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. 2013 Sep;140(17):3613-23.
doi: 10.1242/dev.094953. Epub 2013 Jul 24.

The Insulator Protein Suppressor of Hairy-wing Is an Essential Transcriptional Repressor in the Drosophila Ovary

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Free PMC article

The Insulator Protein Suppressor of Hairy-wing Is an Essential Transcriptional Repressor in the Drosophila Ovary

Alexey A Soshnev et al. Development. .
Free PMC article

Abstract

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)(-/-) sterility, as Rbp9(+/-), su(Hw)(-/-) females are fertile. Eggs produced by Rbp9(+/-), su(Hw)(-/-) females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.

Keywords: Chromatin insulator; Drosophila oogenesis; Neural gene expression; Rbp9; Su(Hw); Transcriptional regulation.

Figures

Fig. 1.
Fig. 1.
Identification of Su(Hw) target genes. (A) Shown are 4- to 6-hour-old DAPI-stained ovarioles isolated from fertile [su(Hw)+/+] and sterile [su(Hw)2/v and su(Hw)Pb/2] females. Scale bars: 25 μm. (B) Microarray analyses identified 175 increased (red bar) and 122 decreased (blue bar) genes, of which 75 and 30 correspond to Su(Hw) target genes, respectively (gray bars). (C) Quantitative PCR validation of target genes. Expression is normalized to the housekeeping gene RpL32 and shown as heat map of fold change values relative to su(Hw)+/+, with blue and red indicating lower and higher expression, respectively. 33/93, heteroallelic combination of two independently generated recombinant chromosomes containing double Cp190H4-1 and mod(mdg4)u1 mutations. The gypsy insulator proteins associated with the SBSs are shown. Three non-target housekeeping genes and su(Hw) were included as controls. Two independent biological samples (1, 2) were analyzed.
Fig. 2.
Fig. 2.
Characterization of Su(Hw) binding sites (SBSs) at target genes. (A) Shown are box plots of fold enrichment of all SBSs (total) and SBSs in activated (red arrow) and repressed (blue arrow) target genes. Within each box, the black line indicates median enrichment, boxes and whiskers represent 25-75 percentile interval and non-outlier range, respectively. P-values of Student’s t-test are indicated. (B) Weblogo of MEME-derived consensus motifs of all SBSs, SBSs at target genes, and the gypsy insulator SBSs. (C) Shown are distributions of SBSs relative to gene features. (D) Shown are enrichments of gypsy insulator proteins at SBSs.
Fig. 3.
Fig. 3.
Su(Hw) is a repressor of CNS-enriched genes. (A) Left to right: target genes ranked by fold changes obtained in microarray analyses, with red corresponding to activated and blue corresponding to repressed genes; genes with SBSs; genes with ovary-enriched expression indicated by green color scale; genes with CNS-enriched expression in three CNS structures [adult brain, thoracoabdominal (t.a.) ganglion and larval CNS] indicated by the orange color scale (Chintapalli et al., 2007). (B) Confocal images of su(Hw)+/+ third instar larval brains stained for Su(Hw) (green, top) and neural markers (Dpn, Repo, ELAV; red, middle), and merged image (bottom). Scale bars: 50 μm. Magnified areas (shown below) are indicated by dotted rectangles. Scale bars: 25 μm. (C) qPCR analyses of activated and repressed target genes in RNA isolated from su(Hw)2/v larval brain and wing disc. Expression was normalized to the housekeeping gene RpL32 and is shown as heat map of fold change values relative to su(Hw)+/+, with blue and red indicating low and high expression, respectively. Three non-target housekeeping genes and su(Hw) were included as controls. Two biological samples (1, 2) were analyzed.
