Skip to main content
. 2014 Mar 12;3:e02313. doi: 10.7554/eLife.02313

Figure 3. Activated KRAS-mediated upregulation of ZNF304 is required for transcriptional silencing of INK4-ARF.

(A) qRT-PCR analysis monitoring INK4A-ARF expression in DLD-1 cells expressing a NS or KRAS shRNA. (B) ChIP analysis monitoring binding of ZNF304, KAP1, SETDB1, and DNMT1 to INK4-ARF promoters in DLD-1 cells expressing a NS or KRAS shRNA. (C) qRT-PCR analysis monitoring INK4A-ARF expression in DLD-1 cells treated with DMSO or manumycin A (Man. A). The results were normalized to DMSO, which was set to 1. (D) ChIP analysis monitoring binding of ZNF304, KAP1, SETDB1, and DNMT1 to INK4-ARF promoters in DLD-1 cells treated with DMSO or Man. A. (E) Immunoblot analysis showing INK4-ARF levels in DLD-1 cells treated with a NS or KRAS shRNA, or DMSO or Man. A. (F) Immunoblot analysis showing INK4-ARF levels in DLD-1 cells treated with DMSO, LY294002, or PI-103. (G) qRT-PCR analysis monitoring ZNF304 expression in DLD-1 cells treated with a NS or KRAS shRNA, or DMSO or Man. A. (H) Immunoblot analysis showing ZNF304 levels in DLD-1 cells treated with Man. A for 24 hr and 0–10 µM MG-132 for 4 hr. (I) PAT-ChIP analysis monitoring binding of ZNF304 to INK4-ARF promoters in matched adjacent normal (N) and KRAS-positive CRC human tumor (T) samples. Results were normalized to normal samples, which were set to 1. Data are represented as mean ± SD. *p≤0.05, **p≤0.01. Results from experiments validating KRAS knockdown efficiency and the role of KRAS in repressing p14ARF expression, as well as experiments validating the role of ZNF304 and its corepressors in INK4-ARF silencing in other KRAS-positive CRC cell lines, are presented in Figure 3—figure supplements 1–4.

DOI: http://dx.doi.org/10.7554/eLife.02313.009

Figure 3.

Figure 3—figure supplement 1. Validation of a role for KRAS in p14ARF transcriptional silencing in DLD-1 cells.

Figure 3—figure supplement 1.

(A and B) Validation of KRAS shRNA knockdown efficiency. qRT-PCR analysis monitoring KRAS expression in DLD-1 cells stably expressing a NS or KRAS shRNA (A) or a second, unrelated KRAS shRNA (B). Values are given relative to expression of KRAS following treatment with a NS shRNA, which was set to 1. (C) qRT-PCR analysis monitoring p14ARF expression in DLD-1 cells stably expressing a second, unrelated KRAS shRNA. Values are given relative to expression of p14ARF following treatment with a NS shRNA, which was set to 1. Data are represented as mean ± SD. *p≤0.05, **p≤0.01.
DOI: http://dx.doi.org/10.7554/eLife.02313.010
Figure 3—figure supplement 2. ZNF304 and its corepressors bind to the INK4-ARF promoters in other KRAS-positive human CRC cell lines.

Figure 3—figure supplement 2.

ChIP analysis monitoring binding of ZNF304, KAP1, SETDB1, and DNMT1 at the promoters of p14ARF, p15INK4B, and p16INK4A in HCT116 (left) and HCT15 (right) cells. As a negative control, binding of the factors was also monitored at an irrelevant DNA region (negative control [NC] DNA). The results were normalized to that obtained with an IgG control antibody, which was set to 1. Data are represented as mean ± SD. *p≤0.05, **p≤0.01.
DOI: http://dx.doi.org/10.7554/eLife.02313.011
Figure 3—figure supplement 3. Validation of a role for ZNF304 and KRAS in INK4-ARF transcriptional silencing in other KRAS-positive human CRC cell lines.

Figure 3—figure supplement 3.

(A) qRT-PCR analysis monitoring expression of p14ARF, p15INK4B, and p16INK4A in HCT116 (left) and HCT15 (right) cells stably expressing an NS, ZNF304, or KRAS shRNA. The results were normalized to that obtained with the NS shRNA, which was set to 1. (B) qRT-PCR analysis monitoring knockdown efficiency of the ZNF304 and KRAS shRNAs in HCT116 (left) and HCT15 (right) cells. (C) qRT-PCR analysis monitoring expression of p14ARF, p15INK4B, and p16INK4A in HCT116 (left) and HCT15 (right) cells treated with DMSO (as a control) or manumycin A (Man. A). The results were normalized to that obtained with DMSO, which was set to 1. Data are represented as mean ± SD. *p≤0.05, **p≤0.01.
DOI: http://dx.doi.org/10.7554/eLife.02313.012
Figure 3—figure supplement 4. The p14ARF promoter is hypermethylated in KRAS-positive human CRC samples.

Figure 3—figure supplement 4.

Bisulfite sequencing analysis of the p14ARF promoter in matched adjacent normal colon (N) and KRAS-positive CRC human tumor samples (T) samples. (Top) Schematic of the promoter; positions of CpGs are shown to scale by vertical lines. (Bottom) Each circle represents a methylated (black) or unmethylated (white) CpG dinucleotide. Each row represents a single clone.
DOI: http://dx.doi.org/10.7554/eLife.02313.013