Incorporation of the noncoding roX RNAs alters the chromatin-binding specificity of the Drosophila MSL1/MSL2 complex

Abstract

The male-specific lethal (MSL) protein-RNA complex is required for X chromosome dosage compensation in Drosophila melanogaster. The MSL2 and MSL1 proteins form a complex and are essential for X chromosome binding. In addition, the MSL complex must integrate at least one of the noncoding roX RNAs for normal X chromosome binding. Here we find the amino-terminal RING finger domain of MSL2 binds as a complex with MSL1 to the heterochromatic chromocenter and a few sites on the chromosome arms. This binding required the same amino-terminal basic motif of MSL1 previously shown to be essential for binding to high-affinity sites on the X chromosome. While the RING finger domain of MSL2 is sufficient to increase the expression of roX1 in females, activation of roX2 requires motifs in the carboxyl-terminal domain. Binding to hundreds of sites on the X chromosome and efficient incorporation of the roX RNAs into the MSL complex require proline-rich and basic motifs in the carboxyl-terminal domain of MSL2. We suggest that incorporation of the roX RNAs into the MSL complex alters the binding specificity of the chromatin-binding module formed by the amino-terminal domains of MSL1 and MSL2.

FIG. 1.

A carboxyl-terminal region of MSL2 that contains proline-rich and basic motifs is important for binding of the MSL complex to the X chromosome. (A) Schematic representation of the msl2 gene constructs used in this study. The numbers in parentheses correspond to the amino acid numbers in full-length MSL2. All constructs were controlled by the Drosophila hsp70 promoter. MSL2(1-193)F has a C-terminal and MSL2(490-773)F has an N-terminal FLAG tag. (B) Protein expression in adult flies was confirmed by Western blotting with anti-MSL2 antibody. (C to N) Female larval salivary gland nuclei from transgenic lines were stained with anti-MSL2 antibody (red) and counterstained with DAPI (blue). All MSL2 protein is expressed from the transgene, as there is no production of MSL2 protein from the endogenous gene in female nuclei. MSL2(1-193)F bound to heterochromatin at the chromocenter (arrowhead) and fourth chromosome (4) (C and D) and to a few other sites, including 8D (asterisk) on the X chromosome (C and E) and 21B on the second chromosome (F). MSL2(1-524) (G), MSL2(1-649) (H), and MSL2(1-684) (I) bound in a similar pattern to MSL2(1-193)F (C), although MSL2(1-684) bound more weakly to chromatin. MSL2(1-743), which contains a proline-rich motif and basic motifs, bound to many sites on the X chromosome (K), as did the control of full-length MSL2 expressed from the hsp70 promoter (J). MSL2(1-743) does not bind to the chromocenter (L) or autosomal sites such as 21B (M). No significant binding to either the X chromosome or chromocenter was detected with just the carboxyl-terminal region of MSL2(490-773)F (N).

FIG. 2.

MSL proteins colocalize with the amino-terminal domain of MSL2 on polytene chromosomes. Female polytene nuclei that express truncated MSL2(1-193) were immunostained with (A) anti-MSL1, (B) anti-MSL3, (C) anti-MLE, (D) anti-MOF, (E) anti-H4K16ac, and (F) anti-HP1 antibodies. MSL1, MLE, MOF, H4K16ac, and HP1 were detected by anti-rabbit Alexa Fluor 594 (red). MSL3 was detected by anti-goat-FITC (green). MSL1, MSL3, MLE, and MOF colocalize with MSL2(1-193) to the chromocenter (arrowhead), the fourth chromosome (4), and sites on the X chromosome (X). H4K16ac staining appears to be largely confined to the chromocenter (E).

FIG. 3.

The N-terminal X chromosome-binding domain of MSL1 is essential for binding of the N-terminal domain of MSL2 to chromatin. (A) Salivary gland nuclei from female larvae that express MSL2(1-193)F in an msl1^L60 null background were stained with anti-MSL2 (red) and counterstained with DAPI (blue). No binding to chromatin was observed. (B and C) Salivary gland nuclei from female larvae that coexpress the amino-terminal domains of MSL2(1-193)F and either MSL1(1-265)HA (B) or MSL1(27-265)HA (C) in an msl1^L60 background were stained with anti-HA antibody (green) and anti-MSL2 antibody (red) and counterstained with DAPI (blue). The amino-terminal domain of MSL1 colocalizes with MSL2(1-193)F (B) to the chromocenter (arrowhead), but the N-terminal basic motif of MSL1 is essential for the binding of both domains to chromatin (C).

FIG. 4.

Overexpression of the amino-terminal domains of MSL1 and MSL2 does not significantly change the chromatin-binding pattern. (A and B) Female salivary gland nuclei from larvae that coexpress the amino-terminal domains of MSL1(1-265)HA and MSL2(1-193)F were stained with anti-HA antibody (green) and anti-MSL2 antibody (red) and counterstained with DAPI (blue). Larvae were raised at 25°C and either given no heat treatment (A) or heat shocked at 37°C for 1 h and then left to recover at 25°C for 4 h (B). Western blot assays with anti-HA (top of panel C), anti-FLAG (top of panel D), or antitubulin (bottom of panels C and D) of protein extracts from untreated (lane 1) or heat-shocked (lane 2) flies. Heat treatment led to a significant increase in the levels of the MSL1NHA and MSL2(1-193)F proteins (arrows).

