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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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A P element insertion in the dmyc locus is a reporter for dmyc activity. (A) A diagram indicating the exons (green rectangles) and introns (thick blue line) of the dmyc gene on the X chromosome. A pGT1 insertion in the second intron (dmycBG02383, top) inserts the Gal4 (blue rectangle) and mini-white (red rectangle) reporters. A second insertion upstream of the dmyc transcription site is also shown (dmycBG00605, bottom) (B) Gal4 and mini-white reporters exhibit dmyc autorepression. RT-PCR amplification products are shown from embryos with dmycBG02383 having Gal4 or Gal4; UAS-dmyc transgenes, and therefore wild type for Myc (wt) or producing ectopic Myc (Myc++), respectively. The numbers under each band indicate the relative band intensities between pairs as indicated by phosphorimaging. (C) Scheme for genetic screen for modifiers of dmyc activity. Females homozygous for dmycBG02383 on the X Chromosome were crossed to males of the Second and Third Chromosome deficiency "kits" (Bloomington). Fly heads are depicted in profile, cartooning the changes in eye color expected when a fly has both dmycBG02383 and either a mutation in a dmyc activator (upper right) or a mutation in a dmyc repressor (lower right).
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Figure 1: A P element insertion in the dmyc locus is a reporter for dmyc activity. (A) A diagram indicating the exons (green rectangles) and introns (thick blue line) of the dmyc gene on the X chromosome. A pGT1 insertion in the second intron (dmycBG02383, top) inserts the Gal4 (blue rectangle) and mini-white (red rectangle) reporters. A second insertion upstream of the dmyc transcription site is also shown (dmycBG00605, bottom) (B) Gal4 and mini-white reporters exhibit dmyc autorepression. RT-PCR amplification products are shown from embryos with dmycBG02383 having Gal4 or Gal4; UAS-dmyc transgenes, and therefore wild type for Myc (wt) or producing ectopic Myc (Myc++), respectively. The numbers under each band indicate the relative band intensities between pairs as indicated by phosphorimaging. (C) Scheme for genetic screen for modifiers of dmyc activity. Females homozygous for dmycBG02383 on the X Chromosome were crossed to males of the Second and Third Chromosome deficiency "kits" (Bloomington). Fly heads are depicted in profile, cartooning the changes in eye color expected when a fly has both dmycBG02383 and either a mutation in a dmyc activator (upper right) or a mutation in a dmyc repressor (lower right).

Mentions: Pc binds to methylated histone H3 at lysine 27 (H3K27), mediating repression of many genetic loci by Myc [18]. In an investigation of other PcG gene products potentially involved in this repression by Myc and Pc, we chose three candidates to test: Posterior sex combs (Psc), which is in a core repressive complex with Pc [34]; E(z), which is a histone methyltransferase that methylates histone H3 lysine 27 and recruits Pc repression [35-37]; and Pho, which is one of two DNA-binding proteins of the group and recruits E(z) [28,30-32]. Our strategy for testing Psc, E(z) and Pho involved a genetic test for modification of expression at the dmyc locus. Each of two different P element insertions in the dmyc locus, dmycBG02383 and dmycBG00605, provides two reporters for dmyc expression: a promoterless yeast Gal4 gene that is expressed by the upstream dmyc promoter, and a Drosophila eye color mini-white gene whose expression is influenced by the regulation of the locus (Figure 1A) [38,39].


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)

A P element insertion in the dmyc locus is a reporter for dmyc activity. (A) A diagram indicating the exons (green rectangles) and introns (thick blue line) of the dmyc gene on the X chromosome. A pGT1 insertion in the second intron (dmycBG02383, top) inserts the Gal4 (blue rectangle) and mini-white (red rectangle) reporters. A second insertion upstream of the dmyc transcription site is also shown (dmycBG00605, bottom) (B) Gal4 and mini-white reporters exhibit dmyc autorepression. RT-PCR amplification products are shown from embryos with dmycBG02383 having Gal4 or Gal4; UAS-dmyc transgenes, and therefore wild type for Myc (wt) or producing ectopic Myc (Myc++), respectively. The numbers under each band indicate the relative band intensities between pairs as indicated by phosphorimaging. (C) Scheme for genetic screen for modifiers of dmyc activity. Females homozygous for dmycBG02383 on the X Chromosome were crossed to males of the Second and Third Chromosome deficiency "kits" (Bloomington). Fly heads are depicted in profile, cartooning the changes in eye color expected when a fly has both dmycBG02383 and either a mutation in a dmyc activator (upper right) or a mutation in a dmyc repressor (lower right).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1887537&req=5

Figure 1: A P element insertion in the dmyc locus is a reporter for dmyc activity. (A) A diagram indicating the exons (green rectangles) and introns (thick blue line) of the dmyc gene on the X chromosome. A pGT1 insertion in the second intron (dmycBG02383, top) inserts the Gal4 (blue rectangle) and mini-white (red rectangle) reporters. A second insertion upstream of the dmyc transcription site is also shown (dmycBG00605, bottom) (B) Gal4 and mini-white reporters exhibit dmyc autorepression. RT-PCR amplification products are shown from embryos with dmycBG02383 having Gal4 or Gal4; UAS-dmyc transgenes, and therefore wild type for Myc (wt) or producing ectopic Myc (Myc++), respectively. The numbers under each band indicate the relative band intensities between pairs as indicated by phosphorimaging. (C) Scheme for genetic screen for modifiers of dmyc activity. Females homozygous for dmycBG02383 on the X Chromosome were crossed to males of the Second and Third Chromosome deficiency "kits" (Bloomington). Fly heads are depicted in profile, cartooning the changes in eye color expected when a fly has both dmycBG02383 and either a mutation in a dmyc activator (upper right) or a mutation in a dmyc repressor (lower right).
Mentions: Pc binds to methylated histone H3 at lysine 27 (H3K27), mediating repression of many genetic loci by Myc [18]. In an investigation of other PcG gene products potentially involved in this repression by Myc and Pc, we chose three candidates to test: Posterior sex combs (Psc), which is in a core repressive complex with Pc [34]; E(z), which is a histone methyltransferase that methylates histone H3 lysine 27 and recruits Pc repression [35-37]; and Pho, which is one of two DNA-binding proteins of the group and recruits E(z) [28,30-32]. Our strategy for testing Psc, E(z) and Pho involved a genetic test for modification of expression at the dmyc locus. Each of two different P element insertions in the dmyc locus, dmycBG02383 and dmycBG00605, provides two reporters for dmyc expression: a promoterless yeast Gal4 gene that is expressed by the upstream dmyc promoter, and a Drosophila eye color mini-white gene whose expression is influenced by the regulation of the locus (Figure 1A) [38,39].

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