Catherine Regnard,
Annalisa Izzo,
Irene Vetter, and
Peter B. Becker*
Adolf-Butenandt-Institut, Molekularbiologie, Schillerstr. 44, 80336 München, Germany
Received 23 January 2004/ Returned for modification 13 January 2005/ Accepted 29 April 2005
| ABSTRACT |
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| INTRODUCTION |
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Our knowledge of the molecular functions of the MSL proteins is rather limited. Each of them is required for faithful association of the DCC with the X chromosome. MSL2 is exclusively expressed in males and required for accumulation of roX RNA, an essential component of DCC (29). Interaction of MSL2 with MSL1 is a mutual requirement for interaction of the two proteins with the X chromosome and for formation of the complete DCC (9, 10, 22). MSL1 appears to be a scaffolding protein since it is able to associate not only with MSL2 but also with MOF and MSL3 (27, 32). MLE, the homolog of human RNA helicase A, appears responsible for stabilizing roX RNA and for its incorporation into DCC (26). A core complex will form in the absence of roX RNA or of any of the three RNA binding subunits MLE, MOF, or MSL3, but its association with only a subset of X-chromosomal sites does not lead to faithful dosage compensation (for review, see reference 14). Histone H4 acetylation by MOF is likely to play a crucial role in the modification of chromatin structure that regulates transcription. In addition, its acetylase activity is important for the distribution of the DCC over the X chromosome (17). The fact that MOF can acetylate MSL1 and MSL3 suggests possible involvement of nonhistone targets as well (8, 27).
MSL3 (15) contains an MRG domain, a domain found throughout eukaryotes from yeasts to humans, with as yet unknown function (5, 25). Interestingly, several members of this "MRG family" are associated with HATs in complexes known or suspected to be involved in transcriptional regulation. Prominent examples are the MSL3-relative Eaf3p, which interacts with the acetyltransferase Esa1 in the yeast NuA4 complex (13), and human MRG15, which can be found associated with the HAT Tip60 in a human complex highly related to the yeast NuA4 (12).
MSL3 interacts with roX RNA, and its association with the X chromosome is sensitive to RNase treatment (8). It can also bind DNA and chromatin and forms a salt-resistant complex with MSL1 in vitro (27). The reconstitution of a four-subunit MSL complex recently led to insight into functional interactions of MSL3 (27). Remarkably, the association of MSL3 with the C terminus of MSL1 in complex with MOF resulted in a dramatic activation of acetyltransferase activity on nucleosome substrates. At the same time, binding of MSL3 led to a refinement of substrate specificity: in the absence of MSL3, the rudimentary acetylation activity of MOF was entirely directed towards MSL1 and did not modify nucleosomes. However, in contact with MSL3, MSL1 was no longer subject to modification, but robust acetylation of nucleosomal H4 was observed. This finding led us to hypothesize the existence of a molecular "checkpoint" that serves to restrict the H4K16ac mark to the X chromosome. According to this model, association of MOF with MSL1 (27) leads to its sequestration on the X chromosome, but only completion of the DCC by incorporation of MSL3 unleashes its H4 acetylase activity. Whether the MOF that is expressed in female cells in the absence of a functional DCC has a lower activity toward histone H4 or profits from an equally forthcoming molecular environment remains to be explored.
The direct interaction of MSL3 with MOF in vitro is comparably weak and does not result in an activation of its HAT activity. Rather, the stimulatory effect of MSL3 on MOF requires simultaneous contact of MSL3 and MOF with the C terminus of MSL1 (27). Since MSL3 can bind DNA and chromatin in vitro (27), its presence in a complex with MOF may stabilize the interaction of the enzyme with the chromatin substrate. It is also possible that contact of MSL3 with MSL1 indirectly affects acetyltransferase activity through conformational changes. In order to address these possibilities, we explored the functional interactions of MSL3s domains. Using a set of mutants that are no longer able to bind nucleic acids or MSL1, we found that the stimulation of MDFs HAT activity, as well as targeting to the X-chromosomal territory, depends on interactions with MSL1 via the MRG signature of MSL3. In contrast, the nucleic acid binding N terminus including the chromo-related domain (CRD) was not a primary determinant of targeting MSL3 to the X-chromosome territory, nor did it contribute to stimulation of MOF's HAT activity in vitro or establishment of the accumulation of the H4K16 acetylation mark on the X chromosome in vivo.
