Fig. 3
CaCrz1 binds in vitro and in vivo to two motifs in the promoter of UTR2. (a) Locations of two potential Crz1-binding motifs (boxed) in the UTR2 promoter. The 5′-TGAGGCGTTG-3′ region in the complementary sequence of the 5′-C(− 342)AACGCCTCA(− 333)-3′ site is the potential Crz1 binding motif predicted in our study, and the 5′-G(− 376)GGCT(− 372)-3′ region is the putative Crz1 binding motif identified previously (28). Locations of EMSA Probe 1 [EMSA_UTR2_F/R(H)] and Probe 2 [EMSA_UTR2_F/R(M)] are indicated with dark lines above their corresponding sequences, and EMSA Probe 3 [EMSA_UTR2_F/R(HM)] is indicated with a dark line under its corresponding sequence. Locations of the ChIP PCR primer pair [CHIP_UTR2_F和CHIP_UTR2_R] are indicated with broken lines above and under their corresponding sequences, respectively. (b) DIG-labelled probe 1 [EMSA_UTR2_F/R (H)] was added into samples in Lanes 1–3. DIG-labelled probe 2 [EMSA_UTR2_F/R(M)] was added into samples in Lanes 4–6. DIG-labelled probe 3 [EMSA_UTR2_ F/R(HM)] was added into samples in Lanes 7–9. Purified His6-Crz1 protein of 1 μg was added into Lanes 2, 3, 5, 6, 8 and 9. Unlabelled probes 1, 2 and 3 were added into samples in Lanes 3, 6 and 9, respectively. Only purified His6-Crz1 protein, but not probe DNA, were added into the sample in Lane 10. (c) Detection of Crz1 binding to the UTR2 promoter in vivo by ChIP analysis. The wild-type strain expressing Crz1-HA and the control strain integrated with CIp10 vector (no tag control) were exposed to 0.2 M CaCl2 for 1 h, and their cells were treated with formaldehyde. Whole cell extractions were obtained from collected cells, and immunoprecipitation was done with anti-HA monoclonal antibodies. Immunoprecipitated pellets were used as templates for PCR with the primer pair ChIP_UTR2_F/R. PCR products were separated on 1% agarose gel