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, 6 (10), e26057

Cell-type Independent MYC Target Genes Reveal a Primordial Signature Involved in Biomass Accumulation

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Cell-type Independent MYC Target Genes Reveal a Primordial Signature Involved in Biomass Accumulation

Hongkai Ji et al. PLoS One.

Abstract

The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schema of the strategy to identify the MYC core target gene signature.
Figure 2
Figure 2. Direct Myc target genes in P493-6 B cells defined by ChIP-chip, array-based nuclear run-on, and gene expression changes.
Venn diagram illustrating Myc bound target genes identified with Santa Cruz (SC) or Epitomics (Epit) anti-Myc antibodies. Bound genes whose expression changed are indicated for the overlap between SC and Epit. Enriched gene ontologies for these genes are shown.
Figure 3
Figure 3. Trophoblastic differentiation of H9 hESCs is associated with reduced Myc.
(A) Photomicrographs of H9 hESCs following exposure to BMP4 for the indicated times. Representative images are shown. (B) Flow cytometry of BMP4 treated cells stained for KRT-PE, a marker of trophoblastic differentiation. Immunoblot at right shows that Myc protein levels are drastically reduced in BMP4-treated H9 cells. Tubulin immunoblot shows equal loading of samples. (C) Expression of the indicated stem cell genes and those enriched in trophoblasts was determined by realtime PCR normalized to B-ACTIN.
Figure 4
Figure 4. Myc target genes common to P493-6 B cells and H9 human embryonic stem cells.
(A) Venn diagram of Myc bound genes in P493-6 and H9 hES cells with the overlap containing 80 upregulated genes. The ontology of these shared genes is shown. (B) RT-PCR validation of Myc-bound target genes in P493-6 and H9 cells with anti-Myc or control IgG signals shown. (C) Validation of Myc-bound targets in H9 hESCs by RT-PCR.
Figure 5
Figure 5. Examples of cell type independent, human ES cell and B cell specific Myc targets.
(A) Gene expression levels (at log2 scale) of FBL, LIN28 and BLMH in the absence and presence of Myc induction in P493-6 B cells (measured by Affymetrix Exon arrays and Affymetrix U133 Plus 2.0 arrays), and log2 gene expression fold changes between hESC and trophoblasts (measured by Agilent microarrays) are shown. Error bars correspond to standard deviations of replicate samples. (B) ChIP-chip binding signals in human P493-6 B cells and H9 ES cells, and ENCODE ChIP-seq binding signals in three human cancer cell lines. For ChIP-chip, TileMap moving average statistic m was computed for each probe using normalized log2 probe intensities, and 2m was displayed as the intensity measure. For ChIP-seq, a 100 bp sliding window was used to scan the genome. Read count in each window was shown at a 25 bp step size. E-box motifs CACGTG (black) and CANNTG (red) were mapped to peak regions and are also shown. (C) ChIP-chip and ChIP-seq binding signals in mouse ES cells.
Figure 6
Figure 6. Myc core target gene signature increases with the neoplastic switch to frank lymphoma in vivo.
(A) Heatmap showing expression levels of the 51 Myc core target genes in wild-type, pre-malignant Eμ-Myc (4–6 week old) littermates and of Eμ-Myc lymphoma. (B) Clustering of human molecular Burkitt's lymphoma (mBL) and non-mBL samples using the 51 core target genes. (C) and (D) Heatmaps showing expression levels of B cell restricted (C) or ES cell restricted (D) upregulated Myc target genes in the mouse B cell samples. Green – low expression; Red – high expression.
Figure 7
Figure 7. Correlation of MYC and Myc core target gene signature (MCS) expression among 8129 samples.
(A) Correlation plot of MCS versus MYC expression across 8129 samples on the Affymetrix human U133A platform. Correlation coefficient = 0.47. p-value was determined based on correlation between MYC and randomly chosen genes (see Methods S1 ). Samples from a few cell or tissue types exhibiting patterns of interest are highlighted in color. (B) Correlation plot of MCS versus RXRA expression across 8129 samples on the Affymetrix U133A platform. (C) Total percentage and samples counts of each significantly enriched (FDR<5%) high MYC high MCS cell or tissue type in MYC+ MCS+ samples compared to in all samples. (D) Comparison of total percentage of tumor samples in all of the data (57.7%) to the total percentage of MYC+ MCS+ samples that are tumors (95.9%).

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