| MATERIALS AND METHODS |
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260-393, and
328-433 constructs, respectively. Construct 270-512 was created by insertion of the msl3 XhoI-HindIII fragment into pGEM7z. Subcloning of the BamHI-BclI and BamHI-NarI inserts from Flag-MSL3-pFastBac into pFastBac1 yielded the 1-219 and 1-140 constructs, respectively. The 91-512 construct was obtained by replacement of wild-type coding sequence of msl3 (fragment NdeI-HindIII) with a PCR product bearing NdeI and HindIII sites at its 5' and 3' ends, respectively, and encompassing the msl3 sequence from codon 91 to codon 512.
Enhanced green fluorescent protein (EGFP) fusion proteins were obtained by inserting PCR products coding for full-length MSL3 or MSL3181-512 bearing 5' KpnI and 3' AgeI sites in frame with EGFP into pEGFP-1 (Clonetech) downstream of an HSP70 promoter. The sequences coding for MSL3
260-393 and MSL3
328-433 were introduced into the same vector by replacement of an NarI-HincII fragment in the wild-type construct by a NarI-HincII fragment from pGEM-(MSL3
260-393) and pGEM-(MSL3
328-433). All clones were sequenced.
Expression, purification, and functional analysis of MSL3 proteins. Baculovirus-mediated expression and the purification of flag-tagged MSL3 or mutant derivatives, as well as acetyltransferase assays on reconstituted nucleosome arrays, were performed as described previously (27).
Nucleic acid binding assay. (i) Production of DNA affinity beads. RoX2 cDNA (1,061 bp; clone 78.2.2 obtained from M. Kuroda [3]) was linearized by XbaI and BanII and immobilized on paramagnetic beads (Dynal) according to reference 31. After immobilization, part of the DNA beads was heated at 95°C for 5 min according to the manufacturer's instructions to obtain single-stranded DNA (ssDNA) on beads. The complementary strand released in the supernatant was analyzed by agarose gel electrophoresis to estimate the efficiency of the denaturation.
(ii) Production of RNA affinity beads. Sense or antisense roX2 RNAs were transcribed from either strand of the roX2 cDNA in vitro with T3 or T7 RNA polymerase (Promega) in the presence of 2.5 mM rNTPs (but 2.2 mM of UTP), 0.25 mM biotin-UTP, and 40 units of RNasin (Promega). This reaction yields RNAs that are biotinylated throughout the sequence. After 3 h of incubation at 37°C, the sample was treated with DNase (RQ1 at 10 units; Promega) and precipitated. The RNA pellet was dissolved in water. Hsp26 RNA (30) was transcribed following the same procedure.
RNA and DNA were quantified spectrophotometrically and immobilized on paramagnetic beads (Dynal) at a concentration of 10 pmol of nucleic acid for 1D mg of Dynabeads according to Sandaltzopoulos and Becker (31).
(iii) MSL3 binding assay. DNA or RNA beads were suspended at 10 ng of nucleic acids per µl in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 0.1 mM EDTA, 1 mM MgCl2, 0.1 mg/ml bovine serum albumin, 0.05% NP-40, and 1 mM dithiothreitol. Fifty to 100 ng of immobilized nucleic acids was incubated for 10 min on ice with various MSL3 derivatives (30 to 100 ng) in a 10-µl final volume of reaction buffer, diluted to 60 µl in the same buffer, and incubated for 1 h at 4°C on a rotating wheel. For competition experiments, 150 to 500 ng of competitor DNA (unbiotinylated roX2 cDNA) or RNA (transcribed from roX2 cDNA without biotin-UTP) was added to the immobilized nucleic acids before the MSL3 proteins. The beads were then processed as described previously (27), and bound MSL3 was detected by Western blotting with an antibody obtained from M. Kuroda. Quantification of chemoluminescence (ECLplus; Amersham) was done with the Fluorchem 8900 system (Alpha Innotech).
