X-chromosome targeting and dosage compensation are mediated by distinct domains in MSL-3.

Abstract

In Drosophila, dosage compensation of X-linked genes is achieved by transcriptional upregulation of the male X chromosome. Genetic and biochemical studies have demonstrated that male-specific lethal (MSL) proteins together with roX RNAs regulate this process. Here, we show that MSL-3 is essential for cell viability and that three domains in the protein have distinct roles in dosage compensation. The chromo-barrel domain (CBD) is not necessary for MSL targeting to the male X chromosome but is important for male viability and equalization of X-linked gene transcription. The polar region cooperates with the CBD in MSL-3 function, whereas the MRG domain is responsible for targeting the protein to the X chromosome. Our results demonstrate that MSL-3 localization to the male X chromosome and transcriptional upregulation of X-linked genes are two separable functions of the MSL-3 protein.

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X-chromosome targeting and dosage compensation
are mediated by distinct domains in MSL-3
Alessia Buscaino, Gae ¨lle Legube & Asifa Akhtar+
Gene Expression Programme, EMBL, Heidelberg, Germany
In Drosophila, dosage compensation of X-linked genes is achieved
by transcriptional upregulation of the male X chromosome.
Genetic and biochemical studies have demonstrated that male-
specific lethal (MSL) proteins together with roX RNAs regulate
this process. Here, we show that MSL-3 is essential for cell
viability and that three domains in the protein have distinct roles
in dosage compensation. The chromo-barrel domain (CBD) is not
necessary for MSL targeting to the male X chromosome but is
important for male viability and equalization of X-linked gene
transcription. The polar region cooperates with the CBD in MSL-3
function, whereas the MRG domain is responsible for targeting
the protein to the X chromosome. Our results demonstrate that
MSL-3 localization to the male X chromosome and transcriptional
upregulation of X-linked genes are two separable functions of
the MSL-3 protein.
Keywords: dosage compensation; transcription; MSL-3;
chromo-barrel domain; MRG
EMBO reports (2006) 7, 531–538. doi:10.1038/sj.embor.7400658
INTRODUCTION
In heterogametic organisms, a specific compensatory mechanism,
dosage compensation, is established to equalize X-chromosomal
gene products between the two sexes. In Drosophila melanogaster,
dosage compensation is achieved by doubling the level of
expression of genes from the single male X chromosome. In
Drosophila, this is mediated by the products of at least five
protein-coding genes, collectively named male-specific lethal
(MSL) proteins (MSL-1, MSL-2, MSL-3, MOF and MLE), and two
non-coding RNAs (roX1 and roX2). These factors function together
in a complex that is termed the dosage compensation complex
(DCC) or MSL complex (reviewed by Lucchesi et al, 2005; Taipale
& Akhtar, 2005). The DCC is specifically assembled in males,
where it binds to hundreds of sites on the X chromosome. DCC
binding to the X chromosome correlates with hyperacetylation of
histone H4 at lysine 16 (H4K16Ac), mediated by the MYST histone
acetyltransferase MOF (Akhtar & Becker, 2000; Smith et al,
2000). Protein–RNA and protein–protein interactions among DCC
components are important in regulating dosage compensation. For
example, MLE, MOF and MSL-3 bind to RNA, and their
localization to the X chromosome is sensitive to RNase treatment
(Richter et al, 1996; Akhtar et al, 2000; Buscaino et al, 2003).
MSL-1, MSL-3 and MOF interact in vitro and this interaction
activates MOF acetyltransferase activity (Buscaino et al, 2003;
Morales et al, 2004). In addition, MOF is able to acetylate MSL-1
and MSL-3 (Buscaino et al, 2003; Morales et al, 2004).
MSL-3 is a member of the MRG family of proteins,
characterized by an amino-terminal chromo-barrel domain
(CBD) flanked by a positively charged polar region and a
carboxy-terminal MRG domain (Bertram & Pereira-Smith, 2001).
The CBD is a protein motif structurally related to chromodomains
(Nielsen et al, 2005). Different chromo-related domains have been
shown to be involved in the recognition and binding of distinct
chromatin components. For example, the chromodomains of HP1
and Polycomb interact with methylated histone tails by means of a
conserved aromatic pocket (Lachner et al, 2001; Fischle et al,
2003) and their deletion in vivo abolishes chromosomal binding
and results in lethality in flies (Messmer et al, 1992; Platero et al,
1995). In contrast, MOF and MSL-3 CBD are involved in RNA
binding in vitro (Akhtar et al, 2000; Morales et al, 2005).
