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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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In situs for SxlPe transcripts in embryos from wild type, cav2248/TM3, Sb, or Su(var)2055/CyO parents.Comparisons of the same-sized area of images taken at 40×. (A) SxlPe transcripts are present only in Ore R wild type females (containing 2 dots per nucleus) during cycle 12 to 14. SxlPe transcripts are present in female embryos from cav2248/TM3 parents as early as cycle 10. SxlPe transcripts are also frequently present in male embryos from cav2248/TM3 parents (single dot per nucleus, usually near nuclear periphery where the dosage compensated X chromosome resides [62]. Not much signal is detected before cycle 13 in males. (B) Poor SxlPe expression is observed in embryos from Su(var)2055/CyO parents in comparison to simultaneously stained embryos from Ore R wild type parents.
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pgen-1002122-g003: In situs for SxlPe transcripts in embryos from wild type, cav2248/TM3, Sb, or Su(var)2055/CyO parents.Comparisons of the same-sized area of images taken at 40×. (A) SxlPe transcripts are present only in Ore R wild type females (containing 2 dots per nucleus) during cycle 12 to 14. SxlPe transcripts are present in female embryos from cav2248/TM3 parents as early as cycle 10. SxlPe transcripts are also frequently present in male embryos from cav2248/TM3 parents (single dot per nucleus, usually near nuclear periphery where the dosage compensated X chromosome resides [62]. Not much signal is detected before cycle 13 in males. (B) Poor SxlPe expression is observed in embryos from Su(var)2055/CyO parents in comparison to simultaneously stained embryos from Ore R wild type parents.

Mentions: The rescue of cav2248 male viability by the loss of Sxl and the antagonistic maternal effects of cav and Su(var)205 on the viability of their Sxl−-bearing female progeny, suggested a role for maternal HOAP and HP1 in regulating SxlPe. To directly assess the effect of reduced HOAP or HP1 on SxlPe, in situ hybridizations were performed with probes that distinguish the early from maintenance Sxl mRNAs on 0–4 hr embryos from cav2248 and Su(var)2055 heterozygous parents. For SxlPe, wild type embryos show two dots on the chromosomes, one for each female X, beginning in cycle 12 through to early cycle 14. Cycle 14 is also when the maintenance promoter, SxlPm, is activated and the autoregulatory splicing loop set in motion in females. In embryos from cav2248 heterozygous parents, two key changes were observed (Figure 3A). First, expression of SxlPe in females began two cycles earlier than normal – cycle 10, and overall expression appeared more robust than in wild type embryos. Second, male embryos also showed SxlPe activation (single in situ dot), although the expression was not as strong or as early as in females. The level of expression in male embryos was more variable both between embryos and at the level of individual nuclei. The majority had sporadic or scattered positive nuclei, while others had much greater numbers, as shown in Figure 3A. A count of early cycle 14 male embryos suggests >95% of the male embryos (n = 33) had at least some positive nuclei. In embryos from Su(var)2055 heterozygous parents, SxlPe expression was not observed in male embryos (single in situ signal) and was weaker and more variable than normal in females (Figure 3B). Analysis of the female embryos indicates that ∼85% express the promoter weakly during cycles 12 and 13 (n = 14–20 for each cycle) and less than half the embryos have normal levels of expression at cycle 14 (n>25). These results are consistent with the early expression of SxlPe being heavily reliant on the maternal deposit of HP1 protein and/or mRNA, with the zygotic contribution becoming more apparent at cycle 14. Expression of the maintenance SxlPm transcripts did not show significant changes in embryos from either genotype (Figure S3). Although HP1 binds to SxlPm in 4–18 hr embryos, at cellular blastoderm the promoter does not appear to be sensitive to a reduction in maternal HP1.


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)

In situs for SxlPe transcripts in embryos from wild type, cav2248/TM3, Sb, or Su(var)2055/CyO parents.Comparisons of the same-sized area of images taken at 40×. (A) SxlPe transcripts are present only in Ore R wild type females (containing 2 dots per nucleus) during cycle 12 to 14. SxlPe transcripts are present in female embryos from cav2248/TM3 parents as early as cycle 10. SxlPe transcripts are also frequently present in male embryos from cav2248/TM3 parents (single dot per nucleus, usually near nuclear periphery where the dosage compensated X chromosome resides [62]. Not much signal is detected before cycle 13 in males. (B) Poor SxlPe expression is observed in embryos from Su(var)2055/CyO parents in comparison to simultaneously stained embryos from Ore R wild type parents.
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Related In: Results  -  Collection

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pgen-1002122-g003: In situs for SxlPe transcripts in embryos from wild type, cav2248/TM3, Sb, or Su(var)2055/CyO parents.Comparisons of the same-sized area of images taken at 40×. (A) SxlPe transcripts are present only in Ore R wild type females (containing 2 dots per nucleus) during cycle 12 to 14. SxlPe transcripts are present in female embryos from cav2248/TM3 parents as early as cycle 10. SxlPe transcripts are also frequently present in male embryos from cav2248/TM3 parents (single dot per nucleus, usually near nuclear periphery where the dosage compensated X chromosome resides [62]. Not much signal is detected before cycle 13 in males. (B) Poor SxlPe expression is observed in embryos from Su(var)2055/CyO parents in comparison to simultaneously stained embryos from Ore R wild type parents.
Mentions: The rescue of cav2248 male viability by the loss of Sxl and the antagonistic maternal effects of cav and Su(var)205 on the viability of their Sxl−-bearing female progeny, suggested a role for maternal HOAP and HP1 in regulating SxlPe. To directly assess the effect of reduced HOAP or HP1 on SxlPe, in situ hybridizations were performed with probes that distinguish the early from maintenance Sxl mRNAs on 0–4 hr embryos from cav2248 and Su(var)2055 heterozygous parents. For SxlPe, wild type embryos show two dots on the chromosomes, one for each female X, beginning in cycle 12 through to early cycle 14. Cycle 14 is also when the maintenance promoter, SxlPm, is activated and the autoregulatory splicing loop set in motion in females. In embryos from cav2248 heterozygous parents, two key changes were observed (Figure 3A). First, expression of SxlPe in females began two cycles earlier than normal – cycle 10, and overall expression appeared more robust than in wild type embryos. Second, male embryos also showed SxlPe activation (single in situ dot), although the expression was not as strong or as early as in females. The level of expression in male embryos was more variable both between embryos and at the level of individual nuclei. The majority had sporadic or scattered positive nuclei, while others had much greater numbers, as shown in Figure 3A. A count of early cycle 14 male embryos suggests >95% of the male embryos (n = 33) had at least some positive nuclei. In embryos from Su(var)2055 heterozygous parents, SxlPe expression was not observed in male embryos (single in situ signal) and was weaker and more variable than normal in females (Figure 3B). Analysis of the female embryos indicates that ∼85% express the promoter weakly during cycles 12 and 13 (n = 14–20 for each cycle) and less than half the embryos have normal levels of expression at cycle 14 (n>25). These results are consistent with the early expression of SxlPe being heavily reliant on the maternal deposit of HP1 protein and/or mRNA, with the zygotic contribution becoming more apparent at cycle 14. Expression of the maintenance SxlPm transcripts did not show significant changes in embryos from either genotype (Figure S3). Although HP1 binds to SxlPm in 4–18 hr embryos, at cellular blastoderm the promoter does not appear to be sensitive to a reduction in maternal HP1.

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