575.
9.
Uenoyama T, Uchida S, Fukunaga A, Oishi K: Studies on the sexspecific lethals of Drosophila melanogaster. IV. Gynandromorph analysis of three male-specific lethals, mle, msl-2(27) and mle(3)132. Genetics 1982, 102:223-231.
10. Bashaw GJ, Baker BS: The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development 1995, 121:3245-3258. 11. Kelley RL, Solovyeva I, Lyman LM, Richman R, Solovyev V, Kuroda MI: Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 1995, 81:867-877. 12. Zhou S, Yang Y, Scott MJ, Pannuti A, Fehr KC, Eisen A, Koonin EV, Fouts DL, Wrightsman R, Manning JE et al.: Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sexspecific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster. EMBO J 1995, 14:2884-2895. Current Opinion in Genetics & Development 2003, 13:161–169
168 Chromosomes and expression mechanisms
13. Gorman M, Franke A, Baker BS: Molecular characterization of the male-specific lethal-3 gene and investigations of the regulation of dosage compensation in Drosophila. Development 1995, 121:463-475. 14. Kuroda MI, Kernan MJ, Kreber R, Ganetzky B, Baker BS: The maleless protein associates with the X chromosome to regulate dosage compensation in Drosophila. Cell 1991, 66:935-947. 15. Hilfiker A, Hilfiker-Kleiner D, Pannuti A, Lucchesi JC: mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila. EMBO J 1997, 16:2054-2060. 16. Amrein H, Axel R: Genes expressed in neurons of adult male Drosophila. Cell 1997, 88:459-469. 17. Franke A, Baker BS: The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. Mol Cell 1999, 4:117-122. 18. Meller VH, Wu KH, Roman G, Kuroda MI, Davis RL: roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell 1997, 88:445-457. 19. Jin Y, Wang Y, Johansen J, Johansen KM: JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associated with the male specific lethal (MSL) Dosage Compensation Complex. J Cell Biol 2000, 149:1005-1010. 20. Bone JR, Lavender J, Richman R, Palmer MJ, Turner BM, Kuroda MI: Acetylated histone H4 on the male X chromosome is associated with dosage compensation in Drosophila. Genes Dev 1994, 8:96-104. 21. Akhtar A, Becker PB: Activation of transcription through histone H4 acetylation by MOF, an acetyl transferase essential for dosage compensation in Drosophila. Mol Cell 2000, 5:367-375. 22. Bashaw GJ, Baker BS: Dosage compensation and chromatin structure in Drosophila. Curr Opin Genet Dev 1996, 6:496-501. 23. Kelley RL, Meller VH, Gordadze PR, Roman G, Davis RL, Kuroda MI: Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 1999, 98:513-522. 24. Meller VH, Gordadze PR, Park Y, Chu X, Stuckenholz C, Kelley RL, Kuroda MI: Ordered assembly of roX RNAs into MSL complexes on the dosage- compensated X chromosome in Drosophila. Curr Biol 2000, 10:136-143. 25. Smith ER, Allis CD, Lucchesi JC: Linking global histone acetylation to the transcription enhancement of Xchromosomal genes in Drosophila males. J Biol Chem 2001, 276:31483-31486. Using chromatin immunoprecipitation experiments it was demonstrated here that histone H4 acetylation on dosage compensated genes correlates with coding regions rather than the promoter regions on the X chromosome, implying that the DCC may function to promote transcription elongation. 26. Kageyama Y, Mengus G, Gilfillan G, Kennedy HG, Stuckenholz C, Kelley RL, Becker PB, Kuroda MI: Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site. EMBO J 2001, 20:2236-2245. The authors of this paper performed the first detailed characterisation of roX1 chromatin entry site and showed that a 217 bp region within roX1 is sufficient to attract dosage compensation proteins to an autosome. 27. Lyman LM, Copps K, Rastelli L, Kelley RL, Kuroda MI: Drosophila male-specific lethal-2 protein: structure/function analysis and dependence on MSL-1 for chromosome association. Genetics 1997, 147:1743-1753. 28. Gu W, Wei X, Pannuti A, Lucchesi JC: Targeting the chromatinremodelling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities. EMBO J 2000, 19:5202-5211. 29. Richter L, Bone JR, Kuroda MI: RNA-dependent association of the Drosophila maleless protein with the male X chromosome. Genes Cells 1996, 1:325-336. Current Opinion in Genetics & Development 2003, 13:161–169
30. Akhtar A, Zink D, Becker PB: Chromodomains as RNA interaction modules. Nature 2000, 407:405-409. We showed here that the MOF HAT is tethered to the male X chromosome by RNA and that histone acetylation on the X chromosome has a high turnover. We also presented evidence that chromodomains of MOF and MSL-3 interact with RNA. 31. Meller V, Rattner BP: The roX genes encode redundant male specific lethal transcripts required for targeting of the MSL complex. EMBO J 2002, 21:1084-1091. Even though roX RNAs seem to have redundant function, it is convincingly shown here that deletion of roX1 and roX2 genes in flies causes malespecific lethality and mistargeting of the MSL proteins as well as histone H4 lysine 16 acetylation. 32. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SIS, Martienssen RA: Regulation of heterochromatic silencing and histone H3 lysine9 methylation by RNAi. Science 2002, 297:1833-1837. 33. Muchardt C, Guilleme M, Seeler JS, Trouche D, Dejean A, Yaniv M: Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1a. EMBO Rep 2002, 3:975-981. 34. Maison C, Bailly D, Peter AHFM, Quivy JP, Roche D, Taddei A, Lachner M, Jenuwein T, Almouzni G: Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat Genet 2002, 30:329-334. 35. Narlikar GJ, Fan HY, Kingston RE: Cooperation between complexes that regulate chromatin structure and transcription. Cell 2002, 108:475-487. 36. Langst G, Becker PB: Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors. J Cell Sci 2001, 114:2561-2568. 37. Deuring R, Armstrong JA, Sarte M, Papoulas O, Prestel M, Daubresse G, Verardo M, Moseley SL, Berloco M, Tsukiyama T et al.: The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol Cell 2000, 5:355-365. 