Table 1.
Fission yeast strains used in this study.
Table 2.
Oligonucleotides used in this study.
Table 3.
Plasmids used in this study.
Figure 1.
The N-termini of Cbf11 and Cbf12 are important for their nuclear localization.
(A) Schematic representation of all Cbf11 and Cbf12 variants used in this study. The N-terminal Rel homology region ('RHR-N', green), beta trefoil domain ('BTD', red), and position of the point mutation affecting DNA binding activity are indicated. (B) A western blot confirming that all N-terminally EGFP-tagged CSL variants are expressed and produce a band of the expected size. The anti-PSTAIRE (Cdc2) western blot serves as a loading control. (C) All EGFP-CSL derivatives localize to the nucleus with the exception of variants devoid of the whole unstructured N-terminal region. Scale bar is 10 µm. The bottom panel shows longitudinal fluorescence intensity profiles of the cells marked with an asterisk.
Figure 2.
Mutation of a conserved arginine residue abolishes CSL DNA binding activity in vitro.
(A) Substitution of the arginine residue at position 318 with histidine ('DBM') or a complete removal of the unstructured N-terminus abrogate the binding of Cbf11 to a DNA probe ('RBP') with the canonical CSL response element in an EMSA experiment. (B) The EMSA band corresponding to the DNA binding activity of the full-length non-mutated Cbf11 can be competed with an excess of the unlabelled RBP probe but not with the mutated DEL2 probe. (C) Full-length non-mutated Cbf12 displays very weak but specific DNA binding activity towards the RBP probe in EMSA. The Cbf12 band shift can be competed with an excess of the unlabelled RBP probe but not with the mutated DEL2 probe. No DNA binding activity was observed when the arginine residue at position 644 was substituted with histidine ('DBM'). A lane with low amounts of Cbf12(Δ1-394), previously shown to bind the RBP probe [15], is shown for comparison (n.b., during native electrophoresis, protein migration speed is not simply proportional to protein mass since native charge and shape also play important roles). (D) Supershift experiments with GFP-tagged Cbf12 variants. For both full-length Cbf12 and Cbf12(Δ1-394) the addition of an anti-GFP antibody, but not an anti-TAP antibody, interferes with protein-DNA complex formation. 'w'–wells; 'p'–free probe. (E, F, G) Western blots of cell extracts from (A), (C) and (D), respectively, confirming that all N-terminally HA-tagged and GFP-tagged CSL variants were expressed properly and that equal amounts of cell extracts were used.
Figure 3.
Cbf11 binds to and activates a reporter gene in vivo.
(A) 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) 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. (C) β-galactosidase activity in the Δcbf11 Δcbf12 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. (D) 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. (E, F) 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. (G) 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.