N-Terminus of Drosophila Melanogaster MSL1 Is Critical for Dosage Compensation

Valentin Babosha
Natalia Klimenko
Anastasia Revel-Muroz
Evgeniya Tikhonova
Pavel Georgiev author has email address
Oksana Maksimenko author has email address

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilova St., Moscow 119334, Russia
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilova St., Moscow 119334, Russia

https://doi.org/10.7554/eLife.93241.1

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Figures and data

Testing the functional activity of MSL1 mutants.
(A) Schematic presentation of the MSL1 deletion proteins expressed in flies. Alignment of the N-terminal domain (1-85 aa) of MSL1 among Drosophilidae. (B) Immunoblot analysis of protein extracts prepared from adult males y¹w¹¹¹⁸ (control) and msl-1⁻; Ubi:msl-1^WT/ Ubi:msl-1^WT(M1[WT]), and from adult females y¹w¹¹¹⁸ (control), ⁻ msl-1⁻ (msl-1^L60/ msl-1^γ269), M1[WT] (msl-1⁻; Ubi:msl-1^WT/Ubi:msl-1^WT) and M1[WT]+MSL2 (msl-1⁻; Ubi:msl-2^WT-FLAG / Ubi:msl-1^WT). Immunoblot analysis was performed using anti- MSL1, anti-MSL2 and anti-lamin Dm0 (internal control) antibodies. (С) Immunoblot analysis of protein extracts prepared from adult females expressing different MSL1 protein variants (MSL1^WT, MSL1^Δ1-85 MSL1^Δ1-15, MSL1^Δ8-20 MSL1^Δ24-39, MSL1^Δ41-85, MSL1^Δ41-65, MSL1^Δ66-85) and y¹w¹¹¹⁸ females (control). Immunoblot analysis was performed using anti-MSL1, and anti- lamin Dm0 (internal control) antibodies. (D) Viability of males expressing MSL1 variants in the msl-1-null background (marked as M1[*]). Viability (as a relative percentage) of msl-1⁻(msl-1^L60/ msl-1^γ269); Ubi:msl-1*/ Ubi:msl-1* males to msl-1⁻; Ubi:msl-1*/Ubi:msl-1* females obtained in the progeny of crosses between females and males with the msl-1⁻/ CyO, GFP; Ubi:msl-1*/Ubi:msl- 1* genotype. Ubi:msl-1* is any transgene expressing one of the tested MSL1 variants. The results are expressed as the mean of three independent crosses, with error bars showing standard deviations. (E) Immunoblot analysis of protein extracts prepared from adult female flies expressing MSL2-FLAG in combination with different MSL1 protein variants (MSL1^WT, MSL1^Δ1-85 MSL1^Δ1-15, MSL1^Δ8-20 MSL1^Δ24-39, MSL1^Δ41-85, MSL1^Δ41-65, MSL1^Δ66-85) and y¹w¹118 females (control). Immunoblot analysis was performed using anti-MSL1, anti-MSL2, and anti- lamin Dm0 (internal control) antibodies.

Testing the role of MSL1 mutants in recruiting the MSL complex on the X chromosome in a female model system.
Distribution of the MSL complex on the polytene chromosomes from 3rd-day female larvae expressing both MSL1* variants and the MSL2^WT- FLAG protein. Panels show the immunostaining of proteins using rabbit anti-MSL1 antibody (green) and mouse anti-FLAG antibody (red). DNA was stained with DAPI (blue). M1[WT](♀ msl-1⁻; Ubi:msl-2^WT-FLAG/ Ubi:msl-1^WT); M1[Δ1-85] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/Ubi:msl-1^Δ1-85); M1[Δ1-15] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/ Ubi:msl-1^Δ1-15); M1[Δ8-20] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/ Ubi:msl-1^Δ8-20); M1[Δ24-39] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/ Ubi:msl-1^Δ24-39); M1[Δ41-85] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/ Ubi:msl-1^Δ41-85); M1[Δ41-65] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/ Ubi:msl-1^Δ66-85); M1[Δ66-85] (♀ msl-1⁻; Ubi:msl-2^WT-FLAG/Ubi:msl-1^Δ66-85).