38. Corona DF, Clapier C, Becker PB, Tamkun JW: Modulation of ISWI function by site-specific histone acetylation. EMBO Rep 2002, 3:242-247. Here, genetics and biochemistry are nicely combined to show that ISWI and histone H4 acetylation on the X chromosome counteract each other. ISWI-lacking mutants have altered chromatin structure, which can be rescued by lowering acetylation levels on the X chromosome. 39. Akhtar A, Becker PB: The histone H4 acetyl transferase MOF uses a C2HC zinc finger for substrate recognition. EMBO Rep 2001, 2:113-118. We here characterised MOF substrate specificity and showed that the MOF zinc finger region is important for interaction with histone H4 tail on a nucleosomal substrate. 40. Goldmark JP, Fazzio TG, Estep PW, Church GM, Tsukiyama T: The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p. Cell 2000, 103:423-433. 41. Fazzio TG, Kooperberg C, Goldmark JP, Neal C, Basom R, Delrow J, Tsukiyama T: Widespread collaboration of Isw2 and Sin3Rpd3 chromatin remodeling complexes in transcriptional repression. Mol Cell Biol 2001, 21:6450-6460. 42. Hiebert JC, Birchler JA: Effects of the maleless mutation on X and autosomal gene expression in Drosophila melanogaster. Genetics 1994, 136:913-926. 43. Birchler JA, Bhadra U, Bhadra MP, Auger DL: Dosage-dependent gene regulation in multicellular eukaryotes: implications for dosage compensation, aneuploid syndromes, and quantitative traits. Dev Biol 2001, 234:275-288. 44. Jin Y, Wang Y, Walker DL, Dong H, Conley C, Johansen J, Johansen KM: JIL1: A novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Mol Cell 1999, 4:129-135. 45. Wang Y, Zhang W, Jin Y, Johansen J, Johansen KM: The Jil-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 2001, 105:433-443. www.current-opinion.com
Dosage compensation Akhtar 169
Here, hypomorphic mutant alleles of JIL-1 the histone H3 kinase were created to show that JIL-1 is required for global chromatin structure. The X chromosome was show to be severely affected. However, despite the altered appearance the dosage compensation members were still found on the male X chromosome. 46. Henry RA, Tews B, Li X, Scott MJ: Recruitment of the male specific lethal (MSL) dosage compensation complex to an autosomally integrated roX chromatin entry site correlates with an increased expression of an adjacent reporter gene in male Drosophila. J Biol Chem 2001, 276:31953-31958. In this paper, the authors show that insertion of either roX1 or roX2 DNA sequences upstream of a lacZ reporter increases transcriptional activation. Surprisingly, the minimal entry site defined in [26] was not sufficient for twofold transcription increase, suggesting that additional sequences may be required for transcription regulation. 47. Wutz A, Rasmussen TP, Jaenisch R: Chromosomal silencing and localisation are mediated by different domains of Xist RNA. Nat Genet 2002, 30:1-8. In this study, the authors made a series of deletions of Xist RNA and by using an elegant inducible ES cell expression system showed that different functional domains can be identified in Xist RNA. A silencing feature within Xist seems to reside within a conserved repeat sequence at the 50 end of the RNA.
52. Mak W, Silva J, Newall AE, Otte AP, Brockdorff N: Mitotically stable association of polycomb group proteins eed and enx1 with the inactive X chromosome in trophoblast stem cells. Curr Biol 2002, 12:1016-1020. Here the authors show that eed and Enx1 (SET domain containing protein) are localised to the inactive X chromosome in female trophoblast stem cells. This nicely complements [51]. 53. Chadwick BP, Willard HF: Cell cycle-dependent localisation of macro H2A in chromatin of the inactive X chromosome. J Cell Biol 2002, 157:1113-1123. 54. Ganesan S, Silver DP, Greenberg RA, Avni D, Drapkin R, Miron A, Mok SC, Randrianarison V, Brodie S, Salstrom S et al.: BRCA1 supports XIST RNA concentration on the inactive X chromosome. Cell 2002, 111:393-405. An interesting new connection between the well-studied BRCA1 gene and X inactivation is made in this paper. Using a combination of knockout cell lines as well as RNA interference, BRCA1 is shown to play an important role in Xist RNA localisation, therefore leading to new insights into X inactivation. 55. Neal KC, Pannuti A, Smith ER, Lucchesi JC: A new human member of the MYST family of histone acetyl transferases with high sequence similarity to Drosophila MOF. Biochim Biophys Acta 2000, 1490:170-174.
48. Cohen DE, Lee JT: X-chromosome inactivation and the search for chromosome-wide silencers. Curr Opin Genet Dev 2002, 12:219-224.
56. Marin I, Baker BS: Origin and evolution of the regulatory gene male-specific lethal-3. Mol Biol Evol 2000, 17:1240-1250.
49. Avner P, Heard E: X-chromosome inactivation: counting, choice and initiation. Nat Rev Genet 2001, 2:59-67.
57. Scott MJ, Pan LL, Cleland SB, Knox AL, Heinrich J: MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila. EMBO J 2000, 19:144-155.
50. Brockdorff N: X-chromosome inactivation: closing in on proteins that bind Xist RNA. Trends Genet 2002, 18:352-358. 51. Wang J, Chen Y, Schneider E, Cross JC, Nagy A, Magnuson T: Imprinted X inactivation maintained by a mouse Polycomb group gene. Nat Genet 2001, 28:371-375. This study showed that a member of the mouse polycomb group, eed, is important for trophoblast development in female embryos. The authors implicate eed protein in maintaining imprinted X inactivation. See also annotation [52].
www.current-opinion.com
58. Palmer MJ, Mergner VA, Richman R, Manning JE, Kuroda MI, Lucchesi JC: The male-specific lethal-one (msl-1) gene of Drosophila melanogaster encodes a novel protein that associates with the X chromosome in males. Genetics 1993, 134:545-557. 59. Smith ER, Pannuti A, Gu W, Steurnagel A, Cook RG, Allis CD, Lucchesi JC: The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol Cell Biol 2000, 20:312-318.
