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Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision.

Li H, Rodriguez J, Yoo Y, Shareef MM, Badugu R, Horabin JI, Kellum R - PLoS Genet. (2011)

Bottom Line: RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability.Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences.In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, Sxl(Pe), is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (Sxl(Pm)) in both sexes. HOAP mutants show inappropriate Sxl(Pe) firing in males and the concomitant inappropriate splicing of Sxl(Pm)-derived transcripts, while females show premature firing of Sxl(Pe). HP1 mutants, by contrast, display Sxl(Pm) splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl(Pe).

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Chromatin immunoprecipitation (ChIP) assays show association of both HOAP and HP1 with the Sxl locus.(A) Relative enrichment (ChIP/input-Sxl sequence/ChIP/input-RpA-70) of specific Sxl locus sequences in HOAP- and HP1-ChIP fractions from 1–3 hr embryos plotted against the molecular map of the Sxl locus (HOAP in red; HP1 in blue). The positions of Sxl fragments monitored for enrichment are shown relative to the SxlPe initiation site, below the map. The positions of E-box binding sites for positive (blue diamonds) and negative (red squares) bHLH factors [63] and Sxl transcripts are also indicated below the map. (p(no enrichment)<0.05*) (B) Bar graphs show enrichment of Sxl sequences in HOAP- (red bars) and HP1- (blue bars) ChIP fractions during development (1–3 hr, 2–3 hr, and 4–18 hr staged embryos). (p(no enrichment)<0.05*; change between 1–3 hr and 2–3 hr embryos, p(no effect) p(no change)<0.10(#), <0.05#).
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pgen-1002122-g004: Chromatin immunoprecipitation (ChIP) assays show association of both HOAP and HP1 with the Sxl locus.(A) Relative enrichment (ChIP/input-Sxl sequence/ChIP/input-RpA-70) of specific Sxl locus sequences in HOAP- and HP1-ChIP fractions from 1–3 hr embryos plotted against the molecular map of the Sxl locus (HOAP in red; HP1 in blue). The positions of Sxl fragments monitored for enrichment are shown relative to the SxlPe initiation site, below the map. The positions of E-box binding sites for positive (blue diamonds) and negative (red squares) bHLH factors [63] and Sxl transcripts are also indicated below the map. (p(no enrichment)<0.05*) (B) Bar graphs show enrichment of Sxl sequences in HOAP- (red bars) and HP1- (blue bars) ChIP fractions during development (1–3 hr, 2–3 hr, and 4–18 hr staged embryos). (p(no enrichment)<0.05*; change between 1–3 hr and 2–3 hr embryos, p(no effect) p(no change)<0.10(#), <0.05#).

Mentions: This establishment promoter of Sxl, SxlPe, is critical to the sex determination decision which is made early in embryogenesis (see Figure 4A for an overview of the Sxl locus). SxlPe is only transcribed in females, which have two X chromosomes. In counting the X chromosome number, also known as the X∶A ratio, SxlPe responds to five X-linked activating genes (sisterless-a, sisterless-b, runt, myc and unpaired) working in conjunction with positive maternal factors such as Daughterless. These activating components have their dose measured against the negative effect of maternal factors, such as Groucho and Extramacrochetae, and genes on the autosomes (deadpan is the only known member). Firing of SxlPe generates functional SXL protein which initiates the female-mode of splicing of transcripts from the maintenance promoter, SxlPm. SxlPm is transcribed in both sexes, soon after SxlPe shuts down and its mRNAs are being turned over. In female embryos, the SXL protein from SxlPe transcripts inhibits inclusion of the male-specific exon, which would otherwise prematurely terminate translation, of SxlPm transcripts. This autoregulatory splicing loop maintains SXL expression for the rest of the female life cycle. As males do not activate SxlPe they make no SXL protein and the splicing of SxlPm transcripts includes the male exon by default. Through this autoregulation, the binary sex determination decision is maintained.


Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision.