Testing the functional role of N-terminal amino acids 3-7 of MSL1.
(A) Schematic presentation of the mutations at the N-terminus of MSL1. (B) Immunoblot analysis of protein extracts prepared from adult female flies expressing different MSL1* variants in the msl-1⁻(msl-1^L60/ msl-1^γ269) background (M1[WT]: MSL1^WT; M1[2A]: MSL1^F5A ^W7A, M1[3S]: MSL1^{K3S R4S} ^K6S; M1[GS]: MSL1^K3S ^R4S ^F5G ^K6S ^W7G). (C) Immunoblot analysis of protein extracts prepared from adult female flies expressing MSL2-FLAG in combination with different MSL1* variants. Immunoblot analysis performed using anti-MSL1, anti-MSL2, anti-MSL3 and anti-lamin Dm0 (internal control) antibodies. (D) Viability of males expressing MSL1 variants (marked as M1[*]) in the msl-1⁻ (msl-1^L60/ msl-1^γ269) background. Viability (as a relative percentage) of msl-1⁻; Ubi:msl-1*/ Ubi:msl-1* males to msl-1⁻; Ubi:msl-1*/Ubi:msl-1* females obtained in the progeny of cross between females and males with msl-1⁻/ CyO, GFP; Ubi:msl-1*/Ubi:msl-1* genotype. Ubi:msl-1* is any transgene expressing one of the tested MSL1* variants. (E) Viability of males expressing MSL2^WT and MSL1* variants (marked as M1[*]) in the msl-1⁻ background. Viability (as a relative percentage) of msl-1⁻; Ubi:msl-2^WT-FLAG /Ubi:msl-1* males to msl-1⁻; Ubi:msl-2^WT-FLAG /Ubi:msl-1* females obtained in the progeny of cross between males msl-1⁻/ CyO, GFP; Ubi:msl-1*/Ubi:msl-1* and females msl-1⁻; Ubi:msl-2/Ubi:msl-2. The results are expressed as the mean of three independent crosses, with error bars showing standard deviations. (F) Distribution of the MSL complex on the polytene chromosomes from 3rd-day female larvae expressing both MSL1* variants and the MSL2-FLAG protein. Panels show the merged results of immunostaining of MSL1 (green, rabbit anti-MSL1 antibody) and MSL2 (red, mouse anti- FLAG antibody). DNA was stained with DAPI (blue).

Testing the functional activity of mutant variants of the MSL1 protein.
(A) Immunoblot analysis of protein extracts prepared from S2 cells expressing MSL2-FLAG and different MSL1-HAx3 protein variants (M1[WT]: MSL1^WT; M1[Δ*]: MSL1^Δ*; M1[2A]: MSL1^2A, M1[3S]: MSL1^3S; M1[GS]: MSL1^GS). Immunoblot analysis performed using anti-HA (MSL1-HAx3 variants), anti-FLAG (MSL2-FLAG) and anti-lamin Dm0 (internal control) antibodies. (B) Protein extracts from Drosophila S2 cells co-transfected with different MSL1-HAx3 variants and MSL2-FLAG were immunoprecipitated with antibodies against FLAG or HA or nonspecific mouse IgG as a negative control. The immunoprecipitates were analyzed by immunoblotting for the presence of FLAG-tagged proteins in immunoprecipitated samples. (C) Distribution of the MSL1*-HA and MSL2-FLAG on the polytene chromosomes from 3rd-day female or male larvae expressing one of MSL1*-HA variants and MSL2-FLAG at the wild-type background (msl-1⁺; Ubi:msl-2^WT-FLAG /Ubi:msl-1*). Panels show the merged results of immunostaining of MSL1*-HA (green, mouse anti-HA antibody) and MSL2 (red, rabbit anti- MSL2 antibody). DNA was stained with DAPI (blue).

Testing role of the N-terminal region in recruiting of roX2 by MSL1.
Extraction of RNA and RNP from adult flies, immunoprecipitation followed by RNA extraction were prepared as described in the Materials and Methods section and briefly schematically explained in the drawing. (A) Expression levels of the roX2 RNA in females of the y¹w¹¹¹⁸, M1[WT] (msl-1⁻; Ubi:msl-1^WT-HA/ Ubi:roX2), M1[GS] (msl-1⁻; Ubi:msl-1^GS-HA/ Ubi:roX2), M1[Δ41-85] (msl-1⁻; Ubi:msl-1^Δ41-85-HA/ Ubi:roX2) and msl1 ⁻(msl-1⁻;Ubi:roX2/TM6,Tb). Individual transcript levels were determined by RT-qPCR with primers for roX2 gene normalized relative to RpL32 for the amount of input cDNA. The error bars show standard deviations of triplicate measurements. (B) RNP extracts from adult females were immunoprecipitated with antibodies against MSL1 (IP-MSL1) or nonspecific rabbit IgG (IP-IgG) as a negative control. The efficiency of immunoprecipitation was tested by immunoblot analysis for the presence of HA-tagged MSL1 protein in immunoprecipitate samples. (С) Total RNA was extracted from immunoprecipitates (α-MSL1 or IgG) and analyzed for the presence of roX2 RNA by RT-PCR. RpL32 was used as negative control. The results of immunoprecipitations are presented as the percentage of input cDNA. ‘output’ – supernatant after immunoprecipitation; ‘IP’ – immunoprecipitated sample. The error bars indicate SDs from three independent biological samples.