Current Opinion in Genetics & Development 2003, 13:161–169
Dosage compensation: an intertwined world of RNA and chromatin remodelling Asifa Akhtar Dosage compensation mechanisms in flies and mammals provide an exquisite example of chromatin associated RNAs in chromosome-wide transcription regulation. Recent progress shows that chromatin modifications are also closely linked to these processes. Concerted action of the RNA/chromatinmodifying enzymes may play a crucial role in determining transcriptional output. Furthermore, non-coding RNAs appear to play a dual role, being targeting modules as well as encoding for target sites for complex recognition. Addresses European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:161–169 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by John Tamkun and David Stillman 0959-437X/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(03)00016-9
Abbreviations DCC dosage compensation complex ES embryonic stem HAT histone acetyltransferase MLE maleless MOF males absent on the first MSL male specific lethal roX RNA on the X chromosome Xic X inactivation centre Xist X inactive specific transcript
females [3] (Figure 1). Themes common to these different processes is the presence of factors that specifically decorate the compensated X chromosome and the recent findings that chromatin modifications may play an important role [4,5,6,7]. In this review, I focus on what is known about Drosophila dosage compensation and discuss the interesting parallels with mammalian X inactivation.
Drosophila: XX = XY In D. melanogaster, the single male X is hyper-transcribed approximately twofold in comparison to females. Several genetic screens, scoring for male-specific lethality, have identified proteins required for dosage compensation [8,9]. Male specific lethal (MSL) proteins include MSL-1, MSL-2 and MSL-3, maleless (MLE) and males absent on the first (MOF) [10–15]. In addition to these proteins, two non-coding RNAs, roX1 and roX2 (‘RNA on the X chromosome’) have also been identified as members of the dosage compensation complex (DCC) [16–18]. An essential histone H3 kinase, JIL-1, also associates with the dosage compensation members [19] (Figure 2; Table 1). The dosage-compensated X chromosome has a more decondensed chromatin structure relative to the autosomes that correlates with hyperacetylation of histone H4 at lysine 16 residue (H4K16) along the length of the X chromosome by MOF histone acetyltransferase (HAT) [4,15,20,21]. The DCC preferentially associates at hundreds of sites on the male X but fails to assemble in females as a result of a translation block of the MSL-2 subunit by a protein called Sexlethal [22]. In the absence of MSL-2, MSL proteins and the roX RNAs, except MOF, are expressed in very low amounts in females [10,11].
Chromatin entry sites on the X chromosome Introduction In diploid species, the dosage-compensation mechanisms have evolved to equalise for the difference in gene dosage occurring caused by unequal number of sex chromosomes between males and females. Failure to compensate leads to lethality early in embryo development. Dosage compensation has been studied in mammals, fruit flies and nematode worms, and, interestingly, each of these species tackles the problem differently. In mammals, it is achieved by inactivation of one of the two female X chromosomes in comparison to the XY males [1]. In Caenorhabditis elegans hermaphrodites, transcription from the two X chromosomes is repressed by half to equalise for the expression from the XO males [2]. By contrast, in Drosophila melanogaster, transcription is upregulated twofold in XY males to equalise for the expression from XX www.current-opinion.com
An intact RNA/protein DCC is required for hyper-transcription of genes on the X chromosome. However, in backgrounds where MOF, MSL-3 or MLE have been mutated, a partial complex containing MSL-1 and MSL-2 can be still detected on 35 ‘chromatin entry sites’ along the X chromosome. Chromatin entry sites act as initial docking sites for the complex assembly from where the complex spreads in cis and paints the rest of the X chromosome [23,24]. To date, two of these presumed entry sites have been identified and, remarkably, they include the genes encoding roX1 and roX2 [23,24,25]. The detailed characterisation shows that the roX1 cDNA is sufficient for attraction of the complex on an autosome. Furthermore, active transcription from the roX1 gene is not a prerequisite Current Opinion in Genetics & Development 2003, 13:161–169
162 Chromosomes and expression mechanisms
Figure 1
Mammals
XY
XX
Dosage compensation C. elegans
XO
X-Inactivation
Drosophila
XX
XY
Repression 2×
Hyperactivation 2×
XX
X
X
X
X
X X
X
XX
Current Opinion in Genetics & Development
Mechanisms of dosage compensation. X-linked gene expression is equalised in males and females by either transcriptional silencing of one of the female X chromosomes relative to males (mammals); by transcription repression so that transcription from both X chromosome of an hermaphrodite is repressed by half relative to males (C. elegans); or by transcriptional activation of the single male X chromosome twofold in comparison to females (Drosophila). The size of the X chromosome symbols represents transcription activity.
for the complex assembly. Within the roX1 locus, a 217 bp region is sufficient for attracting the complex. What seems remarkable is that, despite being entry sites, roX1 and roX2 DNAs bear no homology except for a 30 bp sequence at the 30 end of the DNA [26]. The significance of this 30 bp region at the DNA or RNA level still remains unclear; it may contain a protein-binding site for either one of the complex members or for an unidentified protein. Analysis of the chromatin entry site using MSL mutants shows that, unlike other entry sites, roX1 and roX2 entry sites are sensitive to the absence of MLE because a partial DCC complex is unable to form in these mutants [24,27]. These results suggest that different entry sites may have different component requirements and therefore may play specialised roles in the context of global male X chromosome regulation.
