Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells

Abstract

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation.

Figure 1.

Transcriptome analysis of 12 cell populations. (A) Identification of LEGs, MEGs, and HEGs. Illumina BeadChip arrays were used to analyze RNA from the 12 cell populations. The RNAs were then classified into low expressed genes (0.05 ≤ P-value ≤ 1), middle expressed genes (0 < P-value < 0.05), and highly expressed genes (P-value = 0). Percentages of the LEGs, MEGs, and HEGs are shown relatively to the total number of genes on the Illumina platform for all 12 cell populations. (B) Comparison of different cell populations. All possible pairwise comparisons between the genes in each category (the sets of LEGs, MEGs, and HEGs) were made, and then the percentage overlap was calculated relative to the average number of genes in each category. The upper and lower quartiles of the box plots are the 75^th and 25^th percentiles, respectively. The whisker top and bottom are 90^th and 10^th percentiles, respectively.

Figure 2.

Comparison of repression mechanisms for 12 cell types. The percentage of LEGs (A) or MEGs (B) bound by H3me3K27, POLR2A, H3me3K9, or 5-meC is shown for each of the 12 cell populations. In panel C, box and whisker plots demonstrate the utilization of the four different marks in all 12 cell populations as shown for LEGs, MEGs, and HEGs. The upper and lower quartiles of the box plots are the 75^th and 25^th percentiles, respectively. The whisker top and bottom are 90^th and 10^th percentiles, respectively.

Figure 3.

Promoters are generally repressed by a single mechanism. Shown are pie charts indicating the percentage of LEGs that are bound by only one of the repression marks, by each of the possible different combinations of two marks, or by any three or four marks; also shown is the percentage of LEGs that are not bound by any of the repression marks.

Figure 4.

Cluster analysis of gene ontology categories in the sets of LEGS repressed by different mechanisms. The sets of LEGs for each cell population that were bound by H3me3K27 (A), POLR2A (B), H3me3K9 (C), 5-meC (D), or no marks (E) were analyzed using the DAVID gene ontology program. The functional annotations that passed the cutoff criteria (see Methods) for each cell population were compared by transforming the lists into a binary data set (if a functional category was present, then its P-value was assigned to 1; if it was absent, then its P-value was assigned to 0). We then performed hierarchical cluster analysis and plotted the dendograms in R (http://www.R-project.org) using hclust function.

Figure 5.

Mechanistic analysis of transitioning populations. The subsets of genes that showed expression changes and a >2000 position change in the target gene list for POLR2A, H3me3K27, H3me3K9, or 5-meC were identified by comparing G0/G1 to S phase 3T3 cells and mESc to EBs. For example, an up-regulated gene in S phase or EBs was required to move 2000 positions higher in the POLR2A list of ranked targets or 2000 positions lower in the H3me3K27, H3me3K9, or 5-meC list. Conversely, a down-regulated gene in S phase or EBs was required to move at least 2000 positions lower in the POLR2A list or 2000 positions higher in the H3me3K27, H3me3K9, or 5-meC list. In addition, we eliminated all genes that were not true targets of each mark (but were simply changing positions in the very bottom of the ranked lists) by requiring that the enrichment value be at least 0.7(on a log2 scale) in the appropriate set. For ease of display of the data, we combined the sets of promoters regulated by changes in H3me3K27, H3me3K9, or 5-meC and identified this set as “regulated by changes in chromatin structure.” We also identified the set of genes that showed changes in RNA levels during the G0 to S phase progression or after differentiation of the mES to EB but which were bound by high levels of POLR2A in both cell states (promoters had a >1.0 [on a log2 scale]), enrichment for POLR2A in both cell states, and lacked silencing marks in either cell state (enrichments <0.7 [on a log2 scale] for H3me3K27, H3me3K9, or 5-meC).