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Coordinated regulation of Myc trans-activation targets by Polycomb and the Trithorax group protein Ash1.

Goodliffe JM, Cole MD, Wieschaus E - BMC Mol. Biol. (2007)

Bottom Line: We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation.We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1.Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. jmgoodli@uncc.edu

ABSTRACT

Background: The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis.

Results: To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1.

Conclusion: Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.

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Myc targets of activation are methylated at both H3K4 and H3K27. (A) (Left) A graph showing basal levels of 680 Myc activated targets (blue, left bar, each gene is represented by a thin line) followed by their levels upon ectopic Myc activation (blue, right bar, same genes as in the left bar). (Right) Basal levels of 57 Myc repression targets are shown (red, left bar, each gene is represented by a single thin line), and their levels following the activation of ectopic Myc (red, right bar, same genes as in the left bar). Compare basal levels of the activation targets versus repression targets. (B) Myc activation targets that are also regulated by Pc, Pho and Ash1 are methylated at both H3K4 and H3K27. DNA purified from chromatin immunoprecipitation reactions provided the template for PCR of CG16712 (upper) and CG18108 (lower), and the ChIP antibodies used are indicated above each lane. The right two lanes show results from sequential ChIP of chromatin by one antibody and then another. Input chromatin DNA is shown the far left lane, used in a 1:1000 dilution. (C) The enrichment by IP of chromatin containing 6 genes was calculated by dividing the levels of PCR product by that of the background, no antibody control. CG16712, aTub67C and fs(1)N are all Myc induced, Pc, Ash1 and Pho repressed, and show methylation of H3K4 and K3K27 together. Atg8a is not affected by any of the four regulators, though it is highly expressed in the embryo and methylated at H3K4 (blue bar). Cyp309a1 and CG18108 are Myc/Pc repressed, and show both H3K4 and H3K27 methylation in wild type embryos, but not at the same locus by sequential ChIP. (D) A target of activation by Myc is depicted, with low levels of expression, in a domain of chromatin bearing H3K27 methylation and H3K4 methylation. A growth signal or other signal, including increased Myc accumulation, allows Myc/Max to bind to a binding site, recruiting activators and inducing transcription. Ash1 is shown directly or indirectly repelling PcG repression and maintaining an active H3K4 methylation state. (E) A target of Myc repression is depicted, with high levels of expression mediated by an unknown transcription factor (TF). A cellular signal and increased Myc accumulation allow Pho and Pc required repression, propagating a repressed chromatin state characterized by H3K27 methylation.
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Figure 8: Myc targets of activation are methylated at both H3K4 and H3K27. (A) (Left) A graph showing basal levels of 680 Myc activated targets (blue, left bar, each gene is represented by a thin line) followed by their levels upon ectopic Myc activation (blue, right bar, same genes as in the left bar). (Right) Basal levels of 57 Myc repression targets are shown (red, left bar, each gene is represented by a single thin line), and their levels following the activation of ectopic Myc (red, right bar, same genes as in the left bar). Compare basal levels of the activation targets versus repression targets. (B) Myc activation targets that are also regulated by Pc, Pho and Ash1 are methylated at both H3K4 and H3K27. DNA purified from chromatin immunoprecipitation reactions provided the template for PCR of CG16712 (upper) and CG18108 (lower), and the ChIP antibodies used are indicated above each lane. The right two lanes show results from sequential ChIP of chromatin by one antibody and then another. Input chromatin DNA is shown the far left lane, used in a 1:1000 dilution. (C) The enrichment by IP of chromatin containing 6 genes was calculated by dividing the levels of PCR product by that of the background, no antibody control. CG16712, aTub67C and fs(1)N are all Myc induced, Pc, Ash1 and Pho repressed, and show methylation of H3K4 and K3K27 together. Atg8a is not affected by any of the four regulators, though it is highly expressed in the embryo and methylated at H3K4 (blue bar). Cyp309a1 and CG18108 are Myc/Pc repressed, and show both H3K4 and H3K27 methylation in wild type embryos, but not at the same locus by sequential ChIP. (D) A target of activation by Myc is depicted, with low levels of expression, in a domain of chromatin bearing H3K27 methylation and H3K4 methylation. A growth signal or other signal, including increased Myc accumulation, allows Myc/Max to bind to a binding site, recruiting activators and inducing transcription. Ash1 is shown directly or indirectly repelling PcG repression and maintaining an active H3K4 methylation state. (E) A target of Myc repression is depicted, with high levels of expression mediated by an unknown transcription factor (TF). A cellular signal and increased Myc accumulation allow Pho and Pc required repression, propagating a repressed chromatin state characterized by H3K27 methylation.

Mentions: In many of our experiments, Pc, Pho and Ash1 proteins facilitate Myc's activity as a repressor, but also appear to oppose its activity as an activator. In an attempt to understand how the same proteins cooperate with Myc to repress one set of targets and conversely work to repress a different set of Myc activation targets, we have to consider a major difference between Myc's targets of activation and repression: before Myc acts upon them, repression targets are necessarily being expressed while activation targets are largely unexpressed (Figure 8A).