Assembly and spreading of the dosage compensation complex The Drosophila dosage compensation appears to be a twostep process (Figure 3a). The first step includes assembly of the complex on chromatin entry sites mediated by MSL-1 and MSL-2 [23]. Whether MSL-1 and MSL-2 recognise some specific feature of the DNA sequence or a special chromatin structure around an entry site in preference to other X chromosomal regions remains unknown. Characterisation of additional entry sites will be an important step in identifying these features. Current Opinion in Genetics & Development 2003, 13:161–169
Once correctly assembled, the DCC proteins seem to occupy many additional distinct regions along the chromosome whereas other regions seem devoid of the complex [27]. It is therefore an enigma how the DCC gets targeted to entry sites, spreads from these relatively well defined sites and occupies distinct X-chromosomal regions. Whether there are unidentified boundary elements dispersed along the X chromosome that prevent the access of DCC from certain X chromosomal regions remains unclear. A similar problem is faced in the mammalian system where, despite downregulation of most X linked genes, some X chromosomal regions escape inactivation. The potential mammalian complex is thus allowed access to most but not all of the X chromosome. In either MOF or MLE mutant males — where the catalytic sites of these enzymes have been mutated — partial complexes containing MSL-1, MSL-2 and MSL-3 are found restricted to chromatin entry sites, suggesting these enzymes affect the second step namely ‘spreading’ along the X chromosome [15,28]. It is interesting to note that, MLE but not MOF protein affects stability of roX RNAs, which implies that even though MLE and MOF work in concert to help the complex spread, they are clearly required for distinct steps during this process [26]. Whether MOF’s participation in the spreading phenomenon is merely to create a more fluid chromatin environment by hyper-acetylation or in addition, MOF can also post-translationally modify other DCC members to modulate their activity remains an interesting issue to be explored.
Targeting or being targeted by roX RNAs At least for some of the DCC members, roX RNAs play a role in X-chromosome-specific targeting. MLE helicase was the first protein within the DCC to have RNA sensitive association to the X chromosome [29]. It was later shown that MLE is also important for stability of roX RNAs [28]. A clue as to how specific targeting may be achieved in the case of MOF came from the finding that MOF is also an RNA binding protein, it binds RNA via its chromodomain, and that it is also tethered to the X chromosome via RNA [30]. In accord with this, deletion of roX1 and roX2 show mis-localisation of the MSL proteins, in particular MSL-2 and H4K16 acetylation to the autosomes [31]. Furthermore, in MOF mutant females overexpressing MSL-2, the roX RNAs are dispersed around the nucleoplasm, suggesting that the MOF/RNA complex is important for correct targeting to the X chromosome [28]. Hence, there seems to be an
(Figure 2 Legend) Linearised depiction of the domain structure of dosage compensation associated factors in (a) Drosophila and (b) mammals. The different domains are indicated as cylindrical shapes. The domain size and location are drawn to indicate their relative position within the proteins. The total length of the proteins and RNAs is indicated: aa, amino acid; kb, kilobase. www.current-opinion.com
Dosage compensation Akhtar 163
Figure 2
(a)
MSL-1: 1039 aa
S/TP rich potential phosphorylation sites
Acidic region (25% D or E)
MSL-2: 773 aa
RING finger
Coiled coil domain
Metallothionein-like domain PHD
MSL-3: 512 aa
2 Chromodomains
MLE: 1293 aa
Helicase domain
Double strand RNA binding domain
9 G-rich repeats
MOF: 827 aa
Chromodomain Zn finger HAT domain
JIL-1: 1207 aa Kinase domain 1
roX1RNA: 3.7 kb
(b)
Kinsae domain 2
roX2 RNA: 0.6 kb
Macro H2A: 372 aa
H2A domain
Eed: 535 aa
WD40
Enx1: 747 aa
SANT
SANT
SET
BRCA1: 1812 aa
RING finger
BRCT
Xist RNA: 17 kb
Current Opinion in Genetics & Development
www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:161–169
164 Chromosomes and expression mechanisms
Table 1 Functional regions within the DCC proteins identified by interaction studies. Functional region
References
MSL-1 Residues 1–84; important for X chromosomal localisation. Residues 85–186 important for in vitro interaction with MSL-2. Residues 759–1039 important for interaction with MOF and MSL-3.
[57,58]
MSL-2 Residues 1–190, containing ring finger (37–87), are important for X chromosomal localisation and interaction with MSL-1.
[12,27,58]
MSL-3 MSL-3 directly interacts with MSL-1. Residues 437–516 containing chromodomain interact with RNA in vitro.
[57] [30]
MOF Residues 518–827 containing zinc finger and HAT domain (MYST domain) are sufficient for HAT activity. Point mutation in the HAT domain (G691E ) abolishes HAT activity. C2HC zinc finger within the MYST domain is important for interaction with chromatin especially histone H4 tail. Point mutations C557G, Y572G, L578/Y580 abolish HAT activity and chromatin interaction. MOF specifically interacts with roX2 RNA in vivo. Chromodomain in MOF interacts with RNA in vitro. Point mutations W426G, Y416D abolish RNA interaction both in vitro and in vivo. MLE Point mutations in helicase domain GKT to GET (ATPase A motif) or DEIH-DQIH. (ATPase B motif) reduce ATPase/Helicase activity, and affect male viability. Residues 940–1293 containing GGY domain: important for targeting MLE to the male X via RNA. JIL-1 Kinase domain 1 and 2: catalytic domains; important for kinase activity. Both kinase domains also interact with MSL-1 and MSL-3.
[59] [15,21] [39] [30]
[29]
[19]
Summary of functional regions identified by protein–protein and protein–RNA interaction studies in Drosophila proteins associated with dosage compensation. Point mutations are indicated by the amino acid of interest, its position within the protein followed by the replaced amino acid.
