Functional role of the c-terminal domains of the msl2 protein of drosophila melanogaster

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Abstract

Dosage compensation complex, consisting of five proteins and two non-coding RNAs roX, specifically binds to the X chromosome in males, providing a higher level of gene expression, which is necessary to compensate for the monosomy of the sex chromosome in male Drosophila compared to two X chromosomes in females. The MSL2 protein contains an N-terminal RING domain, which acts as an E3 ligase in the ubiquitination of proteins and is the only subunit of the complex that is expressed only in males. The functional role of two C-terminal domains of the MSL2 protein, enriched with proline (P-domain) and basic amino acids (B-domain), was investigated. As a result, it was shown that the B-domain destabilizes the MSL2 protein, which is associated with the presence of two lysines whose ubiquitination is under the control of the RING domain of MSL2. The unstructured proline-rich domain stimulates transcription of the roX2 gene, which is necessary for the effective formation of the dosage compensation complex.

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About the authors

E. A. Tikhonova

Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: maksog@mail.ru
Russian Federation, Moscow

P. G. Georgiev

Institute of Gene Biology, Russian Academy of Sciences

Email: maksog@mail.ru
Russian Federation, Moscow

O. G. Maksimenko

Institute of Gene Biology, Russian Academy of Sciences

Email: maksog@mail.ru
Russian Federation, Moscow

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2. Fig. 1. Structural organization of the MSL2 protein. a – Scheme of the MSL2 protein. The main domains are shown: RING, CXC, CLAMP-interacting, P and B. b – Alignment (Clustal Omega) of the C-terminal part of the MSL2 protein in well-studied Drosophilidae species. The P-domain is highlighted in orange, the B-domain – in green.

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3. Fig. 2. Generation of transgenic lines expressing mutant MSL2 proteins. a – Scheme of the expression vector used. Shown are the promoter and 5′-UTR of the Ubiquitin-63E gene, the last intron, 3′-UTR and polyadenylation signal (polyA) of the dctcf gene, as well as the polyadenylation signal from the SV40 virus. MSL2 variants are shown below the vector scheme; the dashed lines indicate the sites of the introduced deletions. b – Immunoblot analysis of protein extracts obtained from adult flies expressing different MSL2 variants tagged with the FLAG epitope (WT, ΔP, ΔB). Immunoblot analysis was performed using antibodies specifically recognizing FLAG and GAF ​​(material application control). c – Comparison of MSL2WT-FLAG, MSL2ΔRING-FLAG and MSL2ΔB-FLAG protein expression in S2 cells. Immunoblot analysis was performed using antibodies specifically recognizing FLAG and lamin (material application control). d – Comparison of viability (in relative units) of adult msl2γ227/msl2γ227 males expressing MSL2-3xFLAG variants (WT, ΔP, ΔB). The ratio of msl2γ227/CyO males expressing MSL2 variants was used as an internal control demonstrating normal viability. The ratio of adult y1w1118; +/+ males to y1w1118; +/CyO males was used as an index of wild-type strain survival. The histogram shows the mean values ​​with standard deviations obtained from the results of three independent experiments; * p-value < 0.05. d – Viability (in relative units) of females homozygous for the transgene relative to adult males expressing MSL2 variants. The histogram shows the mean values ​​with standard deviations obtained from three independent experiments; ** p-value < 0.01

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4. Fig. 3. Analysis of CDK binding to chromatin in transgenic fly lines. a – Comparison of MSL1 and MSL2 binding to polytene chromosomes of msl2γ227 male larvae expressing different MSL2 variants (MSL2WT, MSL2ΔP). b – Comparison of MSL1 and MSL2 binding to polytene chromosomes of female larvae heterozygous for the transgene expressing one of the MSL2 variants (MSL2WT, MSL2ΔP, MSL2ΔB). The photographs show immunostaining with mouse anti-FLAG antibodies (MSL2, green) and rabbit anti-MSL1 antibodies (red). DNA staining is with DAPI (blue). c – Comparison of MSL2 protein distribution along the polytene X chromosome in females heterozygous for the transgene expressing one of the MSL2 variants (MSL2WT, MSL2ΔP). Two independent stainings are shown; MSL2 staining – mouse anti-FLAG antibodies (red), DNA – DAPI (blue). d – Comparison of MSL1, MSL2 and CLAMP protein binding to the SPP in males expressing MSL2 variants (WT and ΔP) in the msl2γ227 background. Red letters indicate the sites to which MSL2 is able to bind directly, according to the data of Villa et al. [23]. The results are presented as percentages of DNA enrichment after immunoprecipitation relative to the original DNA (% of original material), normalized to the corresponding positive control binding sites of MSL1 (26E3), MSL2 (25A3) and CLAMP (39A1) on autosomes. The histograms show a comparison of the level of MSL2ΔP protein binding with the level of MSL2WT binding (scaled to "1"). Whiskers show standard deviations for three independent experiments; *p < 0.05

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5. Fig. 4. Comparison of roX1 and roX2 in females expressing MSL2WT, MSL2ΔP, MSL2ΔB. a – Comparison of MSL1, MSL2, CLAMP protein binding in lines expressing MSL2WT, MSL2ΔB MSL2ΔP, in the regions of the roX1 and roX2 genes; * p < 0.05; ** p < 0.01. b – Expression levels of roX1 and roX2 RNA in male and female larvae of the y1w1118 fly line (wild type) and flies expressing MSL2WT, MSL2ΔP, MSL2ΔB, against the background of the msl2γ227 null mutation. Histograms show the change in the mRNA level of the tested roX genes in the lines expressing MSL2WT, MSL2ΔP, MSL2ΔB, compared to the expression level in males of the y1w1118 line (corresponds to the mark "1" on the scale). Whiskers show standard deviations for three independent measurements; * p < 0.05

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