However, the observation that the MSL-3 CBD contains an
aromatic pocket similar to that responsible for the recognition and
binding of methyl groups in the chromodomains of HP1 and Pc
raises the possibility that the MSL-3 CBD could also recognize
methyl groups (Nielsen et al, 2005).
The positively charged polar region of MSL-3 contains lysine
116, acetylated by MOF in vitro (Buscaino et al, 2003).
Interestingly, the MOF CBD and the HP1 chromodomain are
both flanked by a positively charged region (Muchardt et al, 2002;
Nielsen et al, 2005). In HP1, the polar region, termed ‘hinge
region’, can interact with RNA and cooperates with the
chromodomain for targeting the protein to heterochromatic loci
(Muchardt et al, 2002). These studies suggest that in different
chromodomain protein families, the polar region cooperates with
the chromodomain in a similar way.
The MRG domain is a novel protein motif of unknown function
(Bertram & Pereira-Smith, 2001). Strikingly, all the characterized
MRG family proteins are part of large chromatin remodelling
Received 4 October 2005; revised 8 February 2006; accepted 10 February 2006;
published online 10 March 2006
+Corresponding author. Tel: þ49 6221 387 550; Fax: þ49 6221 387 518;
E-mail: akhtar@embl.de
Gene Expression Programme, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
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complexes, all of which contain MYST family acetyltransferases
(Doyon et al, 2004).
Although DCC localization to the X chromosome and male
viability are correlated, it is not yet clear whether DCC binding to
the male X chromosome is sufficient to achieve the twofold
upregulation of X-linked genes or whether targeting and dosage
compensation are two distinct events. To address this question, we
have conducted a structure/function analysis of MSL-3 in vivo. Our
results demonstrate that MSL-3 targeting and dosage compensation
are mediated by distinct MSL-3 domains. We show that the CBD,
unlike other chromo-related domains, is not important for DCC
targeting to the X chromosome but is required for the proper
compensation of X-linked genes and for male viability. We show the
polar region cooperates with the CBD, whereas the MRG domain is
responsible for targeting MSL-3 to the male X chromosome.
RESULTS AND DISCUSSION
Characterization of msl-3083allele
Both the Oishi and the Baker labs identified msl-3 as a gene, the
product of which is required for male viability in Drosophila
(Uchida et al, 1981; Gorman et al, 1995). However, none of the
identified alleles has been molecularly characterized in detail.
Therefore, we first carried out a detailed characterization of an
msl-3 allele, msl-3083. Sequencing the genomic locus from msl-3083
homozygous adult female flies showed that the msl-3083has a
point mutation in the splicing junction of the first intron, which is
mutated owing to a deletion of T104 (supplementary Fig S1A
online). Western blot analysis using crude extract prepared from
msl-3083homozygous embryos with an MSL-3-specific antibody
raised against the full-length protein demonstrated that msl-3083
flies do not produce any full-length or truncated protein
(supplementary Fig S1B online). These results were confirmed by
immunofluorescence studies on polytene chromosomes (supple-
mentary Fig S1C online) and they demonstrate that msl-3083is
a null allele of the msl-3 gene. Next, we were interested to
determine why mutations in msl-3 gene lead to male lethality and
whether the absence of MSL-3 produces the same phenotypes in
different tissues. For this purpose, msl-3083mutant clones were
generated in the eyes of adult flies with the Minute FLP/FRT system
and in wing imaginal discs with the FLP/FRT system. Loss of MSL-3
in the eyes led to a male-specific phenotype, as clones of mutant
cells are much smaller in males versus females (supplementary
Fig S1D online). This phenotype indicated that msl-3 is required
for cell proliferation or survival. To examine whether msl-3
mutation leads to cell death, msl-3 mutant clones were generated
in wing discs using Hs-FLP, and cell death was examined by
staining discs with an antibody against activated caspase 9. As
shown in supplementary Fig S1E online, we could detect msl-3083
mitotic clones in female imaginal discs. In males, we could not
identify any msl-3083mutant clones. Instead, a few apoptotic cells
were present (supplementary Fig S1E online). These data show that
msl-3 gene is equally required in different tissues and they suggest
that the lack of the msl-3 gene induces cell death.