intimate connection between MOF and roX RNAs. However, the detailed mechanism is still poorly understood. Apart from MLE and MOF, MSL-3 is the third RNAbinding component within the complex, underscoring the importance of RNAs in integrity and specificity of the DCC [30]. Future experiments will tell us whether the specificity for RNA comes from folding of these RNAs into a distinct structure, which act as a scaffold for protein assembly and subsequent targeting, or whether RNAs act as flags signalling for the X chromosome. Alternatively, the DCC proteins themselves may remodel the RNA structure by interaction and act instead as chaperones for the RNA to be transported to and along the X chromosome. Interesting links between the RNA interference machinery and heterochromatic silencing in Schizosaccharomyces pombe and the involvement of RNA in regulation of pericentric heterochromatin have been made recently [32–34]. These findings further reinforce the versatile nature of non-coding RNAs in various regulatory processes. Detailed functional studies will be instrumental in deciphering the true mechanisms operating behind regulation using non-coding RNAs in the chromatin context. Current Opinion in Genetics & Development 2003, 13:161–169
Association with additional chromatin remodelling factors Chromatin remodelling factors come in two flavours: either enzymes that utilise ATP to mobilise nucleosomes or enzymes that modify chromatin structure by posttranslational modification of histones [35]. Recent work on the chromatin remodelling enzyme ISWI ATPase [36] has shed light into new possible interactions between the DCC and ISWI. In the fly, ISWI is essential
(Figure 3 Legend) Dosage compensation in (a) Drosophila and (b) mammals. (a) Dosage compensation proteins assemble on 35 distinct ‘chromatin entry sites’ on the male X chromosome. The two identified entry sites include roX1 and roX2 RNA encoding genes. The assembly of a functional complex leads to hyperacetylation of the surrounding chromatin at histone H4 lysine 16 residue followed by spreading of the complex along the X chromosome at numerous additional sites. Dosage compensation in Drosophila leads to transcription enhancement of most X-linked genes. (b) In mammals, X inactivation initiates at a single site, the X inactivation centre (Xic), which includes the Xist RNA encoding gene. An as yet unidentified RNA/protein complex spreads bidirectionally from Xic. The onset of X inactivation proceeds with hypoacetylation of chromatin and hypermethylation of histone H3 lysine 9 residue. During the maintenance phase of X inactivation, macro H2A, a histone variant, is incorporated into the silent chromatin. X inactivation in mammals leads to transcriptional repression of most X-linked genes. www.current-opinion.com
Dosage compensation Akhtar 165
Figure 3
(a)
K16
K16
6
K1
K16 K16
K1
? K1 6
6
K16
K16
6
K1
K16
K16
K16
K16
Entry site
MLE
MOF
MSL-3
MSL-1/MSL-2
roX1/roX2
(b) ? ? ?
?
? ?
?
K9 K9
K1
?
?
6
K9
K16
K9
K16
K9
6
K1
K16
K9
K9 K16
K9
?
?
K1 K9
6
K9
K9
K9
Xic
K9
K9
K9
HAT
K9
K9
HDAC
K9
K9
K9
HMTase
K9
BRCA1/BARD1
K9
macroH2A
Xist Current Opinion in Genetics & Development
www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:161–169
166 Chromosomes and expression mechanisms
for organism viability. In the mutant, the dying male larvae display a dramatically altered X chromosome [37]. This phenotype probably occurs due to an imbalance caused by hyperacetylation of the male X chromosome by MOF that works towards a more open chromatin structure and ISWI that may lead to a more condensed chromatin state. In the absence of ISWI, this opposing activity is lost and therefore the X chromosome appears extremely decondensed. In agreement with this, the ATPase activity of ISWI on histone H4 substrate, which is also a substrate for MOF interaction on nucleosomes, is inversely correlated with its acetylation status [38,39]. Taken together with the observation that ISWI staining does not coincide with RNA polymerase II staining and that yeast ISW2 functions to repress genes induced early in meiosis [40,41], it will be interesting to find out whether or not ISWI functions to downregulate gene expression also on the X chromosome. Association of a chromatin-remodelling factor that may function as a putative repressive activity corroborates the model which proposes that the DCC functions to modulate the action of MOF histone acetyl transferase so that the autosomal genes are not over transcribed which may occur due to the presence of a single copy of the X chromosome in males [42,43]. Like ISWI, JIL-1 kinase also plays a more general role in chromosome architecture. JIL-1 has been shown to phosphorylate histone H3 at Serine 10 residue [19]. The connection between the DCC and JIL-1 originates from the following observations. JIL-1 immunostaining shows a twofold enrichment on the X chromosome relative to the autosomes [19]. Hypomorphic JIL-1 mutants show a variety of nuclear defects with alterations in chromosome morphology, the X chromosome seems most severely affected. In addition, these mutants also show a skewed male to female ratio [44,45] (Figure 2; Table 1). The exact role of JIL-1, similar to ISWI, in the context of dosage compensation, remains to be defined as targeting of the DCC members is not affected in JIL-1 mutants [19]. An attractive hypothesis is that JIL-1 may regulate DCC by post-translational modification of the DCC members. H4K16 acetylation on the X chromosome seems to have a high turnover [30] because loss of MOF from the X chromosome leads to a rapid deacetylation of the X chromosome. General histone deacetylase activity in Drosophila may be responsible for this effect, MOF being required on the X chromosome to maintain the hyperacetylated status. Alternatively, a specific histone deacetylase may associate with the complex to regulate dosage compensation. The interplay between this putative histone deacetylase and MOF histone acetyl transferase may have important consequences for the function of the complex. Current Opinion in Genetics & Development 2003, 13:161–169
It appears that, in addition to the well-defined components tailored for male specific functions, the DCC may also interact with additional chromatin-remodelling factors. This larger protein–protein and protein–RNA network might function to fine tune the X chromosomal transcriptional activity.
Function of the dosage compensation complex To date, it has remained a mystery how the dosagecompensation complex functions at the molecular level to activate transcription. It may function at the level of chromatin remodelling, transcription initiation or transcription elongation. Apart from the close correlation between hyper-acetylation and transcriptional upregulation, evidence that dosage compensation may play an active role in transcription regulation comes from several recent observations. Tethering of a chromatin entry site like roX1 or roX2 genes adjacent to a b-galactosidase reporter at an autosomal location increases transcription activation of an heterologous promoter [46]. Interestingly, the exon sequence rather than genomic sequence was more efficient in transcription activation. Furthermore, it was observed that even though a 217 bp region within roX1 is sufficient for complex targeting [26], surrounding sequences within roX1/2 entry sites may have additional function in transcriptional modulation. A similar question was posed by studying the chromatin status along dosage-compensated genes in vivo using chromatin immunoprecipitation experiments [25]. The X-linked dosage compensated genes Phosphogluconate dehydrogenase and Zwischenferment showed higher acetylation levels in the coding region than in the promoter region. These results suggest that the DCC might enhance transcription elongation. It will be interesting to know whether this increased acetylation correlates with increased efficiency of the polymerase read through and whether the dosage-compensation machinery interacts with basal transcription factors.
