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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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Aberrant sex-specific SxlPm-derived transcripts are observed in mutants for HOAP and in mutants for HP1.(A) RT-PCR was used to specifically amplify male- or female-specific SxlPm transcripts. The P1/P3 primer pair amplifies a 482 bp product from the female-specific transcript and a 672 bp product from the male-specific transcript in wild type animals (labeled wt in all three panels). The P2/P3 pair amplifies a 284 bp product from a male-specific transcript in males only. (B) Pools of RNA from individually sexed male or female cav1 homozygous or cav2248 heterozygous embryos failing to progress to the larval stage were used in RT-PCR assays with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs, revealing the presence of aberrant female-specific SxlPm transcripts in male embryos (arrowhead). RT-PCR assays of RNA from four different individually sexed Su(var)2054/Su(var)2055 female (♀) or male (♂) larvae, each with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs revealed the presence of aberrant sex-specific SxlPm transcripts in both sexes. The methods used to genotype and sex individual embryos and larvae are described in Materials and Methods.
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pgen-1002122-g002: Aberrant sex-specific SxlPm-derived transcripts are observed in mutants for HOAP and in mutants for HP1.(A) RT-PCR was used to specifically amplify male- or female-specific SxlPm transcripts. The P1/P3 primer pair amplifies a 482 bp product from the female-specific transcript and a 672 bp product from the male-specific transcript in wild type animals (labeled wt in all three panels). The P2/P3 pair amplifies a 284 bp product from a male-specific transcript in males only. (B) Pools of RNA from individually sexed male or female cav1 homozygous or cav2248 heterozygous embryos failing to progress to the larval stage were used in RT-PCR assays with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs, revealing the presence of aberrant female-specific SxlPm transcripts in male embryos (arrowhead). RT-PCR assays of RNA from four different individually sexed Su(var)2054/Su(var)2055 female (♀) or male (♂) larvae, each with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs revealed the presence of aberrant sex-specific SxlPm transcripts in both sexes. The methods used to genotype and sex individual embryos and larvae are described in Materials and Methods.

Mentions: To determine if the enhanced male lethality of cav mutants is associated with inappropriate SXL activity in males, RT (reverse transcriptase)-PCR assays were used to monitor sex-specific splicing of SxlPm transcripts in heterozygous cav2248 and homozygous cav1 male embryos that failed to progress to the larval stage. Individual embryos of the appropriate genotype were identified through a GFP-marked wild type balancer chromosome (described more fully in Materials and Methods) and sexed by PCR with Y-linked primers (Figure S2). RNA was then purified from pools of male or female embryos and used in RT-PCR assays with Sxl primer pairs (P1/P3 and P2/P3) designed to discriminate between SxlPm transcripts spliced in the male vs. female mode (Figure 2A). Assays of RNA from wild type (y1w67c23) embryos showed correct sex-specific splicing of SxlPm transcripts in both males and females (Figure 2B). By contrast, a minor fraction of transcripts had undergone the female-mode of splicing in heterozygous cav2248 and homozygous cav1 male embryos (arrowhead in Figure 2B).


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)

Aberrant sex-specific SxlPm-derived transcripts are observed in mutants for HOAP and in mutants for HP1.(A) RT-PCR was used to specifically amplify male- or female-specific SxlPm transcripts. The P1/P3 primer pair amplifies a 482 bp product from the female-specific transcript and a 672 bp product from the male-specific transcript in wild type animals (labeled wt in all three panels). The P2/P3 pair amplifies a 284 bp product from a male-specific transcript in males only. (B) Pools of RNA from individually sexed male or female cav1 homozygous or cav2248 heterozygous embryos failing to progress to the larval stage were used in RT-PCR assays with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs, revealing the presence of aberrant female-specific SxlPm transcripts in male embryos (arrowhead). RT-PCR assays of RNA from four different individually sexed Su(var)2054/Su(var)2055 female (♀) or male (♂) larvae, each with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs revealed the presence of aberrant sex-specific SxlPm transcripts in both sexes. The methods used to genotype and sex individual embryos and larvae are described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111545&req=5

pgen-1002122-g002: Aberrant sex-specific SxlPm-derived transcripts are observed in mutants for HOAP and in mutants for HP1.(A) RT-PCR was used to specifically amplify male- or female-specific SxlPm transcripts. The P1/P3 primer pair amplifies a 482 bp product from the female-specific transcript and a 672 bp product from the male-specific transcript in wild type animals (labeled wt in all three panels). The P2/P3 pair amplifies a 284 bp product from a male-specific transcript in males only. (B) Pools of RNA from individually sexed male or female cav1 homozygous or cav2248 heterozygous embryos failing to progress to the larval stage were used in RT-PCR assays with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs, revealing the presence of aberrant female-specific SxlPm transcripts in male embryos (arrowhead). RT-PCR assays of RNA from four different individually sexed Su(var)2054/Su(var)2055 female (♀) or male (♂) larvae, each with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs revealed the presence of aberrant sex-specific SxlPm transcripts in both sexes. The methods used to genotype and sex individual embryos and larvae are described in Materials and Methods.
Mentions: To determine if the enhanced male lethality of cav mutants is associated with inappropriate SXL activity in males, RT (reverse transcriptase)-PCR assays were used to monitor sex-specific splicing of SxlPm transcripts in heterozygous cav2248 and homozygous cav1 male embryos that failed to progress to the larval stage. Individual embryos of the appropriate genotype were identified through a GFP-marked wild type balancer chromosome (described more fully in Materials and Methods) and sexed by PCR with Y-linked primers (Figure S2). RNA was then purified from pools of male or female embryos and used in RT-PCR assays with Sxl primer pairs (P1/P3 and P2/P3) designed to discriminate between SxlPm transcripts spliced in the male vs. female mode (Figure 2A). Assays of RNA from wild type (y1w67c23) embryos showed correct sex-specific splicing of SxlPm transcripts in both males and females (Figure 2B). By contrast, a minor fraction of transcripts had undergone the female-mode of splicing in heterozygous cav2248 and homozygous cav1 male embryos (arrowhead in Figure 2B).

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