Cbf11 binds to and activates a reporter gene <i>in vivo</i>.

<p>(<b>A</b>) Schematic representation of expression reporter plasmids with the β-galactosidase gene under the control of a minimal promoter with three copies of either the canonical ('RBP') or mutated ('DEL2') CSL response element (not to scale). (<b>B</b>) β-galactosidase activity in wild-type and CSL deletion mutant strains harbouring the RBP reporter plasmid. Activation of the reporter is dependent on endogenous Cbf11 but not Cbf12. (<b>C</b>) β-galactosidase activity in the <i>Δcbf11 Δcbf12</i> strain harbouring the RBP reporter plasmid and overexpressing the indicated CSL protein variants (HA-tagged) from a plasmid. The presence of high levels of both Cbf11 and Cbf12 activates, to a different degree, the RBP reporter. This activation is abolished in both CSL proteins by the 'DBM' point mutation in the BTD domain. (<b>D</b>) β-galactosidase activity in strains with chromosomally TAP-tagged Cbf11 or Cbf12 harbouring either the RBP or mutated DEL2 reporter plasmid. Both strains show RBP reporter activation similar to the untagged wild-type strain in (B) and no activation of the DEL2 reporter with mutated CSL binding sites. (<b>E, F</b>) Western blots of cell extracts from strains used in (C) and (D), respectively, confirming that all N-terminally HA-tagged and C-terminally TAP-tagged CSL variants were expressed properly. (<b>G</b>) Chromatin immunoprecipitation of TAP-tagged Cbf11 and Cbf12 from strains described in (D). Cbf11 binds strongly the promoter region of the RBP reporter but not the mutated DEL2 reporter. No binding to either reporter could be detected for Cbf12. All data represent mean values±standard deviations from at least three independent experiments.