Mammals: Xx = XY Despite the difference in transcriptional output, the X inactivation process in mammals, similar to flies, requires several steps that include choice of the X chromosome to inactivate followed by initiation and then spreading of inactivation along the X chromosome (Figure 3b). The choice and initiation of inactivation are early steps during embryo development and occur when the totipotent embryonic cells start to differentiate. Unlike in flies, inactivation in mammals is initiated at a single site at the X inactivation centre (Xic), and from there propagates bidirectionally. The Xist gene is located within the Xic, which encodes a non-coding, spliced, polyadenylated RNA. Xist RNA is expressed exclusively from the inactive www.current-opinion.com
Dosage compensation Akhtar 167
X chromosome and coats the X chromosome. Therefore, similar to flies, the ‘targeting and being targeted’ roles also seem valid for Xist. However, Xist RNA only spreads in cis, whereas roX RNAs can be also be targeted in trans. Furthermore, Xist RNA is essential for the onset of inactivation but not for maintainance [47,48–50].
regulation. It is possible that, despite the evolutionary distance, some aspects of the targeting mechanism remain similar between these two organisms. Combination of genomics and proteomics approaches will yield exciting new insights into these chromosome-wide transcription regulatory processes.
Because of the complexity of the mammalian system, an analogous X inactivation complex has not been identified yet, but several recent observations make it a possibility (Figure 2). The inactive X chromosome has long been known to be hypoacetylated, but interestingly it has been reported that methylation at the histone H3 lysine 9 (H3K9) residue constitutes an early mark on the inactive X chromosome [5,6]. At least for imprinted inactivation, the eed/enx1 complex appears to be a good candidate for the histone methyltransferase complex because, in trophectoderm cells, these proteins are localised on the inactive X chromosome, and eed mutants fail to maintain imprinted X inactivation [50,51,52].
Acknowledgements
The inactive X (Xi) chromatin is enriched in a histone variant macro H2A, within a nuclear structure known as the macrochromatin body. However, macro H2A associates with the inactive Xi 5–6 days after the onset of X inactivation and therefore appears to be important in the later step of maintenance of Xi chromatin structure. Macro H2A association also appears to be sensitive to cell-cycle progression [50,53]. Unexpectedly, an exciting new connection between BRCA1 and Xist RNA was reported recently [54]. BRCA1 is a tumour suppressor gene involved in breast and ovarian tumours. BRCA1 seems to colocalise with Xist RNA in female somatic cells and loss of BRCA1 leads to defects in X inactivation, altered Xist RNA appearance, loss of H3K9 methylation as well as loss of Macro H2A association. BRCA1 also appears to interact with a particular region of Xist RNA via its ring finger domain. Whether proteins associated with BRCA1 constitute an analogous DCC in mammals remains to be seen. It is noteworthy that some of the Drosophila dosage compensation members are also conserved in mammals, with as yet undefined function, in particular, chromodomaincontaining proteins MSL-3 and MOF [55,56]. Whether these proteins have evolved into a new and distinct function instead of dosage compensation remains to be determined.
Conclusions Evolutionarily distinct dosage compensation mechanisms in Drosophila and mammals seem to have evolved interesting parallels with noncoding RNAs and chromatin modifications. Recent advances in Drosophila suggest that the dosage compensation complex is a dynamic entity with multiple interactions with additional chromatin machinery, and RNAs play a very integrated role in this www.current-opinion.com
I thank Peter Becker, Elisa Izaurralde, Stefan Kass and Mikko Taipale for critical reading of the manuscript. I am also grateful to Mikko Taipale for help with the artwork.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Lyon M: Gene action in the X-chromosome of the mouse (Mus musculus). Nature 1961, 190:372-373.
2.
Meyer BJ, Casson LP: Caenorhabditis elegans compensates for the difference in X chromosome dosage between the sexes by regulating transcript levels. Cell 1986, 47:871-881.
3.
Mukherjee AS, Beermann W: Synthesis of ribonuleic acid by the X-chromosomes of Drosophila melanogaster and the problem of dosage compensation. Nature 1965, 207:785-786.
4.
Turner BM, Birley AJ, Lavender J: Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 1992, 69:375-384.
5.
Heard E, Rougeulle C, Arnaud D, Avner P, Allis DC, Spector DL: Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation. Cell 2001, 107:727-738. See annotation [6]. 6.
Mermoud JE, Popova B, Peters AH, Jenuwein T, Brockdorff N: Histone H3 lysine 9 methylation occurs rapidly at the onset of random X chromosome inactivation. Curr Biol 2002, 12:247-251. By using specific antibodies raised against methylated peptides in embryonic stem (ES) cells, these studies [5,6] were the first documenting that histone H3 lysine 9 methylation constitutes an early mark on the female inactive X chromosome. 7.
Fong Y, Bender L, Wang W, Strome S: Regulation of the different chromatin states of autosomes and X chromosomes in the germ line of C. elegans. Science 2002, 296:2235-2238. This study showed that in C. elegans eed1/enx1 homologues MES-6 and MES-2 also form a stable complex required for gene silencing and germline development, and this also correlates with histone H3 lysine 9 methylation. 8.
Belote JM, Lucchesi JC: Control of X chromosome transcription by the maleless gene in Drosophila. Nature 1980, 285:573-575.
9.
Uenoyama T, Uchida S, Fukunaga A, Oishi K: Studies on the sexspecific lethals of Drosophila melanogaster. IV. Gynandromorph analysis of three male-specific lethals, mle, msl-2(27) and mle(3)132. Genetics 1982, 102:223-231.
