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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in <it>Drosophila</it>. 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 <it>ash1 </it>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 <it>Pc </it>and <it>pho </it>transcripts, as determined by concurrent microarray analysis of <it>Pc </it>and <it>pho </it>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.</p> <p>Conclusion</p> <p>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.</p

Myc Localizes to Histone Locus Bodies during Replication in Drosophila

Myc is an important protein at the center of multiple pathways required for growth and proliferation in animals. The absence of Myc is lethal in flies and mice, and its over-production is a potent inducer of over-proliferation and cancer. Myc protein is localized to the nucleus where it executes its many functions, however the specific sub-nuclear localization of Myc has rarely been reported. The work we describe here began with an observation of unexpected, punctate spots of Myc protein in certain regions of Drosophila embryos. We investigated the identity of these puncta and demonstrate that Myc is co-localized with coilin, a marker for sub-nuclear organelles known as Cajal Bodies (CBs), in embryos, larvae and ovaries. Using antibodies specific for U7 snRNP component Lsm11, we show that the majority of Myc and coilin co-localization occurs in Histone Locus Bodies (HLBs), the sites of histone mRNA transcription and processing. Furthermore, Myc localizes to HLBs only during replication in mitotic and endocycling cells, suggesting that its role there relates to replication-dependent canonical histone gene transcription. These results provide evidence that sub-nuclear localization of Myc is cell-cycle dependent and potentially important for histone mRNA production and processing

Su(z)2 antagonizes auto-repression of Myc in Drosophila, increasing Myc levels and subsequent trans-activation.

All tumor cell lines that have been tested are defective for Myc auto-repression, and have high levels of Myc produced from wild type loci and re-arranged loci. Like mammalian Myc auto-repression, Myc protein represses the expression of its gene, dmyc, in Drosophila. This activity requires Polycomb (Pc), since RNAi for Pc in the embryo eliminates Myc auto-repression. We have observed that upon depletion of Polycomb in the embryo, levels of one of 18 different chromatin-binding genetic regulators, Su(z)2, rise dramatically. We pursued the possibility that increased levels of this protein, Su(z)2, interfere with Myc auto-repression, potentially explaining the loss of auto-repression upon Pc RNAi. We report that embryos expressing both ectopic Myc and ectopic Su(z)2 fail in Myc auto-repression. Surprisingly, histone H3K27 tri-methylation at the dmyc locus is inversely correlated with the presence of auto-repression. We show phenotypic consequences of potent dmyc auto-repression, and their complete reversal by ectopic Su(z)2: dmyc auto-repression induced a diminutive (dm) phenotype, and upon elimination of auto-repression by Su(z)2, overall levels of Myc increased and completely rescued the phenotype. We show that this increase in Myc levels caused dramatic activation of Myc activation targets. These data suggest that Su(z)2 is capable of increasing the potency of Myc activity by eliminating Myc's feedback regulation by auto-repression. Although Su(z)2 eliminated Myc auto-repression, we found that Myc repression of other genes is not affected by Su(z)2. These data suggest a unique antagonistic role for Su(z)2 in Myc auto-repression, and a potential mechanism for cancer-cell specific loss of Myc auto-repression

Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila

Aberrant accumulation of the Myc oncoprotein propels proliferation and induces carcinogenesis. In normal cells, however, an abundance of Myc protein represses transcription at the c-myc locus. Cancer cells often lose this autorepression. We examined the control of myc in Drosophila and show here that the Drosophila ortholog, dmyc, also undergoes autorepression. We find that the developmental repressor Polycomb (Pc) is required for dmyc autorepression, and that this Pc-dMyc-mediated repression spreads across an 875-kb region encompassing the dmyc gene. To further investigate the relationship between Myc and Polycomb, we used microarrays to identify genes regulated by each, and identify a striking relationship between the two: A large set of dMyc activation targets is normally repressed by Pc, and 73% of dMyc repression targets require Pc for this repression. Chromatin immunoprecipitation confirmed that many dMyc-Pc-repressed loci have an epigenetic mark recognized by Pc. Our results suggest a novel relationship between Myc and Polycomb, wherein Myc enhances Polycomb repression in order to repress targets, and Myc suppresses Polycomb repression in order to activate targets

dmyc auto-repression reduces total Myc protein levels compared to wild type, which is rescued by combining ectopic Myc with ectopic Su(z)2 expression.

