Figure 4

SpPKC1 is a direct target of SpRunt-1. (A) Schematic of the 5' flanking sequence of SpPKC1, showing locations and sequences of the Runx consensus binding elements (underlined bases mutated for reporter gene studies). (B) EMSA of recombinant Runt domain (RD)-CBFβ heterodimers using probes consisting of target sequences from CyIIIa [12] and the distal and proximal Runx sites from SpPKC1. A 100-fold excess of unlabeled CyIIIa sequence was used as a competitor to test the specificity of the interactions. "C", complex between Runt domain and probe; "FP", free probe. (C) Chromatin immunoprecipitation (ChIP) from 48 h embryos using either an anti-SpRunt-1 [12] or nonimmune IgG. CyIIIa serves as a positive control, whereas histone H4 sequences (present as several hundred copies per haploid genome) serves as a negative control for specificity. (D) Relative normalized expression of wild-type (WT) PKC1-luc and mutant PKC1-luc, in which the proximal and distal Runx target sites have been mutated individually or in combination to abolish SpRunt-1 binding (PM, DM, and PM+DM, respectively), expression in control embryos and embryos injected with SpRunt-1 MASO. Fold difference in expression levels of each construct and of the basal luciferase vector alone (V) compared to the WT construct was measured by quantitative RT-PCR [38]. Each bar represents the averages and standard deviations from three to five different experiments. (E) Pathway summarizing the experimental approaches and results showing that SpRunt-1 promotes cell survival via transcriptional activation of SpPKC1.