CBD and MRG domains are required for male viability
To determine the function of the different MSL-3 domains in vivo,
we generated transgenic flies expressing MSL-3 derivatives under
the control of a heat shock or tubulin promoter, tagged at the C
terminus with an enhanced green fluorescent protein (EGFP) or
FLAG epitope (Fig 1A). The expression levels of all derivatives
were compared with the wild-type (wt) MSL-3 protein by
performing western blot analysis of the heads of adult males
(supplementary Fig S2A–D online). To assess the function of the
mutant proteins, the transgenic lines were assayed for their ability
to rescue the male-specific lethal phenotype of msl-3083(Fig 1B).
As expected, a transgenic construct encoding EGFP- or FLAG-
tagged MSL-3 can efficiently rescue the msl-3083phenotype
(Fig 1B, lanes 1–3).
This analysis showed that a point mutation in the aromatic
pocket of the MSL-3 CBD (F56A-EGFP) did not severely affect
MSL-3 function (Fig 1B, lane 5). This demonstrated that the
proposed methyl binding properties of MSL-3 are not necessary for
dosage compensation and male viability.
A recent report has suggested that the CBD is dispensable for
MSL-3 function (Morales et al, 2005). It was therefore surprising
that the complementation test showed that the CBD is essential for
male viability because DCBD–EGFP male flies died as pupae and
only a few males survived until adulthood (Fig 1B, lane 4). The
flies that survived showed a range of phenotypic abnormalities
(rough eyes, missing bristles and misshapen wings; Fig 1C).
We also found that a point mutation in K116 (K116G-FLAG) or
deletion of the positively charged polar region (DPOLAR–EGFP)
does not affect the function of the protein (Fig 1B, lanes 6,7).
Similar results have been observed in other contexts where
mutations of lysine residues known to be acetylated in vitro do not
produce any visible phenotypes in vivo, suggesting that other
lysines are also acetylated in vivo and/or that there are several
lysines that can be acetylated in the absence of the real substrate
(Megee et al, 1990; Zhang et al, 1998; Feng et al, 2005).
Interestingly, we found that similar to HP1, the positively
charged polar region cooperates with the CBD in MSL-3 function,
as MRG–EGFP male flies died earlier than DCBD–EGFP male
flies and they never reached adulthood (Fig 1B, lane 8). Finally,
we found that the MRG domain is essential for male viability
because DMRG–EGFP male flies die as first/second instar larvae
(Fig 1B, lane 9).
MRG domain is required for targeting to the male X
Having documented that all MSL-3 domains are important for
male viability, we next investigated whether the reason for loss
of function of these derivatives was mistargeting of MSL-3
and/or other DCC components from the male X chromosome.
Immunostaining and RNA fluorescence in situ hybridization
(FISH) was performed on male third instar larvae expressing
MSL-3 derivatives and homozygous mutant for msl-3083except for
MRG–EGFP and DMRG–EGFP, where owing to early lethality the
immunofluorescence was performed on male third instar larvae
expressing the fusion proteins in a wt background. This analysis
showed that deletion of the MRG domain impairs MSL-3 targeting
to the male X chromosome without affecting its nuclear
localization (Fig 2A,B) and that the MRG domain is sufficient to
target the protein to the male X chromosome (Fig 3D). MSL-3
interacts with MSL-1 by means of its C terminus in vitro (Morales
et al, 2005) and it is therefore likely that this interaction is
important for MSL-3 localization to the X chromosome.
Interestingly, and in accord with a previous report (Morales
et al, 2005), mutations in the CBD or in the polar region do not
affect DCC localization to the male X chromosome (Fig 3A,C;
MSL-3 and dosage compensation
A. Buscaino et al
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100% 50% 0%
Transgenic construct insertion
analysed
Rescue frequency
1tub–MSL-3–EGFP2 78.3% (290/370)
2
tub–MSL-3–FLAG276.8% (203/264)
3
hs–MSL-3–EGFP271.8% (354/493)
4
hs–∆CBD–EGFP
2 8.3% (39/468)
5
tub–F56A–EGFP263.9% (273/427)
6
hs–∆POLAR–EGFP
2 71.48% (594/831)
7
tub–K116G–FLAG2 69.3% (1,197/1,727)
8 hs–MRG–EGFP1 0% (0/105)
hs–∆MRG–EGFP
29 0% (0/242)
MSL-3 (1–513 aa)
1 35 72 151513175490
CBD
MRG region Polar region
Polar region mutant
CBD Mutant
MRG Mutant
MRG (262–513 aa)
∆POLAR (∆ 88–153 aa)
F56A
∆MRG (1–261 aa)
EGFP
wt
EGFP
EGFP
EGFP
EGFP
EGFP
FLAG
FLAG
K116G
CBD + Polar region mutant
MSL-3 (1–513 aa)
∆CBD (∆ 34–89 aa)
B
A
Female
abcd
klij
efgh
C
Male
Fig 1|Generation of MSL-3 derivatives and rescue test. (A) Schematic representation of MSL-3 domains and transgenic flies generated for this study.