<p>Similarly aged embryos of four genotypes are beside one another, with the genotypes of embryos indicated above each column. Anti-Myc staining is green.</p

Increased Su(z)2 provides for activation of Myc targets, and a reduction in their H3K27 tri-methylation.

<p>A) RT-PCR analysis showing expression of three Myc activation targets (indicated on the left side of the gel pictures) in embryos of 4 different genotypes (indicated above the lanes). Average band intensities (Quantity 1) of biological triplicates are plotted on the right, with relative standard deviations indicated. B) ChIP assay showing histone H3K27 tri-methylation at five Myc activation targets (indicated to the left of the gel pictures) in embryos of genotypes indicated on top. Data shown on the left are plotted in a stacked column chart; the y-axis is the density of each PCR product divided by the density of the input sample PCR product, and the values for each gene are stacked together for each of the four genotypes. These data show a representative set of biological triplicates.</p

Pc RNAi results in an increase in Su(z)2 levels.

<p>A) We suggest two possible scenarios for the role of Pc in Myc auto-repression: Pc is either directly involved with Myc protein in repression (left), or required to repress a third player, gene Y (right), whose protein product interferes with Myc repression (right). B) A candidate for gene Y. See Goodliffe et al., 2007, for microarray data generation. Log₂ ratios of changes in levels of 19 PcG and TrxG transcripts are shown, compared to wild type levels (Gal4), in embryos with ectopic Myc (blue), ectopic Myc plus Pc RNAi (red) and ectopic Myc plus pho RNAi (green).</p

Su(z)2 disrupts auto-repression by Myc.

<p>A) RT-PCR analysis of endogenous, ectopic and total Myc expression in embryos of four different genotypes, which are indicated above each lane (Gal4 = arm-Gal4, Gal4-Myc = armGal4; UAS dmyc, Gal4-Su(z)2 = armGal4; Su(z)2XP, Gal4-Myc-Su(z)2 = armGal4; Su(z)2XP; UAS dmyc). A 0–21 hour collection of embryos was used for RNA isolation and for all subsequent assays. Ras64B was used as a loading control. B) A chart showing endogenous dmyc expression, the average of biological triplicates is plotted with standard deviations indicated for four genotypes of embryos. Expression was quantified using Quantity 1 (Bio-Rad). The blue line denotes a statistically significant change in endogenous dmyc levels from Gal4-Myc to Gal4-Myc-Su(z)2. C) Living larvae of the genotypes shown, all grown at low density, aged 4 days after egg laying at room temperature, and photographed simultaneously.</p

Ectopic Su(z)2 does not interfere with Myc repression of targets other than dmyc.

<p>A) A representative gel of RT-PCR data showing the expression of six Myc targets of repression in four genotypes, as indicated above each column of bands. Total dmyc expression and Ras64B expression, a loading and RNA level control, are shown in the bottom two panels. B) The average band intensities indicating levels of expression and relative standard deviation are plotted for the 8 genes shown in A. Genotypes are indicated along the X axis, and the Y axis shows band intensities as quantified by Quantity 1 (Bio-Rad). C) ChIP results showing H3K27 tri-methylation of three of the 8 genes shown in A, in the 4 genotypes of the experiment. Ras64B is a negative control. These data show a representative picture of biological triplicates for all 8 genes.</p

MicroRNA miR-308 regulates dMyc through a negative feedback loop in Drosophila

Summary The abundance of Myc protein must be exquisitely controlled to avoid growth abnormalities caused by too much or too little Myc. An intriguing mode of regulation exists in which Myc protein itself leads to reduction in its abundance. We show here that dMyc binds to the miR-308 locus and increases its expression. Using our gain-of-function approach, we show that an increase in miR-308 causes a destabilization of dMyc mRNA and reduced dMyc protein levels. In vivo knockdown of miR-308 confirmed the regulation of dMyc levels in embryos. This regulatory loop is crucial for maintaining appropriate dMyc levels and normal development. Perturbation of the loop, either by elevated miR-308 or elevated dMyc, caused lethality. Combining elevated levels of both, therefore restoring balance between miR-308 and dMyc levels, resulted in lower apoptotic activity and suppression of lethality. These results reveal a sensitive feedback mechanism that is crucial to prevent the pathologies caused by abnormal levels of dMyc