Transfections and RNA interference. SF4 and SL2 cells were transfected with Effectene (Qiagene) according to the manufacturer's instructions. MSL3 derivatives were expressed as C-terminal EGFP fusions after transfection of Drosophila SF4 or SL2 cells, and their localization was determined as described before (27). For RNA interference (RNAi), SL2 cells were used. Double-stranded RNA (dsRNA) was prepared as described previously (23). The following gene-specific primers were used: TTAATACGACTCACTATAGGGAGAATGACGGAGCTAAGGGACGA and TTAATACGACTCACTATAGGGAGAGTTTGTCTGCCCCGGTTTC in order to obtain dsRNA corresponding to the first 500 nucleotides of MSL3. Primers TTAATACGACTCACTATAGGGAGAATGTCCCCTATACTAGGTTA and TTAATACGACTCACTATAGGGAGAACGCATCCAGGCACATTG served to produce dsRNA for glutathione S-transferase (GST). A total of 2 x 106 cells were incubated with 10 µg of dsRNA for 1 h in serum-free medium and then supplemented with serum-containing medium. After 2 days of incubation at 26°C, cells were treated for 15 min with 5 µg dsRNA and then transfected with 0.5 to 1 µg of expression vectors coding for MSL3-GFP fusion proteins. After 2 more days, the cells were immunostained with anti-MSL3 antibody (a gift of M. Kuroda) for the GST-treated cells or mouse anti-GFP antibody (Molecular Probes) for cells expressing fusion proteins. The X territory was counterstained with anti-acetyl-H4K16 (Upstate), and anti-Mof (a gift of J. Lucchesi). DNA was stained using Hoechst 33258.
| RESULTS |
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In summary, the binding analysis documents that the nucleic acid binding surfaces of MSL3 reside in the N terminus of the protein and do not involve MRG sequences.
MSL3 interacts with MSL1 via the MRG domain.
We previously showed that MSL3 directly interacts with the 66 C-terminal amino acids of MSL1 (27). We next investigated which part of MSL3 is involved in this interaction. We coexpressed a panel of flag-tagged MSL3 derivatives (see Fig. 3) with full-length, untagged MSL1 in Sf9 cells using the baculovirus system. The MSL3 proteins were purified from total cell extracts by flag-mediated pull-down and washed stringently. Associated MSL1 was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (Fig. 3). Intact MSL3 and MSL3 derivatives lacking the N-terminal 140 amino acids interacted efficiently with MSL1, which was readily detected on the gel (Fig. 3, lanes 1, 4, and 5). However, deletion of any part of the MRG similarity essentially abolished MSL1. Complementary experiments monitoring the interaction of immobilized MSL1 with in vitro-translated MSL3 fragments confirmed these results (data not shown). Additional experiments also revealed that deletion of MSL3-specific sequences between the MRG similarities (
328-433) also destroyed the interaction of MSL3 with MSL1 (data not shown). In summary, the deletion analysis failed to identify a small MSL1 interaction surface in MSL3 and suggests that the three separate regions of similarity to the MRG domain contribute to folding the MSL3 C terminus into a single, large domain.
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260-393 after 2 days of RNAi. Two days later, we monitored the X-territory staining of GFP-positive cells as before (Fig. 6C and D). All GFP-fused proteins were expressed properly after RNAi of GST (data not shown) with a transfection efficiency of about 15%. In contrast, only the MSL3181-512-GFP fusion protein was expressed after RNAi of MSL3, showing that the ds RNA corresponding to the 5' end of msl3 interfered only with expression of MSL3-GFP and MSL3
260-393-GFP.
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| DISCUSSION |
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328-433) selectively removed MSL3-specific sequences between two MRG blocks. There is considerable conservation of these sequences in the Drosophila species for which sequence information has recently become available (T. Straub, personal communication), suggesting a conserved function, but whether this sequence contains a dedicated MSL1 interface remains to be explored. In any case, our analysis suggests that the MRG sequence similarity reflects a functional domain. The MRG-MSL1 contact is essential for targeting MSL3 to the X-chromosomal territory, confirming the functional importance of the interactions defined in vitro. We suggest that MRG modules in other MRG family members may also constitute protein-protein interaction units.
Role of nucleic acid binding of MSL3.