FIG. 5.

roX RNA levels in hsp-msl2 lines. (A) Salivary gland nuclei from female larvae that express MSL2(1-193)F were stained with anti-MSL2 (top) and counterstained with DAPI (lower). MSL2(1-193)F bound to 3F and 10C, the locations of the roX1 and roX2 genes, respectively. (B) Northern blot hybridization analysis of RNA isolated from transgenic females and wild-type flies. The membrane was hybridized with ³²P-labeled roX1 probe (top) and reprobed with an rp49 probe (lower) as a loading control. Low levels of roX1 RNA were detected in all lines except MSL2(490-773)F females. (C and D) Quantitative real-time RT-PCR was used to measure the levels of the roX1 (C) and roX2 (D) RNAs in transgenic females and y w (parental strain “wild-type”) flies. The RNAs were isolated from females from the following strains: y w (sample 1), MSL2(1-193)F (sample 2), MSL2(1-524) (sample 3), MSL2(1-649) (sample 4), MSL2(1-683) (sample 5), MSL2(1-743) (sample 6), full-length MSL2 (sample 7), and MSL2(490-773)F (sample 8). Sample 9 was from y w males. Each sample was measured in triplicate. (E) To determine if the amino-terminal domain of MSL2 is sufficient to induce roX1 transcription, RT-PCR was performed with RNA from females that express MSL2(1-193)F and were either homozygous for msl1^L60 (lanes 2 and 3), heterozygous for msl1^L60 (lanes 4 and 5), or wild type for msl1 (lane 6 and 7). RT(+) indicates the presence of reverse transcriptase in cDNA synthesis reaction; RT(−) indicates its absence. M is a molecular weight marker (lane 1). The lower part of the panel shows RT-PCR products with a primer pair for pgd transcripts as a cDNA synthesis control. (F) Real-time quantitative RT-PCR of the RNA samples analyzed in panel E. roX1 transcript was readily detected in msl1⁺ females but was not above background levels in msl1^L60 homozygotes. (G) Quantitative RT-PCR of roX RNA that coimmunoprecipitated (co-IP) with MSL2 antibody relative to control immunoprecipitation with no added antibody (mock). Each RNA sample was analyzed in triplicate, and the average ± the standard deviation is shown. (H) Western blot assay with MSL2 antibody of immunoprecipitated (IP) extracts from which the RNA quantified in panel G was extracted. Bands corresponding to the expected sizes for MSL2(1-683) (lane 1), MSL2(1-743) (lane 2), and MSL2(1-773) (lane 3) were detected in extract immunoprecipitated with MSL2 antibody but not in the controls (lanes 4 to 6).

FIG. 6.

Association of roX1 RNA with polytene chromosomes in hsp70-msl2 lines. (A to H) Single-stranded biotinylated roX1 probe was hybridized to salivary gland nuclei from female larvae that express the indicated version of MSL2 and were detected with fluorescein-avidin (green) and counterstained with DAPI (blue). (D to H) The female nuclei coexpress roX1 RNA from an autosomal hsp83-roX1 transgene. (I to K) Female nuclei that coexpress roX1 RNA and MSL2(1-193)F (I and J) or MSL2(1-649) (K) were immunostained with MSL2 antibody (red) and counterstained with DAPI (blue). The chromocenter (arrowhead), X chromosome (X), and hybridization/binding sites on the X chromosome (arrows) are indicated. (I to K, insets) Binding to sites at the base of the X chromosome, but not the chromocenter, was detected in most nuclei. (L) Quantitative real-time RT-PCR of roX1 RNA levels in females that coexpress roX1 and the indicated truncated version of MSL2. The average of two independent experiments is shown.

FIG. 7.

MLE weakly coimmunoprecipitates with the carboxyl-terminal domain of MSL2. Protein extracts from transformant female flies that co-overexpressed MSL2(490-773)F and MSL1 (A), MSL3 (B), MLE (C), or MOF (D) were immunoprecipitated (IP) with anti-FLAG affinity matrix and detected by Western blotting (WB) with the indicated antibodies. Immunoprecipitated extracts (Ip) are shown in the right lane of each panel, and 10% of the corresponding input (Input) is shown in the left lane. Coimmunoprecipitation experiments were also performed with extracts from females that overexpress MLE and either MSL2(1-193)F (E) or full-length MSL2 (F). A small amount of MLE consistently coimmunoprecipitated with the carboxyl-terminal domain of MSL2 or full-length MSL2 but not with the amino-terminal domain.

FIG. 8.

Proposed model. (A) In female nuclei that express a truncated version of MSL2 that lacks the carboxyl-terminal domain (M2ΔC) and contain a low level of roX RNA, the MSL complex assembles and binds to pericentromeric heterochromatin. roX RNA is not incorporated into the protein complex. Chromatin binding is mediated by the amino-terminal domains of MSL1 (M1) and M2ΔC. (B) However, if roX RNA levels are high, some RNA is incorporated into the complex, which then spreads from the pericentromeric heterochromatin to sites at the base of the X chromosome. (C) Longer versions of MSL2 that contain proline-rich and basic motifs in the C-terminal domain efficiently incorporate low levels of roX RNA into the complex and bind to hundreds of sites along the length of the X chromosome. We suggest that incorporation of roX RNA alters the chromatin-binding specificity of the MSL1/MSL2 complex to recognize features on the X chromosome (B and C). Most of the interactions shown are known from this or previous studies, but it is not known how MLE would associate with the MSL complex that contains M2ΔC and lacks roX RNA (A).