Coordinated regulation of Myc trans-activation targets by Polycomb and the Trithorax group protein Ash1.

Goodliffe JM, Cole MD, Wieschaus E - BMC Mol. Biol. (2007)

Myc targets of activation are methylated at both H3K4 and H3K27. (A) (Left) A graph showing basal levels of 680 Myc activated targets (blue, left bar, each gene is represented by a thin line) followed by their levels upon ectopic Myc activation (blue, right bar, same genes as in the left bar). (Right) Basal levels of 57 Myc repression targets are shown (red, left bar, each gene is represented by a single thin line), and their levels following the activation of ectopic Myc (red, right bar, same genes as in the left bar). Compare basal levels of the activation targets versus repression targets. (B) Myc activation targets that are also regulated by Pc, Pho and Ash1 are methylated at both H3K4 and H3K27. DNA purified from chromatin immunoprecipitation reactions provided the template for PCR of CG16712 (upper) and CG18108 (lower), and the ChIP antibodies used are indicated above each lane. The right two lanes show results from sequential ChIP of chromatin by one antibody and then another. Input chromatin DNA is shown the far left lane, used in a 1:1000 dilution. (C) The enrichment by IP of chromatin containing 6 genes was calculated by dividing the levels of PCR product by that of the background, no antibody control. CG16712, aTub67C and fs(1)N are all Myc induced, Pc, Ash1 and Pho repressed, and show methylation of H3K4 and K3K27 together. Atg8a is not affected by any of the four regulators, though it is highly expressed in the embryo and methylated at H3K4 (blue bar). Cyp309a1 and CG18108 are Myc/Pc repressed, and show both H3K4 and H3K27 methylation in wild type embryos, but not at the same locus by sequential ChIP. (D) A target of activation by Myc is depicted, with low levels of expression, in a domain of chromatin bearing H3K27 methylation and H3K4 methylation. A growth signal or other signal, including increased Myc accumulation, allows Myc/Max to bind to a binding site, recruiting activators and inducing transcription. Ash1 is shown directly or indirectly repelling PcG repression and maintaining an active H3K4 methylation state. (E) A target of Myc repression is depicted, with high levels of expression mediated by an unknown transcription factor (TF). A cellular signal and increased Myc accumulation allow Pho and Pc required repression, propagating a repressed chromatin state characterized by H3K27 methylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Myc targets of activation are methylated at both H3K4 and H3K27. (A) (Left) A graph showing basal levels of 680 Myc activated targets (blue, left bar, each gene is represented by a thin line) followed by their levels upon ectopic Myc activation (blue, right bar, same genes as in the left bar). (Right) Basal levels of 57 Myc repression targets are shown (red, left bar, each gene is represented by a single thin line), and their levels following the activation of ectopic Myc (red, right bar, same genes as in the left bar). Compare basal levels of the activation targets versus repression targets. (B) Myc activation targets that are also regulated by Pc, Pho and Ash1 are methylated at both H3K4 and H3K27. DNA purified from chromatin immunoprecipitation reactions provided the template for PCR of CG16712 (upper) and CG18108 (lower), and the ChIP antibodies used are indicated above each lane. The right two lanes show results from sequential ChIP of chromatin by one antibody and then another. Input chromatin DNA is shown the far left lane, used in a 1:1000 dilution. (C) The enrichment by IP of chromatin containing 6 genes was calculated by dividing the levels of PCR product by that of the background, no antibody control. CG16712, aTub67C and fs(1)N are all Myc induced, Pc, Ash1 and Pho repressed, and show methylation of H3K4 and K3K27 together. Atg8a is not affected by any of the four regulators, though it is highly expressed in the embryo and methylated at H3K4 (blue bar). Cyp309a1 and CG18108 are Myc/Pc repressed, and show both H3K4 and H3K27 methylation in wild type embryos, but not at the same locus by sequential ChIP. (D) A target of activation by Myc is depicted, with low levels of expression, in a domain of chromatin bearing H3K27 methylation and H3K4 methylation. A growth signal or other signal, including increased Myc accumulation, allows Myc/Max to bind to a binding site, recruiting activators and inducing transcription. Ash1 is shown directly or indirectly repelling PcG repression and maintaining an active H3K4 methylation state. (E) A target of Myc repression is depicted, with high levels of expression mediated by an unknown transcription factor (TF). A cellular signal and increased Myc accumulation allow Pho and Pc required repression, propagating a repressed chromatin state characterized by H3K27 methylation.
Mentions: In many of our experiments, Pc, Pho and Ash1 proteins facilitate Myc's activity as a repressor, but also appear to oppose its activity as an activator. In an attempt to understand how the same proteins cooperate with Myc to repress one set of targets and conversely work to repress a different set of Myc activation targets, we have to consider a major difference between Myc's targets of activation and repression: before Myc acts upon them, repression targets are necessarily being expressed while activation targets are largely unexpressed (Figure 8A).

Bottom Line: We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation.We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1.Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. jmgoodli@uncc.edu

ABSTRACT

Background: The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis.

Results: To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1.

Conclusion: Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.

Show MeSH
Related in: MedlinePlus