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. 2010 Jun;38(10):3209-21.
doi: 10.1093/nar/gkq026. Epub 2010 Feb 5.

The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

Torsten Fauth  1 , Felix Müller-Planitz, Cornelia König, Tobias Straub, Peter B Becker
Affiliations

The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

Torsten Fauth et al. Nucleic Acids Res. 2010 Jun.

Abstract

Dosage compensation in Drosophila melanogaster involves the selective targeting of the male X chromosome by the dosage compensation complex (DCC) and the coordinate, approximately 2-fold activation of most genes. The principles that allow the DCC to distinguish the X chromosome from the autosomes are not understood. Targeting presumably involves DNA sequence elements whose combination or enrichment mark the X chromosome. DNA sequences that characterize 'chromosomal entry sites' or 'high-affinity sites' may serve such a function. However, to date no DNA binding domain that could interpret sequence information has been identified within the subunits of the DCC. Early genetic studies suggested that MSL1 and MSL2 serve to recognize high-affinity sites (HAS) in vivo, but a direct interaction of these DCC subunits with DNA has not been studied. We now show that recombinant MSL2, through its CXC domain, directly binds DNA with low nanomolar affinity. The DNA binding of MSL2 or of an MSL2-MSL1 complex does not discriminate between different sequences in vitro, but in a reporter gene assay in vivo, suggesting the existence of an unknown selectivity cofactor. Reporter gene assays and localization of GFP-fusion proteins confirm the important contribution of the CXC domain for DCC targeting in vivo.

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Figures

Figure 1.
Figure 1.
Recombinant MSL derivatives investigated in this study. (A) Schematic representation of MSL2 domain organization and various MSL2 expression constructs. All MSL2 constructs contain a C-terminal FLAG tag. Numbers correspond to the amino acid positions in full-length MSL2. (B) Alignment of orthologue CXC domains from the Drosophila melanogaster MSL2 protein (DmCXC) and from the H. sapiens protein KIAA1585 (HsCXC). Black boxes highlight the conserved cysteines (37). Arrows indicate the introduced point mutations. (C) Coomassie-stained SDS–polyacrylamide gel of purified recombinant MSL proteins.
Figure 2.
Figure 2.
Binding of recombinant MSL proteins to a DNA high-affinity site in vitro. Electrophoretic mobility shift assay. Increasing concentrations (from 5 to 250 nM) of (A) MSL2 and MSL1 or (B) MSL2 and the MSL2–MSL1 complex were incubated with radiolabeled 40 bp DBF12-L15 dsDNA and protein–DNA complexes were separated from unbound DNA in non-denaturing agarose gels. (C) Binding curves obtained from quantification of (A) and (B) and fitting to a standard bimolecular model (see ‘Materials and Methods’ section).
Figure 3.
Figure 3.
Binding of different recombinant MSL2 versions to a DNA high-affinity site in vitro. Electrophoretic mobility shift assay as in Figure 2 with increasing concentrations of MSL2 and MSL2-ΔCXC (A) or MSL2 and MSL2-ΔRING (B) or MSL2 and MSL2-ΔPro/Bas (C). (D) Binding curves obtained from quantification of (A–C) and fitting to a standard bimolecular model.
Figure 4.
Figure 4.
Competition assays to determine the binding preference of MSL2 for different nucleic acids. (A) Electrophoretic mobility shift assay. Fifty nanomolar of MSL2 was incubated with the radiolabeled 40 bp DBF12-L15 dsDNA and then increasing concentrations (from 5 to 2080 nM) of unlabeled competitor nucleic acids were added. Representative examples of competition with ds- and ssDNA are shown. (B) Competition curves obtained from quantification of data such as in (A), but also including ssRNA and dsRNA as competitors and fitting to the model as described in ‘Materials and Methods’ section.
Figure 5.
Figure 5.
Binding of different recombinant MSL2 derivatives to RNA. Electrophoretic mobility shift assays. Increasing concentrations of MSL2 versions were incubated with radiolabeled dsRNA of DBF12-L15 and protein–RNA complexes were separated from unbound RNA in non-denaturing agarose gels. A representative example of MSL2-ΔCXC binding to dsRNA is shown. (B) Binding curves obtained from quantification of EMSAs as in (A), but including MSL2-ΔRING and MSL2-ΔPro/Bas and fitting to a standard bimolecular model.
Figure 6.
Figure 6.
Binding of recombinant MSL2 to different DNA fragments in vitro. Electrophoretic mobility shift assay. Increasing concentrations of MSL2 were incubated with radiolabeled dsDNA of (A) the high-affinity DBF12-L15 element and the mutated version DBF12-L18 or (C) the DBF12-L15 trimer and a random fragment of similar length derived from the multiple cloning site of a vector (mcs). Protein–DNA complexes were separated from unbound DNA in non-denaturing agarose gels. (B) and (D) Binding curves obtained from quantification of (A) and (C) and fitting to a standard bimolecular model.
Figure 7.
Figure 7.
Reporter gene assay to measure transactivation potential of different MSL2 constructs in vivo. (A) Schematic representation of the cotransfected plasmids that constitute the reporter gene system (drawing is not to scale). Drosophila SL2 cells were transiently cotransfected with different MSL2-VP16 constructs together with luciferase reporter gene constructs (pGL3-TK) carrying the model DBF12-L15 or the mutated site DBF12-L18 (40 bp elements). A renilla luciferase expression vector (pRL-TK) served as normalization control. (B) Transactivation potential of each MSL2 derivative is displayed as fold activation (for details see ‘Materials and Methods’ section). The black bars indicate a reporter gene bearing MSL2 binding sites (DBF12-L15), the white bars represent (lack of) activation in the presence of DBF12-L18.
Figure 8.
Figure 8.
In vivo localization of different MSL2 derivatives. Stable Drosophila SL2 cell lines, which express different MSL2 versions fused to GFP, were analyzed by immunofluorescence staining. Localization of MSL2-GFP derivatives were visualized using anti-GFP antibodies (α-GFP). Endogenous MSL1 was detected using anti-MSL1 antibodies (α-MSL1). DNA was counterstained with Hoechst 33258 (DNA). (A) Examples showing single nuclei with proper targeting of MSL2 to the X chromosomal territory (left) or dispersed, non-physiological distribution of the GFP fusion proteins. (B) Representative fields of cells. Cells that express the MSL2-GFP transgene were analyzed for proper X territory staining. The percentage of cells, which show mislocalized, dispersed GFP signals from the total number of GFP positive cells counted are displayed to the right.
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