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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Oncogene. 2019 Jan 8;38(17):3170–3184. doi: 10.1038/s41388-018-0653-x

Figure 6. S27 phosphorylation leads to dissociation of MZF1 from zinc finger transcription factors with transcriptional repressor functions.

Figure 6

(a) MZF1-S27E is constitutively active. MCF7 cells were transfected with the CTSB firefly luciferase reporter, the Renilla luciferase construct, and empty vector (mock) or MZF1-WT or MZF1-S27E Δt-3xHA plasmids. Reporter activity was calculated as the firefly luciferase activity divided by the Renilla luciferase activity, and is presented as the percentage of the mock-transfected control. Values shown are mean ± standard deviation of three independent experiments.Statistical significance was calculated with one-way ANOVA with Dunnett’s correction.

(b) Constitutively active MZF1-S27E binds less efficiently to some zinc-finger TFs than the MZF1-S27A (inactive form). Affinity purification mass spectrometry analysis of nuclear proteins associated with MZF1-S27E (red) and MZF1-S27A (yellow) Strep-tactin baits. The relative interacting protein abundances compared to the MZF1 bait abundance (%) were calculated from the spectral counts. The error bars represent ± the standard deviation of four experiments (SD). The interactor abundance (%) difference between the S27A and S27E mutants are shown for each interacting protein.

(c) S27-phosphorylated MZF1 resides in the nucleus. Immunofluorescence image of p95-ErbB2-MCF7 cells stained with the anti-MZF1-pS27 antibody (pS27-MZF1; green), F-actin (cytoskeleton; red), and Hoechst (nucleus; blue). Image is a representative of three independent experiments.

(d) CTCF inhibits CTSB reporter activity. MCF7 cells were transfected with the CTSB firefly luciferase reporter, the Renilla luciferase construct and empty vector (mock) or Myc-CTCF pcDNA3.1 plasmids. Reporter activity was calculated as in a. Values shown are the mean ± standard deviation of three independent experiments. Statistical significance was calculated with the Student’s t-test (unpaired with Welch’s correction).

(e) MZF1 expression levels do not affect CTCF expression. Quantitative RT-PCR of CTCF expression in CRISPR MZF1-30 cells expressing empty vector control (mock) or MZF1-WT or MZF1-S27A pcDNA 3.1 plasmids. CTCF expression is normalized to the expression of PPIB. Values shown are the mean ± standard deviation of three independent experiments. Statistical significance was calculated as in a.