10. Bashaw GJ, Baker BS: The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development 1995, 121:3245-3258. 11. Kelley RL, Solovyeva I, Lyman LM, Richman R, Solovyev V, Kuroda MI: Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 1995, 81:867-877. 12. Zhou S, Yang Y, Scott MJ, Pannuti A, Fehr KC, Eisen A, Koonin EV, Fouts DL, Wrightsman R, Manning JE et al.: Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sexspecific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster. EMBO J 1995, 14:2884-2895. Current Opinion in Genetics & Development 2003, 13:161–169
168 Chromosomes and expression mechanisms
13. Gorman M, Franke A, Baker BS: Molecular characterization of the male-specific lethal-3 gene and investigations of the regulation of dosage compensation in Drosophila. Development 1995, 121:463-475. 14. Kuroda MI, Kernan MJ, Kreber R, Ganetzky B, Baker BS: The maleless protein associates with the X chromosome to regulate dosage compensation in Drosophila. Cell 1991, 66:935-947. 15. Hilfiker A, Hilfiker-Kleiner D, Pannuti A, Lucchesi JC: mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila. EMBO J 1997, 16:2054-2060. 16. Amrein H, Axel R: Genes expressed in neurons of adult male Drosophila. Cell 1997, 88:459-469. 17. Franke A, Baker BS: The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. Mol Cell 1999, 4:117-122. 18. Meller VH, Wu KH, Roman G, Kuroda MI, Davis RL: roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell 1997, 88:445-457. 19. Jin Y, Wang Y, Johansen J, Johansen KM: JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associated with the male specific lethal (MSL) Dosage Compensation Complex. J Cell Biol 2000, 149:1005-1010. 20. Bone JR, Lavender J, Richman R, Palmer MJ, Turner BM, Kuroda MI: Acetylated histone H4 on the male X chromosome is associated with dosage compensation in Drosophila. Genes Dev 1994, 8:96-104. 21. Akhtar A, Becker PB: Activation of transcription through histone H4 acetylation by MOF, an acetyl transferase essential for dosage compensation in Drosophila. Mol Cell 2000, 5:367-375. 22. Bashaw GJ, Baker BS: Dosage compensation and chromatin structure in Drosophila. Curr Opin Genet Dev 1996, 6:496-501. 23. Kelley RL, Meller VH, Gordadze PR, Roman G, Davis RL, Kuroda MI: Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 1999, 98:513-522. 24. Meller VH, Gordadze PR, Park Y, Chu X, Stuckenholz C, Kelley RL, Kuroda MI: Ordered assembly of roX RNAs into MSL complexes on the dosage- compensated X chromosome in Drosophila. Curr Biol 2000, 10:136-143. 25. Smith ER, Allis CD, Lucchesi JC: Linking global histone acetylation to the transcription enhancement of Xchromosomal genes in Drosophila males. J Biol Chem 2001, 276:31483-31486. Using chromatin immunoprecipitation experiments it was demonstrated here that histone H4 acetylation on dosage compensated genes correlates with coding regions rather than the promoter regions on the X chromosome, implying that the DCC may function to promote transcription elongation. 26. Kageyama Y, Mengus G, Gilfillan G, Kennedy HG, Stuckenholz C, Kelley RL, Becker PB, Kuroda MI: Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site. EMBO J 2001, 20:2236-2245. The authors of this paper performed the first detailed characterisation of roX1 chromatin entry site and showed that a 217 bp region within roX1 is sufficient to attract dosage compensation proteins to an autosome. 27. Lyman LM, Copps K, Rastelli L, Kelley RL, Kuroda MI: Drosophila male-specific lethal-2 protein: structure/function analysis and dependence on MSL-1 for chromosome association. Genetics 1997, 147:1743-1753. 28. Gu W, Wei X, Pannuti A, Lucchesi JC: Targeting the chromatinremodelling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities. EMBO J 2000, 19:5202-5211. 29. Richter L, Bone JR, Kuroda MI: RNA-dependent association of the Drosophila maleless protein with the male X chromosome. Genes Cells 1996, 1:325-336. Current Opinion in Genetics & Development 2003, 13:161–169
30. Akhtar A, Zink D, Becker PB: Chromodomains as RNA interaction modules. Nature 2000, 407:405-409. We showed here that the MOF HAT is tethered to the male X chromosome by RNA and that histone acetylation on the X chromosome has a high turnover. We also presented evidence that chromodomains of MOF and MSL-3 interact with RNA. 31. Meller V, Rattner BP: The roX genes encode redundant male specific lethal transcripts required for targeting of the MSL complex. EMBO J 2002, 21:1084-1091. Even though roX RNAs seem to have redundant function, it is convincingly shown here that deletion of roX1 and roX2 genes in flies causes malespecific lethality and mistargeting of the MSL proteins as well as histone H4 lysine 16 acetylation. 32. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SIS, Martienssen RA: Regulation of heterochromatic silencing and histone H3 lysine9 methylation by RNAi. Science 2002, 297:1833-1837. 33. Muchardt C, Guilleme M, Seeler JS, Trouche D, Dejean A, Yaniv M: Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1a. EMBO Rep 2002, 3:975-981. 34. Maison C, Bailly D, Peter AHFM, Quivy JP, Roche D, Taddei A, Lachner M, Jenuwein T, Almouzni G: Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat Genet 2002, 30:329-334. 35. Narlikar GJ, Fan HY, Kingston RE: Cooperation between complexes that regulate chromatin structure and transcription. Cell 2002, 108:475-487. 36. Langst G, Becker PB: Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors. J Cell Sci 2001, 114:2561-2568. 37. Deuring R, Armstrong JA, Sarte M, Papoulas O, Prestel M, Daubresse G, Verardo M, Moseley SL, Berloco M, Tsukiyama T et al.: The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol Cell 2000, 5:355-365. 