Journal: Nature

Article Title: The sex-specific factor SOA controls dosage compensation in Anopheles mosquitoes

doi: 10.1038/s41586-023-06641-0

Figure Lengend Snippet: a , Representative immunostaining of SOA (orange), RNA polymerase 2 (Pol2; grey) with RNA FISH (green) of a X-linked transcription site ( AGAP000651 intron). DAPI in blue. Scale bar, 10 μm. b , Heatmap showing normalized SOA CUT&Tag coverage for significant peaks (males versus females) and metaplot showing mean enrichment (top). c , Pie chart of the significant SOA peaks versus the A. gambiae genome. P value: one-sided Fisher’s test for overrepresentation of peaks on the X chromosome.UNKN, scaffolds that could not be assigned to any chromosome. d , Bar plot of SOA peak annotations for genomic features. UTR, untranslated region. e , Genome browser snapshots of SOA CUT&Tag coverage. f , Metaplot of SOA CUT&Tag coverage at the TSS ± 1 kb (all genes). Lines reflect gene groups by chromosomal location and expression levels based on RNA-seq of wild-type male pupae. Genes with fewer than ten average read counts across replicates were considered as not expressed. g , Left, metaplot of SOA CUT&Tag coverage at 3 random k -means clusters generated from expressed, X-linked genes ( n = 857 genes, see also f ). The TSS is a reference point to plot 1 kb upstream; gene bodies (TSS to the transcription end site (TES)) were scaled to 5 kb. Right, violin plot of log 2 (TPM) values by RNA-seq of wild-type male pupae. The centre line indicates the median. P value: two-sided Wilcoxon rank-sum comparing combined clusters 1 and 2 versus cluster 3. h , As in g . Heatmap showing the SOA CUT&Tag coverage at expressed X-linked genes. Three random k -means clusters were generated that separated the groups on the basis of SOA binding strength. Biological replicates ( n = 4 male, n = 2 female) were merged for visualization ( b , e – h ).

Article Snippet: This was followed by hybridization in Stellaris RNA FISH hybridization buffer (SMF-WA1-60-BS, LGC Biosearch Technologies) supplemented with 10% with formamide containing 125 nM probe mix targeting the introns of the X-linked gene act5c ( AGAP000651 , sequences in Supplementary Table ), which was incubated overnight in a humidified chamber at 37 °C.

Techniques: Immunostaining, Expressing, RNA Sequencing, Generated, Binding Assay

Journal: Nature

Article Title: The sex-specific factor SOA controls dosage compensation in Anopheles mosquitoes