Our in vitro analysis showed that MSL3 interacted better with single-stranded nucleic acids than with dsDNA. The significance of ssDNA interaction, if any, is unclear at the moment. In contrast, there is evidence that MSL3 interacts with roX RNA in vivo and in vitro (2, 8), but the domain involved in RNA binding had not been defined. Our biochemical analysis demonstrated that the nucleic acid binding structures reside in the N-terminal half of MSL3, which also contains the CRD. Previously, Buscaino et al. (8) suggested that RNA interaction of MSL3 is affected by its acetylation at lysine 116, close to the CRD. In our hands, a fragment comprising the first 140 amino acids (and hence the CRD as well as K116) was not sufficient for nucleic acid binding, but sequences up to amino acid 259 contributed significantly. To what extent the CRD of MSL3 contributes to RNA binding needs to be established. The CRDs of MSL3 and MOF appear more related to each other than to canonical chromodomains. They lack the
-helix supporting the ß-sheet bundle and aromatic residues that may be involved in recognition of methylated histone N termini (7). The CRD of MOF also appears not to be sufficient for RNA binding (A. Buscaino and A. Akhtar, personal communication). A further interesting similarity between MOF and MSL3 is that nucleic acid interactions are not the primary targeting determinant for either MOF (27) or MSL3 (this work). Although impairment of the CRDs leads to somewhat increased binding of the corresponding GFP fusion protein to autosomes, their concentration on the X-chromosomal territory is still obvious. However, the CRDs and noncoding RNA may have functions that are not assayed for in simple recruitment experiments. It is also possible that the CRDs of MOF and MSL3 provide partially redundant functions for DCC assembly. In contrast, mutations in MOF or MSL3 that abrogate their interaction with the C terminus of MSL1 prevent faithful recruitment to the X chromosome. Obviously, the recruitment assay we employed may just reveal the strongest binary interaction that MSL3 or MOF are involved in. However, the fact that overexpression of an MSL3 lacking all nucleic acid binding capacity was able to complement an MSL3 deficiency and to trigger the accumulation of MOF and H4K16 acetylation on the X-chromosomal territory emphasizes the importance of the MSL protein interactions for the assembly of a functional DCC.
MSL complexes can be formed in vitro in the absence of RNA (26; A. Izzo and P.B. Becker, unpublished observations). A deficiency of roX RNA in vivo can be partially overcome by overexpression of the "platform" proteins MSL1 and MSL2 (28). It is possible that transient overexpression of MSL3 overcomes the RNA requirement and that under normal conditions of limiting MSL protein concentrations RNA is required for faithful DCC assembly.
DCC assembly may provide a checkpoint for activation of MOF. The remarkable stimulation of MOF's HAT activity upon association of MSL3 with an MSL1-MOF complex was not due to enhanced binding of MSL3 to nucleic acids but rather required contact of MSL3 with the MSL1 scaffold. MOF and MSL3 are brought into proximity by interaction with adjacent structures in the C terminus of MSL1 (27). It is possible that the MSL1 scaffold stabilizes an otherwise transient and therefore nonproductive direct contact between MSL3 and MOF (27). The existence of such a contact has been inferred from the fact that MSL3 can be acetylated by MOF (8). However, when it comes to acetylation, MSL1 is a much better substrate for MOF than MSL3 (27). The new data reinforce our previous model of an acetylation "checkpoint" built into DCC assembly. Accordingly, the regulatory potential of H4K16 acetylation would only be fully realized upon binding of MOF with MSL1 and the completion of the complex by association of MSL3 (27). Such a checkpoint would render full activation of MOF dependent on proper DCC assembly and hence "maleness" and serve to restrict the critical epigenetic mark to the X chromosome.
| ACKNOWLEDGMENTS |
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We thank I. Dahlsveen, G. Gilfillan, T. Straub, and the reviewers for critical reading of the manuscript and helpful discussions and T. Straub for sequence alignments. We thank M. Kuroda and John Lucchesi for MSL antibodies and A. Buscaino and A. Akhtar for unpublished information.
| FOOTNOTES |
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Present address: Laboratoire de Biologie Moléculaire des Eucaryotes, LBME-CNRS UMR 5099-IFR 109, Université Paul Sabatier, 118 Route de Narbonne, 31062-Toulouse Cedex, France. ![]()
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