38. Corona DF, Clapier C, Becker PB, Tamkun JW: Modulation of ISWI function by site-specific histone acetylation. EMBO Rep 2002, 3:242-247. Here, genetics and biochemistry are nicely combined to show that ISWI and histone H4 acetylation on the X chromosome counteract each other. ISWI-lacking mutants have altered chromatin structure, which can be rescued by lowering acetylation levels on the X chromosome. 39. Akhtar A, Becker PB: The histone H4 acetyl transferase MOF uses a C2HC zinc finger for substrate recognition. EMBO Rep 2001, 2:113-118. We here characterised MOF substrate specificity and showed that the MOF zinc finger region is important for interaction with histone H4 tail on a nucleosomal substrate. 40. Goldmark JP, Fazzio TG, Estep PW, Church GM, Tsukiyama T: The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p. Cell 2000, 103:423-433. 41. Fazzio TG, Kooperberg C, Goldmark JP, Neal C, Basom R, Delrow J, Tsukiyama T: Widespread collaboration of Isw2 and Sin3Rpd3 chromatin remodeling complexes in transcriptional repression. Mol Cell Biol 2001, 21:6450-6460. 42. Hiebert JC, Birchler JA: Effects of the maleless mutation on X and autosomal gene expression in Drosophila melanogaster. Genetics 1994, 136:913-926. 43. Birchler JA, Bhadra U, Bhadra MP, Auger DL: Dosage-dependent gene regulation in multicellular eukaryotes: implications for dosage compensation, aneuploid syndromes, and quantitative traits. Dev Biol 2001, 234:275-288. 44. Jin Y, Wang Y, Walker DL, Dong H, Conley C, Johansen J, Johansen KM: JIL1: A novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Mol Cell 1999, 4:129-135. 45. Wang Y, Zhang W, Jin Y, Johansen J, Johansen KM: The Jil-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 2001, 105:433-443. www.current-opinion.com
Dosage compensation Akhtar 169
Here, hypomorphic mutant alleles of JIL-1 the histone H3 kinase were created to show that JIL-1 is required for global chromatin structure. The X chromosome was show to be severely affected. However, despite the altered appearance the dosage compensation members were still found on the male X chromosome. 46. Henry RA, Tews B, Li X, Scott MJ: Recruitment of the male specific lethal (MSL) dosage compensation complex to an autosomally integrated roX chromatin entry site correlates with an increased expression of an adjacent reporter gene in male Drosophila. J Biol Chem 2001, 276:31953-31958. In this paper, the authors show that insertion of either roX1 or roX2 DNA sequences upstream of a lacZ reporter increases transcriptional activation. Surprisingly, the minimal entry site defined in [26] was not sufficient for twofold transcription increase, suggesting that additional sequences may be required for transcription regulation. 47. Wutz A, Rasmussen TP, Jaenisch R: Chromosomal silencing and localisation are mediated by different domains of Xist RNA. Nat Genet 2002, 30:1-8. In this study, the authors made a series of deletions of Xist RNA and by using an elegant inducible ES cell expression system showed that different functional domains can be identified in Xist RNA. A silencing feature within Xist seems to reside within a conserved repeat sequence at the 50 end of the RNA.
52. Mak W, Silva J, Newall AE, Otte AP, Brockdorff N: Mitotically stable association of polycomb group proteins eed and enx1 with the inactive X chromosome in trophoblast stem cells. Curr Biol 2002, 12:1016-1020. Here the authors show that eed and Enx1 (SET domain containing protein) are localised to the inactive X chromosome in female trophoblast stem cells. This nicely complements [51]. 53. Chadwick BP, Willard HF: Cell cycle-dependent localisation of macro H2A in chromatin of the inactive X chromosome. J Cell Biol 2002, 157:1113-1123. 54. Ganesan S, Silver DP, Greenberg RA, Avni D, Drapkin R, Miron A, Mok SC, Randrianarison V, Brodie S, Salstrom S et al.: BRCA1 supports XIST RNA concentration on the inactive X chromosome. Cell 2002, 111:393-405. An interesting new connection between the well-studied BRCA1 gene and X inactivation is made in this paper. Using a combination of knockout cell lines as well as RNA interference, BRCA1 is shown to play an important role in Xist RNA localisation, therefore leading to new insights into X inactivation. 55. Neal KC, Pannuti A, Smith ER, Lucchesi JC: A new human member of the MYST family of histone acetyl transferases with high sequence similarity to Drosophila MOF. Biochim Biophys Acta 2000, 1490:170-174.
48. Cohen DE, Lee JT: X-chromosome inactivation and the search for chromosome-wide silencers. Curr Opin Genet Dev 2002, 12:219-224.
56. Marin I, Baker BS: Origin and evolution of the regulatory gene male-specific lethal-3. Mol Biol Evol 2000, 17:1240-1250.
49. Avner P, Heard E: X-chromosome inactivation: counting, choice and initiation. Nat Rev Genet 2001, 2:59-67.
57. Scott MJ, Pan LL, Cleland SB, Knox AL, Heinrich J: MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila. EMBO J 2000, 19:144-155.
50. Brockdorff N: X-chromosome inactivation: closing in on proteins that bind Xist RNA. Trends Genet 2002, 18:352-358. 51. Wang J, Chen Y, Schneider E, Cross JC, Nagy A, Magnuson T: Imprinted X inactivation maintained by a mouse Polycomb group gene. Nat Genet 2001, 28:371-375. This study showed that a member of the mouse polycomb group, eed, is important for trophoblast development in female embryos. The authors implicate eed protein in maintaining imprinted X inactivation. See also annotation [52].
www.current-opinion.com
58. Palmer MJ, Mergner VA, Richman R, Manning JE, Kuroda MI, Lucchesi JC: The male-specific lethal-one (msl-1) gene of Drosophila melanogaster encodes a novel protein that associates with the X chromosome in males. Genetics 1993, 134:545-557. 59. Smith ER, Pannuti A, Gu W, Steurnagel A, Cook RG, Allis CD, Lucchesi JC: The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol Cell Biol 2000, 20:312-318.
Current Opinion in Genetics & Development 2003, 13:161–169