Fig. 4.
Fig. 4.
Rbp9 is repressed by Su(Hw). (A) Images of su(Hw)+/+ germarium stained for Su(Hw) (top, green), Rbp9 (bottom, red) and DAPI (white). Developmental regions (R1 to R3) of the germarium and egg chamber stages (S1 to S3) are indicated. Scale bars: 25 μm. (B) su(Hw)+/+ (top), sterile su(Hw)2/v (middle) and fertile su(Hw)f/v (bottom) ovarioles stained for Rbp9 (red) and DAPI (white). Scale bars: 25 μm. (C) UCSC genome browser view of the Rbp9 gene locus, including tracks. Top to bottom: chromosome coordinates, su(Hw)wt ChIP-Seq reads, preimmune serum IP control reads, su(Hw)f ChIP-Seq reads, preimmune serum IP control reads, fragments amplified in qPCR analyses (E), RefSeq gene annotation. (D) ChIP-qPCR analyses of ovary-bound CP190 (top) and Mod67.2 (bottom) at Rbp9 SBSs. Negative controls (1-4) were genomic regions with no SBS (Soshnev et al., 2012). ChIP from a mod(mdg4)u1 mutant background was a negative control. (E) qPCR analyses of promoter-specific Rbp9 transcripts in su(Hw)+/+ (black bars) and su(Hw)2/v (red bars) mutant background. Expression is normalized to housekeeping gene RpL32 and is shown as fold change relative to su(Hw)+/+. Error bars indicate s.d. of three biological samples. (F) qPCR analyses of gene expression changes in su(Hw), Cp190 and mod(mdg4) mutant ovaries. Expression is normalized to the housekeeping gene RpL32 and shown as a fold change relative to su(Hw)+/+. Gapdh2 and β-tubulin are negative controls. R33 and R93 indicate two independently generated recombinant chromosomes containing Cp190H4-1 and mod(mdg4)u1 mutations. Error bars indicate s.d. of two independent biological samples.
Fig. 5.
Fig. 5.
Decreased Rbp9 expression rescues female sterility of su(Hw) null mutants. (A) DAPI-stained ovarioles isolated from su(Hw)2/v and Rbp9P2775/+; su(Hw)2/v females. Asterisks indicate egg chamber apoptosis. Arrowheads indicate late-stage egg chambers. Scale bars: 50 μm. (B) qPCR analyses of su(Hw) and Rbp9 RNA levels in the fertile su(Hw)+/+ (gray), su(Hw)f/v (black), sterile su(Hw)2/v (red) and su(Hw)Pb/2 (pink) and fertile Rbp9P2690/+; su(Hw)2/v (green) and Rbp9P2775/+; su(Hw)2/v (blue) backgrounds. Expression is normalized to housekeeping gene RpL32 and is shown as fold change relative to su(Hw)+/+. Error bars indicate s.d. of three biological samples. (C) qPCR analyses of activated and repressed target genes in ovaries dissected from su(Hw)+/+, su(Hw)2/v, Rbp9P2690/+; su(Hw)2/v and Rbp9P2775/+; su(Hw)2/v females. Expression was normalized to the housekeeping gene RpL32 and is shown as heat map of fold change values relative to su(Hw)+/+, blue and red indicating low and high expression, respectively. Three non-target housekeeping genes were included as controls. Two biological samples (1, 2) were analyzed.
Fig. 6.
Fig. 6.
Rescue of female sterility reveals a somatic function for Su(Hw). (A) SEM image of a su(Hw)+/+ egg and a Rbp9P2775/+, su(Hw)2/v egg. (B) Stage 12 egg chambers from su(Hw)+/+ and Rbp9P2775/+, su(Hw)2/v mutant females, stained with Broad-Z1 (red) and DAPI (white). Scale bars: 50 μm. (C) Left: whole-mount egg chambers stained for Gurken (red) and DAPI (white) dissected from wild-type (top) and Rbp9P2775/+, su(Hw)-/- (bottom) females. Dashed rectangles indicate magnified areas (shown to the right). Arrowheads indicate Gurken-positive vesicles. Scale bars: 50 μm (left) and 5 μm (right).

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