Comparisons of the binding patterns of MSL proteins between 2-3-day adult males and females expressing MSL2.
(A) Average signal (top) and heatmap (bottom) showing the log fold-change (FC) between normalized (RPKM) test signals and nonspecific IgG signals for HAS and autosomal regions (see Materials and Methods). LogFC was calculated after smoothing signals using the Daniell kernel with kernel size 50 and the addition of a pseudocount. On the heatmaps, the peaks are ranked according to the average logFC in the M1[wt] male sample. ♂ M1[wt] indicates 2–3-day-old msl-1⁻; Ubi:msl-1^WT/ Ubi:msl-1^WT males. ♀M1[wt]+MSL2 indicates 2–3-day-old msl-1⁻; Ubi:msl-2 ^WT-FLAG/ Ubi:msl-1^WT females. (B and C) Enrichment of MSL1 (green), MSL2 (red), and MSL3 (yellow) signals associated with the X- linked genes encoding Pp2C1 and the non-coding RNAs roX1 and roX2 in (B) M1[WT] males and (C) M1[WT]+MSL2 females.

Role of the N-terminal regions in the recruitment of the dosage compensation complex to the HAS.
To study the functional role of the N-terminal region of MSL1 for the recruitment of the dosage compensation complex, we compared the profiles of MSL1, MSL2, and MSL3 binding in 2-3-day females expressing MSL2 and one of four MSL1 variants (MSL1^WT, MSL1^GS, MSL1^Δ1-15, MSL1^Δ41-85). y¹w¹¹¹⁸ (wild-type males and females), ♀M1[WT]+MSL2 (msl-1⁻; Ubi:msl-2^WT-FLAG/Ubi:msl-1^WT females), ♀M1[Δ41-85]+MSL2 (msl-1⁻; Ubi:msl-2^WT-FLAG/Ubi:msl-1^Δ41-85 females), ♀M1[Δ1-15]+MSL2 (msl-1⁻; Ubi:msl-2^WT- FLAG/Ubi:msl-1^Δ1-15 females), ♀M1[GS]+MSL2 (msl-1⁻; Ubi:msl-2^WT-FLAG/Ubi:msl-1^GS females). (A) Average log fold-change between normalized (RPKM) test signals and nonspecific IgG signals in HAS (see Materials and Methods). Average log fold-change was calculated after smoothing signals using the Daniell kernel with kernel size 50 and the addition of a pseudocount. Next, the tracks were aggregated using 100-bp bins. (B) Heatmap showing average log fold- change between normalized (RPKM) test signals and nonspecific IgG signals in region ± 500 bps from HAS centers. (C and D) Average signal (RPKM) for (C) MSL1 protein and (D) MSL2 protein in different fly lines at HAS (see Methods). (E) Enrichment of MSL1, MSL2, and MSL3 signals associated with the X-linked genes encoding Pp2C1 and the non-coding RNAs roX1 and roX2 in females expressing MSL2 and one of four MSL1 variants (MSL1^WT, MSL1^GS, MSL1^Δ1-15, MSL1^Δ41-85).

MSL1 shows different scenarios of binding upon N-terminal region modification.
Regions clusterization according to MSLs binding in different female fly lines. Regions are divided into 2 groups: MSL1∩MSL2 overlapping peaks (X chromosome) (A) and MSL1 alone peaks (autosomes and X chromosome) (B) (see Materials and Methods). Left - Heatmap of average logFC between MSL and nonspecific IgG signal in female fly lines, hierarchical clusterization and cluster subdivision is drawn on the left corner (see Materials and Methods). Right - Average logFC profiles of MSL signal in different clusters for female fly lines. Average log fold-change was calculated after smoothing signals using the Daniell kernel with kernel size 100 and the addition of a pseudocount.

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