doi: 10.1038/s41586-023-06641-0

Figure Lengend Snippet: ( a ) Bar plot showing Yob mRNA levels relative to Rp49 in WT and homozygous SOA-R pupae measured by RT-qPCR. Yob mRNA levels confirm the sex of the pupae used in Fig. . The height of the bar plot is the mean of n = 4 biological replicates with overlaid individual data points. ( b ) Representative pictures of SOA immunostaining (orange), RNA Polymerase 2 immunostaining (grey) and co-RNA FISH (green) of a X-linked transcription site ( AGAP000651 ) on salivary gland nuclei of a homozygous male SOA-R L4 larva. The RNA-FISH probes were designed against the introns of the AGAP000651 gene. DAPI is shown in blue, scale bar = 10 μm. ( c ) Representative pictures of SOA immunostaining conducted on homozygous SOA-R male and female adult mosquito tissues. Pictures show nuclei of Malpighian tubules (left, scale bar = 5 μm) or gut (right, scale bar = 10 μm) with SOA in orange and DAPI in blue. The pictures represent a 3D view of a z-stack. Further images in Fig. . ( d ) Pearson correlation clustering of SOA CUT&Tag samples based on affinity scores after peak calling. The experiment was conducted with SOA antibody and IgG in WT and homozygous SOA-R female pupae. The SOA antibody data was filtered using the IgG control and then subjected to clustering. ( e ) Heatmap showing the normalized CUT&Tag coverage on all significant peaks (FDR<0.05, fold-change >0) in SOA-R in comparison with WT female pupae. The mean enrichment is shown as a metaplot on top ( n = 2 biological replicates, merged for visualization). ( f ) Genome browser snapshot of the SOA CUT&Tag enrichment obtained in SOA-R females in comparison with WT males on a representative region of the X-chromosome. Duplicate reads were filtered out and the replicates were merged for visualization. ( g ) CUT&Tag as in ( e ) Metaplot showing the mean CUT&Tag enrichment on expressed X-linked genes (≥10 average read counts), which were further grouped by unsupervised k-means clustering in 3 groups with strong, medium and weak SOA binding strength. The coverage was calculated using the TSS as a reference point with 1 kb upstream and the gene bodies downstream scaled to 5 kb. The replicates were merged for visualization. ( h ) Violin plot with center line representing the median RNA expression in log2 TPM (transcripts per million) from RNA-seq of WT females for each of the 3 clusters (based on binding in SOA-R , see ( g )). p -value: two-sided Wilcoxon rank-sum comparing combined clusters 1 and 2 versus cluster 3. ( i ) Euclidean distance heatmap obtained by DESeq2 representing the similarity of the samples in RNA-seq conducted from WT ( n = 3 biological replicates) and homozygous SOA-R ( n = 4 biological replicates) female pupae. ( j ) RNA-seq as in ( i ) Violin plots showing the log2FC on expressed X-linked genes (≥10 average read counts), which were further grouped by unsupervised k -means clustering in 3 groups with strong, medium and weak SOA binding strength, see ( g ). The center line represents the median log2FC, which equals 0.613, 0.355, and 0.117 (strong, intermediate and weak binding) and corresponds to fold changes of 1.529, 1.279, and 1.084, respectively. ( k ) RNA-seq as in ( j ) but plotting the log2FC for all genes according to the chromosomal location in WT compared to homozygous SOA-R pupae as a violin plot. Each gene with an average read count (baseMean) > 0 was taken into account, irrespective of whether it was scored as DE or not. The Bonferroni-corrected p -value was obtained with a two-sided Wilcoxon rank-sum test comparing X with all autosomes. The center line represents the median (also see Supplementary Table ). ( l ) RNA-seq as in ( i ) but plotting the log2FC distribution of autosomal (grey) and X-linked genes. The X-linked genes were split into two groups based on SOA binding in CUT&Tag (Supplementary Table ). The yellow violin plot shows X chromosomal genes without SOA peaks, the blue violin plot shows peaks that were scored as differentially bound by DiffBind ( SOA-R versus WT females, FDR<0.05, fold>0). Median log2FC values for each group are available in Supplementary Table . The Bonferroni-corrected p -values obtained with a two-sided Wilcoxon rank-sum test comparing all groups between each other are: autosomal versus X-linked genes without SOA peak p = 6.57E-12; autosomal versus X-linked genes with SOA peak p = 1.45E-44; X-linked genes without versus with SOA peak p = 5.48E-06. ( m ) A single culture of SOA-R (males + females) was conducted in parallel to WT males (T4 strain), cultured separately. For both, the developmental timing of each of the 3 genotypes was scored by counting the appearance of pupae. Pupa appearance is represented as a cumulative distribution with dots representing a given time-point when pupa numbers were scored. The t = 0 on the x -axis represents the time when the first pupa appeared in the culture. The data represents one experiment. For comparison, the mean WT male and female pupation timings scored in Fig. (exp 1-2022) are plotted in the panel. A separate experiment with additional n = 3 independent replicate cultures for SOA-R grown together with WT is presented in Fig. . ( n ) Checkerboard plot indicating the relative frequency of SOA+/SOA+ males (colour-coded) after 10,000 generations of selection depending on the selection coefficients s m in males ( x -axis) and s f in females ( y -axis). Fitness is normalized to 1 in SOA−/SOA− males and females. Moreover, we assume that SOA+ is dominant over SOA− in males and recessive in females. Hence, the fitness of SOA+ bearing males is 1 + s m , while the fitness of SOA+/SOA+ females is 1- s f . Even if selection against SOA+ in females is stronger than selection in favour of SOA+ in males, SOA+ is, for most parameter combinations, maintained in the population at considerable frequencies.

Article Snippet: This was followed by hybridization in Stellaris RNA FISH hybridization buffer (SMF-WA1-60-BS, LGC Biosearch Technologies) supplemented with 10% with formamide containing 125 nM probe mix targeting the introns of the X-linked gene act5c ( AGAP000651 , sequences in Supplementary Table ), which was incubated overnight in a humidified chamber at 37 °C.

Techniques: Quantitative RT-PCR, Immunostaining, Control, Comparison, Binding Assay, RNA Expression, RNA Sequencing, Cell Culture, Selection