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P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.

Dahlberg O, Shilkova O, Tang M, Holmqvist PH, Mannervik M - PLoS Genet. (2015)

Bottom Line: Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels.Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes.We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

View Article: PubMed Central - PubMed

Affiliation: Dept. Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

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Pol II occupancy is reduced at genes affected by P-TEFb depletion.(A) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). (B) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. (C-F) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. (C) Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with Sry-α and CG7271 in Cdk9 embryos. (D) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. (E) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. (F) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P<0.05, two-tailed unpaired Student´s t-test (calculations in S4 Table).
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pgen.1004971.g006: Pol II occupancy is reduced at genes affected by P-TEFb depletion.(A) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). (B) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. (C-F) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. (C) Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with Sry-α and CG7271 in Cdk9 embryos. (D) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. (E) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. (F) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P<0.05, two-tailed unpaired Student´s t-test (calculations in S4 Table).

Mentions: P-TEFb phosphorylates elongation factors and the Pol II CTD on Ser2 in order to release the polymerase into active elongation. We immunostained embryos to examine the localization of Ser2 phosphorylated Pol II. During the first nuclear division stages, a Pol II Ser2 signal was detected in the cytoplasm, presumably coming from polymerases not bound to chromatin (Fig. 6A). Interestingly, this signal showed an increase in Cdk9 embryos compared to wild-type. In later stage, cellularizing embryos, nuclear Pol II Ser2 was detected in all somatic cells, but not in the transcriptionally silent pole cells (Fig. 6A). Compared to wild-type embryos, reduced levels of Ser2 phosphorylated Pol II was detected in Cdk9 embryos at this stage (Fig. 6A). Quantification of the embryo stainings demonstrated that 59 +/- 1% of Ser2 phosphorylation remained in Cdk9 embryos. We further prepared extracts from embryos depleted of Cdk9 or Cyclin T and investigated global Pol II CTD Ser2 phosphorylation levels by Western blot. Embryo extracts from either Cdk9 or CycT knock-down cellularizing embryos showed lower levels of Pol II Ser2 phosphorylation (Fig. 6B), compared to the hypophosphorylated Pol IIa form of the CTD (Fig. 6B). The same results were obtained with a different Pol II Ser2 antibody (S1 Fig.). In these experiments, 39% and 43% of the phospho-Ser2 per CTD signal remained in Cdk9 and CycT embryos, respectively (Fig. 6B). Interestingly, despite different levels of Cdk9 and CycT depletion (Fig. 1), we find similar levels of Ser2 phosphorylation in Cdk9 and CycT embryos (Fig. 6B). One possibility is that the active pool of P-TEFb is depleted to the same extent in Cdk9 and CycT embryos, despite different knock-down efficiencies. Another possibility is that the activities of other Ser 2 kinases are affected by either Cdk9 or CycT depletion to the same degree. Regardless, our data show that P-TEFb knock-down during oogenesis did not reduce Pol II Ser2 phosphorylation at this stage. Instead, increased Pol II Ser 2 phosphorylation levels were found in early embryos prior to zygotic transcription. However, during cellularization, reduced amounts of Ser2 phosphorylated Pol II accumulated in the somatic nuclei of P-TEFb depleted embryos.


P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.

Dahlberg O, Shilkova O, Tang M, Holmqvist PH, Mannervik M - PLoS Genet. (2015)

Pol II occupancy is reduced at genes affected by P-TEFb depletion.(A) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). (B) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. (C-F) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. (C) Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with Sry-α and CG7271 in Cdk9 embryos. (D) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. (E) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. (F) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P<0.05, two-tailed unpaired Student´s t-test (calculations in S4 Table).
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pgen.1004971.g006: Pol II occupancy is reduced at genes affected by P-TEFb depletion.(A) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). (B) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. (C-F) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. (C) Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with Sry-α and CG7271 in Cdk9 embryos. (D) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. (E) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. (F) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P<0.05, two-tailed unpaired Student´s t-test (calculations in S4 Table).
Mentions: P-TEFb phosphorylates elongation factors and the Pol II CTD on Ser2 in order to release the polymerase into active elongation. We immunostained embryos to examine the localization of Ser2 phosphorylated Pol II. During the first nuclear division stages, a Pol II Ser2 signal was detected in the cytoplasm, presumably coming from polymerases not bound to chromatin (Fig. 6A). Interestingly, this signal showed an increase in Cdk9 embryos compared to wild-type. In later stage, cellularizing embryos, nuclear Pol II Ser2 was detected in all somatic cells, but not in the transcriptionally silent pole cells (Fig. 6A). Compared to wild-type embryos, reduced levels of Ser2 phosphorylated Pol II was detected in Cdk9 embryos at this stage (Fig. 6A). Quantification of the embryo stainings demonstrated that 59 +/- 1% of Ser2 phosphorylation remained in Cdk9 embryos. We further prepared extracts from embryos depleted of Cdk9 or Cyclin T and investigated global Pol II CTD Ser2 phosphorylation levels by Western blot. Embryo extracts from either Cdk9 or CycT knock-down cellularizing embryos showed lower levels of Pol II Ser2 phosphorylation (Fig. 6B), compared to the hypophosphorylated Pol IIa form of the CTD (Fig. 6B). The same results were obtained with a different Pol II Ser2 antibody (S1 Fig.). In these experiments, 39% and 43% of the phospho-Ser2 per CTD signal remained in Cdk9 and CycT embryos, respectively (Fig. 6B). Interestingly, despite different levels of Cdk9 and CycT depletion (Fig. 1), we find similar levels of Ser2 phosphorylation in Cdk9 and CycT embryos (Fig. 6B). One possibility is that the active pool of P-TEFb is depleted to the same extent in Cdk9 and CycT embryos, despite different knock-down efficiencies. Another possibility is that the activities of other Ser 2 kinases are affected by either Cdk9 or CycT depletion to the same degree. Regardless, our data show that P-TEFb knock-down during oogenesis did not reduce Pol II Ser2 phosphorylation at this stage. Instead, increased Pol II Ser 2 phosphorylation levels were found in early embryos prior to zygotic transcription. However, during cellularization, reduced amounts of Ser2 phosphorylated Pol II accumulated in the somatic nuclei of P-TEFb depleted embryos.

Bottom Line: Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels.Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes.We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

View Article: PubMed Central - PubMed

Affiliation: Dept. Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

Show MeSH