wt, enhanced green fluorescent protein (EGFP)-tagged and FLAG-tagged wild-type MSL-3 were generated as controls; chromo-barrel domain (CBD)
mutants, deletion in the CBD and point mutation in the CBD of MSL-3; polar region mutants, deletion in the polar region and point mutation;
CBDþpolar region mutant, deletion of the CBD and the polar region; MRG mutant, deletion of the MRG motif. (B) Rescue test of msl-3083lethality
by MSL-3 carboxy-terminal-tagged proteins (lanes 1–3), CBD MSL-3 mutant (lanes 4,5), polar region MSL-3 mutants (lanes 6,7), CBD and polar
region MSL-3 mutant (lane 8) and MRG mutant (lane 9). The numbers of insertion analysed, the number of males and females counted and the
average of the rescue frequency are indicated for each mutant. (C) Homozygous males expressing DCBD–EGFP in a homozygous msl-3083mutant
background show different male-specific phenotypic abnormalities, including rough eyes (compare panels b,c,d with panel a); missed bristles
(compare panels f,g,h with panel e) and misshapen wings (compare panels j,k,l with panel i). Panels a,e,i show normal phenotype of homozygous
females expressing DCBD–EGFP.
MSL-3 and dosage compensation
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supplementary Figs S3,S4 online). However, in DCBD–EGFP male
flies, we observed an autosomal mislocalization, which varied in
number and position (Fig 3A). Taken together, these data show
that the MRG domain is necessary and sufficient for targeting of
MSL-3 to the X chromosome, whereas the CBD, although essential
for male viability, is not required for binding to the X chromosome.
CBD deletion impairs expression of X-linked genes
It was intriguing to find that the MSL proteins remained localized
to the male X chromosome in DCBD–EGFP flies and yet these flies
died as pupae (Figs 1B,3A,C). Mistargeting of MSL proteins to
certain autosomal sites (Fig 3A) suggested that the male-specific
lethal phenotype may be a result of transcriptional upregulation of
EGFP
EGFP
MSL-1
MSL-1
Merged
Merged
MSL-3–EGFP
∆MRG–EGFP
A
B
EGFPMSL-1 Merged
∆MRG–EGFP
MSL-1 MergedEGFP
MSL-3–EGFP
Fig 2|The MRG domain is required for MSL-3 localization to the X chromosome. (A) Polytene chromosomes from MSL-3–EGFP (top) or DMRG–
EGFP (bottom) male larvae were stained with antibodies against EGFP and MSL-1. (B) Salivary glands from MSL-3–EGFP (top) or DMRG–EGFP
(bottom) male larvae were stained with antibodies against EGFP and MSL-1.
Fig 3|Deletion of MSL-3 chromo-barrel domain does not affect targeting of MSL proteins to the male X chromosome. (A) Polytene chromosomes from
DCBD–EGFP male larvae were stained with antibodies against EGFP, MSL-1, MOF, MSL-2 and MLE. Mislocalization of MSL proteins to autosomes is
indicated by arrows. (B) Polytene chromosomes from F56A-EGFP male larvae were stained with antibodies against EGFP and MSL-1. (C) Intact
salivary glands from MSL-3–EGFP (top) or DCBD–EGFP (bottom) male larvae were hybridized with roX1- and roX2-specific probes or hybridized with
roX1- and roX2-specific probes and simultaneously stained with MSL-1 antibody. (D) Polytene chromosomes from MRG–EGFP male larvae were
stained with antibodies against EGFP and MSL-1.
c
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Merged MLEMSL-2
∆CBD–EGFP
MSL-1MergedEGFP
MOFMSL-1Merged
MSL-1MSL-2 Merged
EGFP MSL-1Merged
F56A–EGFP
MSL-1Merged roX 1
MSL-1 MergedroX 2
roX2roX1Merged
MSL-3–EGFP
∆CBD–EGFP
EGFP MSL-1Merged
MRG–EGFP
A
B
C
D
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