Fig. 4

MYB is a direct target gene of Snail1-HA. a Schematic representation of MYB exons 1 and 2, and a distal region 85 kb downstream of the transcriptional start site (TSS). The location of Snail1-HA ChIP-seq peaks, PCR amplicons employed in ChIP-qPCR and FAIRE analyses, as well as the positions of EMSA probes are indicated. Distance of PCR amplicons from the TSS is given in kilobase pairs (kb). b ChIP-qPCR experiments with LS174T and HT29 cells stably transduced with Dox-inducible retroviral control and Snail1-HA expression vectors. Cells were treated 0.1 µg ml⁻¹ Dox (LS174T) and 1 µg ml⁻¹ Dox (HT29) for 6 h. PCR amplicons as depicted in a. Shown are the mean and SEM; n = 3. c EMSA demonstrating binding of Snail1-HA to DNA sequences from MYB intron 1 in vitro. Material from in vitro translation reactions programmed with empty vector served as negative control (vector). WT wild type E-box motif, mut mutated E-box motif. d FAIRE analyses of MYB intron 1 and a region +85 kb downstream of the MYB gene in LS174T and HT29 cells stably transduced with Dox-inducible retroviral control and Snail1-HA expression vectors. Cells were left untreated or received 0.1 µg ml⁻¹ Dox (LS174T) and 1 µg ml⁻¹ Dox (HT29) for the times indicated. Data were calculated as relative enrichment of sequences of interest in formaldehyde-crosslinked versus non-crosslinked material. Shown are the mean and SEM; n = 3. e ChIP-qPCR analyses to assess the presence of H3K27Ac, H3K4me1, and H3K4me3 at different regions within intron 1 of the MYB gene and a region +85 kb downstream of the MYB TSS in LS174T and HT29 cells stably transduced with Dox-inducible retroviral control and Snail1-HA expression vectors. Cells were left untreated or received 0.1 µg ml⁻¹ Dox (LS174T) and 1 µg ml⁻¹ Dox (HT29) for the times indicated. Data were calculated as percent of input material. Enrichment was further normalized to histone H3 occupancy to account for regional differences in nucleosome density. Shown are the mean and SEM; n = 3. Statistical significance was calculated using a two-tailed unpaired Students’ t test. ***p-value < 0.001; **p-value < 0.01; *p-value < 0.05; ns: not significant