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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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Ash1 works against Pc/Pho repression to facilitate Myc activation. (A) Changes in expression of 77 genes are shown, log2 of ratios of expression over basal, for transcripts whose levels increase with ectopic Myc (blue bars). For each of the 77 genes, changes in expression are also shown in embryos with ectopic Myc and ash1 RNAi (red bars, next to the blue bar for each gene). (B) A graph showing the expression of 60 genes in response to eight genetic conditions, as clustered by K-means. The log10 scale of normalized expression for each gene is represented as a vertical line, and along the X axis left to right, the 60 are grouped by sample: Gal4 alone, Gal4 Myc, Gal4 Pc RNAi, Gal4 Myc Pc RNAi, Gal4 ash1 RNAi, Gal4 Myc ash1 RNAi, Gal4 pho RNAi, Gal4 Myc pho RNAi. These Myc activated genes fail to be activated with ash1 RNAi (compare 2nd and 6th sets), and they are also repressed by Pc and Pho (compare 1st, 3rd and 7th sets). (C) A Model suggesting a possible regulation scheme for the genes shown in B, "X".
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Figure 7: Ash1 works against Pc/Pho repression to facilitate Myc activation. (A) Changes in expression of 77 genes are shown, log2 of ratios of expression over basal, for transcripts whose levels increase with ectopic Myc (blue bars). For each of the 77 genes, changes in expression are also shown in embryos with ectopic Myc and ash1 RNAi (red bars, next to the blue bar for each gene). (B) A graph showing the expression of 60 genes in response to eight genetic conditions, as clustered by K-means. The log10 scale of normalized expression for each gene is represented as a vertical line, and along the X axis left to right, the 60 are grouped by sample: Gal4 alone, Gal4 Myc, Gal4 Pc RNAi, Gal4 Myc Pc RNAi, Gal4 ash1 RNAi, Gal4 Myc ash1 RNAi, Gal4 pho RNAi, Gal4 Myc pho RNAi. These Myc activated genes fail to be activated with ash1 RNAi (compare 2nd and 6th sets), and they are also repressed by Pc and Pho (compare 1st, 3rd and 7th sets). (C) A Model suggesting a possible regulation scheme for the genes shown in B, "X".

Mentions: We were curious about the inverse, whether Ash1 is required for activation by Myc. Levels of 680 genes increase upon activation of ectopic Myc. In embryos with both ectopic Myc and ash1 RNAi, levels of 76 of these genes fail to increase at the same level (Figure 7A). None of these 76 genes is also regulated by Ash1 alone, suggesting that Ash1's regulation of these genes is limited to activation by Myc. Upon closer examination of the 76 genes, we found a cluster of 60 that are regulated by Pc and Pho (Figure 7B, generated by K-means clustering of Myc activation targets). The regulation of these genes by Pc and Pho is negative, in that the targets have elevated expression with Pc or Pho RNAi (Figure 7B). Therefore, many of these targets are normally repressed by Pc/Pho. Among the named genes in this group, several encode structural constituents, such as Ccp84Ac (larval cuticle), Dhc62B (Dynein Heavy chain 62B) and Actin 88F, and some are involved in metabolism, such as Glycogenin and Lipase1. One possible explanation for the regulation of these Myc targets is that the role of Ash1 is to oppose Pc/Pho repression, allowing Myc activation. In fact, Ash1 opposes Pc repression at homeotic loci [47]. Therefore, our data suggest that Ash1 also opposes Pc repression at loci activated by Myc (Figure 7C).


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)

Ash1 works against Pc/Pho repression to facilitate Myc activation. (A) Changes in expression of 77 genes are shown, log2 of ratios of expression over basal, for transcripts whose levels increase with ectopic Myc (blue bars). For each of the 77 genes, changes in expression are also shown in embryos with ectopic Myc and ash1 RNAi (red bars, next to the blue bar for each gene). (B) A graph showing the expression of 60 genes in response to eight genetic conditions, as clustered by K-means. The log10 scale of normalized expression for each gene is represented as a vertical line, and along the X axis left to right, the 60 are grouped by sample: Gal4 alone, Gal4 Myc, Gal4 Pc RNAi, Gal4 Myc Pc RNAi, Gal4 ash1 RNAi, Gal4 Myc ash1 RNAi, Gal4 pho RNAi, Gal4 Myc pho RNAi. These Myc activated genes fail to be activated with ash1 RNAi (compare 2nd and 6th sets), and they are also repressed by Pc and Pho (compare 1st, 3rd and 7th sets). (C) A Model suggesting a possible regulation scheme for the genes shown in B, "X".
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1887537&req=5

Figure 7: Ash1 works against Pc/Pho repression to facilitate Myc activation. (A) Changes in expression of 77 genes are shown, log2 of ratios of expression over basal, for transcripts whose levels increase with ectopic Myc (blue bars). For each of the 77 genes, changes in expression are also shown in embryos with ectopic Myc and ash1 RNAi (red bars, next to the blue bar for each gene). (B) A graph showing the expression of 60 genes in response to eight genetic conditions, as clustered by K-means. The log10 scale of normalized expression for each gene is represented as a vertical line, and along the X axis left to right, the 60 are grouped by sample: Gal4 alone, Gal4 Myc, Gal4 Pc RNAi, Gal4 Myc Pc RNAi, Gal4 ash1 RNAi, Gal4 Myc ash1 RNAi, Gal4 pho RNAi, Gal4 Myc pho RNAi. These Myc activated genes fail to be activated with ash1 RNAi (compare 2nd and 6th sets), and they are also repressed by Pc and Pho (compare 1st, 3rd and 7th sets). (C) A Model suggesting a possible regulation scheme for the genes shown in B, "X".
Mentions: We were curious about the inverse, whether Ash1 is required for activation by Myc. Levels of 680 genes increase upon activation of ectopic Myc. In embryos with both ectopic Myc and ash1 RNAi, levels of 76 of these genes fail to increase at the same level (Figure 7A). None of these 76 genes is also regulated by Ash1 alone, suggesting that Ash1's regulation of these genes is limited to activation by Myc. Upon closer examination of the 76 genes, we found a cluster of 60 that are regulated by Pc and Pho (Figure 7B, generated by K-means clustering of Myc activation targets). The regulation of these genes by Pc and Pho is negative, in that the targets have elevated expression with Pc or Pho RNAi (Figure 7B). Therefore, many of these targets are normally repressed by Pc/Pho. Among the named genes in this group, several encode structural constituents, such as Ccp84Ac (larval cuticle), Dhc62B (Dynein Heavy chain 62B) and Actin 88F, and some are involved in metabolism, such as Glycogenin and Lipase1. One possible explanation for the regulation of these Myc targets is that the role of Ash1 is to oppose Pc/Pho repression, allowing Myc activation. In fact, Ash1 opposes Pc repression at homeotic loci [47]. Therefore, our data suggest that Ash1 also opposes Pc repression at loci activated by Myc (Figure 7C).

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