Li H, Rodriguez J, Yoo Y, Shareef MM, Badugu R, Horabin JI, Kellum R - PLoS Genet. (2011)

Chromatin immunoprecipitation (ChIP) assays show association of both HOAP and HP1 with the Sxl locus.(A) Relative enrichment (ChIP/input-Sxl sequence/ChIP/input-RpA-70) of specific Sxl locus sequences in HOAP- and HP1-ChIP fractions from 1–3 hr embryos plotted against the molecular map of the Sxl locus (HOAP in red; HP1 in blue). The positions of Sxl fragments monitored for enrichment are shown relative to the SxlPe initiation site, below the map. The positions of E-box binding sites for positive (blue diamonds) and negative (red squares) bHLH factors [63] and Sxl transcripts are also indicated below the map. (p(no enrichment)<0.05*) (B) Bar graphs show enrichment of Sxl sequences in HOAP- (red bars) and HP1- (blue bars) ChIP fractions during development (1–3 hr, 2–3 hr, and 4–18 hr staged embryos). (p(no enrichment)<0.05*; change between 1–3 hr and 2–3 hr embryos, p(no effect) p(no change)<0.10(#), <0.05#).
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pgen-1002122-g004: Chromatin immunoprecipitation (ChIP) assays show association of both HOAP and HP1 with the Sxl locus.(A) Relative enrichment (ChIP/input-Sxl sequence/ChIP/input-RpA-70) of specific Sxl locus sequences in HOAP- and HP1-ChIP fractions from 1–3 hr embryos plotted against the molecular map of the Sxl locus (HOAP in red; HP1 in blue). The positions of Sxl fragments monitored for enrichment are shown relative to the SxlPe initiation site, below the map. The positions of E-box binding sites for positive (blue diamonds) and negative (red squares) bHLH factors [63] and Sxl transcripts are also indicated below the map. (p(no enrichment)<0.05*) (B) Bar graphs show enrichment of Sxl sequences in HOAP- (red bars) and HP1- (blue bars) ChIP fractions during development (1–3 hr, 2–3 hr, and 4–18 hr staged embryos). (p(no enrichment)<0.05*; change between 1–3 hr and 2–3 hr embryos, p(no effect) p(no change)<0.10(#), <0.05#).
Mentions: This establishment promoter of Sxl, SxlPe, is critical to the sex determination decision which is made early in embryogenesis (see Figure 4A for an overview of the Sxl locus). SxlPe is only transcribed in females, which have two X chromosomes. In counting the X chromosome number, also known as the X∶A ratio, SxlPe responds to five X-linked activating genes (sisterless-a, sisterless-b, runt, myc and unpaired) working in conjunction with positive maternal factors such as Daughterless. These activating components have their dose measured against the negative effect of maternal factors, such as Groucho and Extramacrochetae, and genes on the autosomes (deadpan is the only known member). Firing of SxlPe generates functional SXL protein which initiates the female-mode of splicing of transcripts from the maintenance promoter, SxlPm. SxlPm is transcribed in both sexes, soon after SxlPe shuts down and its mRNAs are being turned over. In female embryos, the SXL protein from SxlPe transcripts inhibits inclusion of the male-specific exon, which would otherwise prematurely terminate translation, of SxlPm transcripts. This autoregulatory splicing loop maintains SXL expression for the rest of the female life cycle. As males do not activate SxlPe they make no SXL protein and the splicing of SxlPm transcripts includes the male exon by default. Through this autoregulation, the binary sex determination decision is maintained.

Bottom Line: RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability.Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences.In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, Sxl(Pe), is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (Sxl(Pm)) in both sexes. HOAP mutants show inappropriate Sxl(Pe) firing in males and the concomitant inappropriate splicing of Sxl(Pm)-derived transcripts, while females show premature firing of Sxl(Pe). HP1 mutants, by contrast, display Sxl(Pm) splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl(Pe).

Show MeSH
Related in: MedlinePlus