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. 2018 Feb 7;11:9. doi: 10.3389/fnmol.2018.00009

FIGURE 5.

FIGURE 5

Identification of a novel NRE in Xenopus Zic5. (A) A ∼4 kb region (-4042 to -29) upstream of Zic5 contains a Zic5 promoter. Mutations of two putative canonical CSL binding sites within this 4 kb region did not affect notch’s activation of promoter activity. X’s represent the mutated site. (B) Deletion assay of Zic5 upstream regulatory sequence identified a 15 bp NRE locating between -200 and -186 bp in response to NICD activation. P-values relative to -186/-29 fragment are -200/-29 (P = 0.004); -286/-29 (P = 0.015); -533/-29 (P = 0.013); -1950/-29 (P = 0.004); -4042/-29 (P = 0.014). (C) Diagram of the constructs containing 15 bp CSL sequence and mutations in the promoter constructs. pXZic5-200 represents the wild-type and pXZic5-200M represents the 15 bp CBS mutant plasmid. (D) Mutation of the 15 bp CBS (pXZic5-200M) abolished the activation triggered by NICD in the reporter assay; P = 0.0017. (E) EMSA assay; NICD expression increased protein binding to the 15 bp NRE (see section “Materials and Methods”). The number represents Mean ± SEM, N = 3, ∗P < 0.05 with the post hoc Newman–Keuls test. RLU, relative luminant unit; Nuclear Ext, nuclear extraction; Zic5 UPS, fluorescent-labeling zic5 upstream Notch response element.