Figure 6

SpCBFβ is not involved in Runx-mediated regulation of SpPKC1. (A) Relative expression levels of SpPKC1, CyIIIa, SpCyclinD and SpDri as determined by RT-PCR analysis of RNA isolated from gastrula stage SpRunt-1-knockdown and SpCBFβ-knockdown embryos. Each bar represents the average +/- standard deviation in the level of expression with respect to controls from two or more microinjection experiments. The dashed line indicates a 3-fold reduction of expression, which is taken here to be the minimum level of reduction considered to be significant. Note that whereas the expression of CyIIIa, SpCyclinD and SpDri are similarly affected by depletion of either SpRunt-1 or SpCBFβ, expression of SpPKC1 is much more strongly affected by SpRunt-1 depletion than by SpCBFβ depletion (asterisk). (B) ChIP of CyIIIa, SpPKC1, SpCyclinD and SpCdk4 using anti-SpRunt-1 and anti-SpCBFβ antibodies and chromatin prepared from late gastrula stage (48 h) embryos. A product of the expected size was obtained from the input DNA with each primer set, confirming the efficacy of the primers and PCR conditions (not shown). SpCdk4, which was not recovered by ChIP with either antibody, was chosen as a specificity control because its expression levels are not affected by knockdown of either SpRunt-1 or SpCBFβ.