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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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Model of the interactions between HOAP and HP1 at SxlPe.(A) Maternal HOAP and HP1 cooperate to form a repressive complex which serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic expression of positive (e.g. Sis-a, Sis-b) and negative (e.g. Dpn) regulatory proteins. Pre-loading of RNA pol II requires HP1. (B) Binding of bHLH proteins to SxlPe sequences is dependent on their zygotic dose and binding sites relative to those for HOAP. At 2–3 hr of development, the two X chromosome dose of positive factors in females, is able to result in activation of SxlPe, and allow the pre-loaded RNA pol II to extend. In males, the single X chromosome presumably does not produce enough positive factors to displace (all of) the negative components. For simplicity not all known X∶A factors are shown. The triangle depicts low levels of H3K9 methylation at the promoter.
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pgen-1002122-g006: Model of the interactions between HOAP and HP1 at SxlPe.(A) Maternal HOAP and HP1 cooperate to form a repressive complex which serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic expression of positive (e.g. Sis-a, Sis-b) and negative (e.g. Dpn) regulatory proteins. Pre-loading of RNA pol II requires HP1. (B) Binding of bHLH proteins to SxlPe sequences is dependent on their zygotic dose and binding sites relative to those for HOAP. At 2–3 hr of development, the two X chromosome dose of positive factors in females, is able to result in activation of SxlPe, and allow the pre-loaded RNA pol II to extend. In males, the single X chromosome presumably does not produce enough positive factors to displace (all of) the negative components. For simplicity not all known X∶A factors are shown. The triangle depicts low levels of H3K9 methylation at the promoter.

Mentions: The interdependency of HOAP and HP1 for their binding to the SxlPe proximal region, most notably the dependence of HP1 on HOAP, also indicates both proteins are in this region in, at least, wild type female embryos. In spite of this interdependency, the genetic data show HOAP repression antagonizes HP1 activation. HOAP repression appears to also be partly HP1-dependent; the mutant HOAP protein from the cav1 allele which lacks HP1-binding also antagonizes HP1 activation. This combination of antagonistic and cooperative interactions suggests a model in which maternal HOAP and HP1 first cooperate to repress SxlPe prior to its activation (Figure 6A). The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic XSE levels, ensuring it is only activated in females where an effective ratio of activating to repressing transcription factors exists (Figure 6B). HP1 is retained at SxlPe during its activation in females, where it presumably switches into an activation role. In early embryos constitutive heterochromatin proteins may be more appropriate for such regulation than the Polycomb Group of facultative heterochromatin proteins, as they would not be subject to cross regulatory signals from body plan specification pathways.


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)

Model of the interactions between HOAP and HP1 at SxlPe.(A) Maternal HOAP and HP1 cooperate to form a repressive complex which serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic expression of positive (e.g. Sis-a, Sis-b) and negative (e.g. Dpn) regulatory proteins. Pre-loading of RNA pol II requires HP1. (B) Binding of bHLH proteins to SxlPe sequences is dependent on their zygotic dose and binding sites relative to those for HOAP. At 2–3 hr of development, the two X chromosome dose of positive factors in females, is able to result in activation of SxlPe, and allow the pre-loaded RNA pol II to extend. In males, the single X chromosome presumably does not produce enough positive factors to displace (all of) the negative components. For simplicity not all known X∶A factors are shown. The triangle depicts low levels of H3K9 methylation at the promoter.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111545&req=5

pgen-1002122-g006: Model of the interactions between HOAP and HP1 at SxlPe.(A) Maternal HOAP and HP1 cooperate to form a repressive complex which serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic expression of positive (e.g. Sis-a, Sis-b) and negative (e.g. Dpn) regulatory proteins. Pre-loading of RNA pol II requires HP1. (B) Binding of bHLH proteins to SxlPe sequences is dependent on their zygotic dose and binding sites relative to those for HOAP. At 2–3 hr of development, the two X chromosome dose of positive factors in females, is able to result in activation of SxlPe, and allow the pre-loaded RNA pol II to extend. In males, the single X chromosome presumably does not produce enough positive factors to displace (all of) the negative components. For simplicity not all known X∶A factors are shown. The triangle depicts low levels of H3K9 methylation at the promoter.
Mentions: The interdependency of HOAP and HP1 for their binding to the SxlPe proximal region, most notably the dependence of HP1 on HOAP, also indicates both proteins are in this region in, at least, wild type female embryos. In spite of this interdependency, the genetic data show HOAP repression antagonizes HP1 activation. HOAP repression appears to also be partly HP1-dependent; the mutant HOAP protein from the cav1 allele which lacks HP1-binding also antagonizes HP1 activation. This combination of antagonistic and cooperative interactions suggests a model in which maternal HOAP and HP1 first cooperate to repress SxlPe prior to its activation (Figure 6A). The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic XSE levels, ensuring it is only activated in females where an effective ratio of activating to repressing transcription factors exists (Figure 6B). HP1 is retained at SxlPe during its activation in females, where it presumably switches into an activation role. In early embryos constitutive heterochromatin proteins may be more appropriate for such regulation than the Polycomb Group of facultative heterochromatin proteins, as they would not be subject to cross regulatory signals from body plan specification pathways.

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