A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs

Abstract

c-Myc (Myc) is an important transcriptional regulator in embryonic stem (ES) cells, somatic cell reprogramming, and cancer. Here, we identify a Myc-centered regulatory network in ES cells by combining protein-protein and protein-DNA interaction studies and show that Myc interacts with the NuA4 complex, a regulator of ES cell identity. In combination with regulatory network information, we define three ES cell modules (Core, Polycomb, and Myc) and show that the modules are functionally separable, illustrating that the overall ES cell transcription program is composed of distinct units. With these modules as an analytical tool, we have reassessed the hypothesis linking an ES cell signature with cancer or cancer stem cells. We find that the Myc module, independent of the Core module, is active in various cancers and predicts cancer outcome. The apparent similarity of cancer and ES cell signatures reflects, in large part, the pervasive nature of Myc regulatory networks.

Figure 1. Myc-centered protein-protein interaction network in ES cells

(A) Schematic representation of the strategy for mapping a Myc-centered protein–protein interaction network in ES cells. High-confidence components of multiprotein complexes were identified and listed in the table. Pink cells represent NuA4 complex proteins. (B) Depiction of the features of the Myc-centered protein-protein interaction network. Proteins with green labels are biotin tagged proteins and pink circles indicate NuA4 complex proteins Proteins identified by multiple biotin tagged factors are shown. Entire protein interaction network is shown in Figure S1C. See also Table S1. (C) Validation of the interaction network by co-immunoprecipitation.

Figure 2. Myc-centered protein-DNA interaction network in ES cells

(A) Representative view of Myc, Max, Dmap1, Tip60, E2F4, and Gcn5 occupancy at the target loci. (B) Number of target promoters bound by each factor or associated with each histone modification signature. Blue represents factors or histone signatures involved in PrC complexes. Red represents factors involved in ES cell core factors, and Green represents Myc and Myc-related factors or histone signatures (D–E). See also Figure S2 and Table S2. (C) Relative position of chromosomal target loci of each factor in the Myc cluster (upper panel) and Core cluster (bottom panel) shown in (B) and (C) to the TSS. (D) Target correlation map: The degree of target co-occupancy of each pair of factors (either transcription factor or histone modification signature) is shown. Yellow indicates more frequent colocalization of each pair of factors. (E) Median distance map: Median distances between the loci co-occupied by two tested factors (except PrC complex proteins) shown in (D). Yellow indicates closer colocalization.

Figure 3. Histone modification signatures on the target promoters of Myc cluster proteins

(A) Histone marks on the target promoters of each factor in the Myc cluster. “All” represents all promoters. (B) Histone marks and target co-occupancy of 7 factors in Myc cluster (Myc, Max, nMyc, Dmap1, E2F1, E2F4, and Zfx).

Figure 4. ES cell modules

(A) Gene expression profiles (log₂, left y-axis) upon J1 ES cell differentiation (wild type ES cells: ES, differentiated ES cells for 14 days: dES day 14) are shown as moving window averaged lines (ES; red line, dES day 14; blue line, bin size 100 and step size 1). Randomized genes are sorted (x-axis) by the target co-occupancy of 7 factors in the Myc cluster (right y-axis). Black bar represents target genes co-occupied by at least 6 factors among the 7 factors in the Myc cluster. (B) Enrichment of KEGG pathways. All Myc target genes (gray bars, total 3733 genes) and genes co-occupied by at least 6 factors among the 7 factors tested marked by black bar in (A) (black bars, total 1756 genes). See also Figure S3A, S3B and Table S3. (C) ES cell modules: Three ES cell modules are defined based on the target co-occupancy within each cluster shown in Figure 2D. See also Table S3. (D) GSEA analyses show the gene activity of the three ES cell modules (Core, PrC and Myc modules) as well as the previously defined ESC-like module (Wong et al. 2008a) upon ES cell differentiation (wild type ES cells; ES vs. 14 days differentiated ES cells; dES). (E) Average gene expression values (log₂) of each module (C) are tested upon ES cell differentiation (ES day0, dES day2, dES day7, and dES day14, respectively). Data are represented as mean ± SEM. See also Figure S2B and Figure S3C.

Figure 5. Module activity in various cells

(A) Average gene expression values (log₂) (Sridharan et al., 2009) of ES cell modules (Core, Prc and Myc) and previously defined ESC-like module are tested in ES cells (ES), iPS cells (iPS), MEFs (MEF), and partial iPS cells (piPS). See also Figure S3D. (B) Average gene expression values (log₂) (Bild et al., 2006) of each module tested in (A) upon induction of Myc in human epithelial cells (Myc induction) and in control cells (WT). Human orthologues of genes in three ES cell modules are tested (listed in Table S3) and data are represented as mean ± SEM (A–B).

Figure 6. ES cell modules in mouse MLL myeloid leukemia models

(A) Average gene expression values (log₂) of ES cell modules and the previously defined ESC-like module are tested in various mouse models of acute myeloid leukemia (AML) initiated by MLL-AF9, MLL-ENL, MLL-AF10, MLL-AF1p, and MLL-GAS7 (Somervaille et al., 2009). (B) Average gene expression values (log₂) of each module are tested in a c-kit high MLL-AF10 leukemia cell population (MLL-AF10 c-kit high) and a c-kit low MLL-AF10 leukemia cell population (MLL-AF10 c-kit low) (Somervaille et al., 2009). Data are represented as mean ± SEM (A–B). See also Figure S4.

Figure 7. ES cell modules in human cancers

(A–B) Average gene expression values (log₂) of ES cell modules and previously defined ESC-like module are tested in human bladder carcinoma samples including superficial (SUP), and invasive carcinomas (INV), as well as normal urothelium (NU) as a control group (marked by black bars) (Sanchez-Carbayo et al., 2006). Each column represents one patient sample (total 157 samples) (A). Averaged module activities within the sample group (NU, INV, and SUP) (B). Data represented as mean ± SEM. (C–E) Average gene expression values (log₂) of ES cell Core (C) and Myc (D) module are tested from 97 human breast cancer patient samples (van 't Veer et al., 2002). (C) Core module activities were calculated, and top and bottom 20% of samples (19 samples each) were further analyzed. Bar graph represents the corresponding interval to metastases (months, bottom panel). (D) Samples showing top and bottom 20% of Myc module activity were further analyzed. Bar graph represents the corresponding interval to metastases (months) for each patient (bottom panel). (E) For each tested group (C–D), interval to distant metastases is calculated as mean ± SEM, and p-values are from Student’s T-tests. See also Figure S5.