Fig. 2.

Functional impacts of CTCF ZF missense mutations. A Published and unpublished missense CTCF mutations (red circles) occurring in acute lymphoblastic leukaemia (L309P, R339Q, R337H, G420D—highlighted in black) superimposed on a C2H2 ZF structure: C = cysteine, H = histidine, Zn = Zn²⁺ ion; R339W (underlined) is a previously characterised change-of-function mutation used as a control. The Polyphen score for each mutation is indicated. Numbers (− 6 to + 6) indicate co-ordinates within the DNA-binding portion of the ZF; residues directly contacting DNA at positions − 1, + 2, + 3 and + 6 are indicated (white ring). B Western blot of WT and mutant CTCF expression in transduced K562 cells; anti-HA antibody detects ectopic CTCF, CTCF antibody detects total CTCF; GAPDH is a loading control; size markers indicate MW in kDa. C Immunofluorescence of HA-tagged WT and mutant CTCF in K562 cells using anti-HA antibody, scale bar = 5 μm. D, E Functional assays of CTCF mutants in K562 cells including: D MTT proliferation; and E colony forming assay in Methocult. Data represent the mean ± s.e.m for 3 experiments each performed in triplicate. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons test for pairwise comparisons between control, WT and mutant (ns = not significant; *p < 0.05; **p < 0.01; ****p < 0.0001)