Figure 4.
The DNA-binding domain (DBD) of Brn2 is both required and sufficient for mitotic chromosome binding. (A) Representation of Brn2 protein domains to scale, including the DBD with its POUs and POUh domains. Red stripes mark residues involved in sequence-specific binding that were mutated in live-imaging experiments. (B) Representative captures from live-cell imaging of P19 cells expressing eGFP fusion proteins of full-length Brn2, its truncated DBD, or Brn2 derivatives with mutations in residues within the POUs domain (C311A/R312E) or POUh domain (N406Q/R407G). Control eGFP is also shown. Cells were synchronized in metaphase using proTAME and Apcin and live imaged together with DNA staining Hoechst. (C) Quantifications of mitotic chromosome enrichment levels from different Brn2 derivatives. Data shown as mean ± SD (n = 30 for each condition). One-way ANOVA Tukey's multiple comparison test was performed. (n.s.) P > 0.05, (****) P ≤ 0.0001. Scale bar, 10 µm.