To identify TET2-regulated genes, RNA-seq was performed following 48 hours of TET2 knockdown in asynchronous MCF7 cells. This revealed repression of 2,269 genes and activation of 2,144 genes (p ≤ 0.05) (Figure 4.4). GATA3 mRNA expression was unaffected by TET2 silencing. ER mRNA expression was modestly repressed, although proteomic analysis after 72 hours of TET2 knockdown (Figure 4.3) suggested that this does not translate into an effect on total protein levels. To compare the gene regulatory programme of TET2 with those of ER and GATA3, RNA-seq was also performed 48 hours after knockdown of either ER or GATA3 in MCF7 cells. ER and GATA3 mRNA levels were robustly depleted, with ER transcripts reduced to 11% of control levels and GATA3 transcripts reduced to 20% of control levels by their respective silencing. As expected, TET2 mRNA levels were robustly and significantly repressed by both ER and GATA3 knockdown (to 23% of control levels by ER knockdown, and 43% of control levels by GATA3 knockdown). Overall, 60% of the genes significantly regulated by TET2 knockdown (2,656 out of 4,413 genes, p ≤ 0.05) were also significantly regulated by both GATA3 and ER silencing. Given that TET2 appears to behave as a target gene of both Figure 4.3. TET2 knockdown significantly depletes TET2 protein levels in MCF7 cells, with no effect on total ER or GATA3 protein levels.
MCF7 cells were treated with an siRNA pool (10 nM) targeting TET2 (siTET2) or a non- targeting control (siNT). Cells were harvested at 72 hours after transfection and full proteome analysis performed. Four replicates of each condition were included in an 11plex TMT MS run. Results represent mean ± SD protein intensity, the aggregate of intensities of the individual unique peptides identified for each protein. **** = p ≤ 0.0001.
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ER and GATA3, some of the concordance between the ER, GATA3 and TET2 gene regulatory profiles is therefore likely to be due to secondary effects as a result of TET2 depletion after ER and GATA3 silencing. Taking the 500 most induced and 500 most repressed genes in response to TET2 silencing (in terms of log2 fold change), 60% of these most differentially regulated genes were modulated in the same direction following ER knockdown (visually depicted in the heatmaps in Figure 4.5A). This included repression of key ER target genes such as PGR, CCND1, XBP1, and CXCL12. Importantly, ER/TET2 shared binding sites were enriched adjacent to TET2 target genes (Figure 4.6), further implying genomic co-operation between these two proteins. Interestingly, similar enrichment of ER/TET2 shared sites was observed both at genes induced by TET2 knockdown and those repressed by TET2 knockdown, implying that TET2 may be capable of both activating and repressive transcriptional activity as part of the ER complex. However, more detailed functional analysis would be required to confirm this observation.
To assess whether the genes regulated by TET2 show a tendency towards modulation of any particular pathway, gene ontology (GO) analysis was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery) (Huang et al. 2007; Huang et al. 2009). Genes repressed by TET2 knockdown showed significant enrichment of six functional categories linked to cell division and cell cycle processes (Figure 4.5B). In contrast, the genes induced by TET2 knockdown demonstrated significant enrichment of only two functional categories, linked to cell communication and signal transduction. When examining solely the TET2-regulated genes that also changed in response to GATA3 and ER knockdown, enrichment of the same functional categories was observed (Figure 4.5C). This demonstrates that the pathways most strongly affected by loss of TET2 in these cells are those converged on by GATA3 and ER regulatory processes, and are related to the cell cycle and proliferation. To place these findings into a functional setting, proliferation of MCF7 cells was assessed over a 4-day period after TET2 knockdown. Consistent with repression of cell cycle gene programs, MCF7 growth was inhibited by TET2 knockdown (Figure 4.7).
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Figure 4.4. TET2 knockdown affects gene expression in MCF7 cells.
siRNA-mediated TET2 silencing (48 hours) resulted in repression of 2,269 genes and activation of 2,144 genes compared to control (n=6, p ≤ 0.05, significantly regulated genes highlighted in red).
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Figure 4.5. TET2 depletion affects gene pathways related to the cell cycle.
A) Heatmaps depict the top 500 induced and top 500 repressed TET2-regulated genes according to log2 fold change. Colour scale represents the relative expression (z-score) of genes across the two conditions (control and knockdown), calculated separately within each comparison (siTET2 vs siNT, siESR1 vs siNT and siGATA3 vs siNT). Hierarchical clustering of genes in the leftmost (siTET2) heatmap is preserved across all three heatmaps. Columns represent independent biological replicates (n=6). B) Barplot displaying –log10(FDR) for GO analysis of the top 500 induced and top 500 repressed TET2-regulated genes according to log2 fold change. Only categories with FDR ≤ 0.05 (threshold indicated by dotted line) are shown. C) Barplot displaying –log10(FDR) for GO analysis of the top 500 induced and top 500 repressed TET2-regulated genes according to log2 fold change, sub-selected from genes also significantly (p ≤ 0.05) regulated by both GATA3 and ER silencing. The top 6 enriched categories are shown for repressed genes, and the top 2 for induced genes. Dotted line indicates FDR 0.05. Enriched processes were identified using the Biological Process category level 3 of the GO hierarchy (GOTERM_BP_3).
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Figure 4.6. ER/TET2 shared sites are enriched in the vicinity of TET2-regulated genes compared to unchanging genes.
Graph shows the cumulative fraction of total ER/TET2 shared binding sites (n = 15,442, MCF7 cells) within up to 100 kb of the TSSs of three groups of genes: genes upregulated by TET2 knockdown (n = 2,144, red line), genes downregulated by TET2 knockdown (n = 2,269, blue line) (p ≤ 0.05), and genes unchanging in response to TET2 knockdown (constant genes, grey lines). Constant genes were randomly selected from those with p > 0.5 and mean expression > 1.0. Grey lines indicate analysis based on constant genes: the dotted line indicates analysis matched to the number of downregulated genes, and the solid line indicates analysis matched to number of upregulated genes.
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Figure 4.7. TET2 knockdown inhibits MCF7 cell growth.
Cell growth is expressed as mean ± SD of % confluence, assessed using the Incucyte Zoom™ system. Knockdown was performed at t = 0; cells were seeded 24 hours prior to this. Data points represent mean ± SD of ≥ 6 technical replicates (wells). Results for each biological replicate are plotted separately. ** = p ≤ 0.01, *** = p ≤ 0.001. siTET2 = TET2 knockdown, siER = ER knockdown, siNT = non-targeting control-treated cells. A), B) and C) show growth experiment replicates 1 to 3 demonstrating the effect of TET2 knockdown on growth of MCF7 cells. D) Barplot showing confluence of TET2 knockdown cells as a % of the confluence of corresponding control cells at 24, 48, 72 and 96 hours after treatment. E) Growth experiment demonstrating the effect of ER knockdown on MCF7 growth. F) Barplot showing confluence of ER knockdown cells as a % of the confluence of corresponding control cells at 24, 48, 72 and 96 hours after treatment.
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