Figure 3

CmpBrp specifically binds to rDNA promoter region in vivo. (A) Identification of CmpBrp target gene. ChIP analysis was performed with CmpBrp antibody (CmpBrp Ab.) or rabbit IgG purified from preimmune serum (Pre) using fixed logarithmic growth cells (OD₇₅₀=0.5) under continuous light conditions. Occupancy of CmpBrp was measured at the indicated promoter regions. For rDNA, the 2500-bp downstream region from its TSP (rDNA no. 2) as well as its promoter region (rDNA no. 1) were analysed. Expression types of Pol II-type genes are shown at the bottom. L, light-responsive; L & D, light- & dark-responsive; Cont., constant; -N, N deprivation-responsive. Values are averages of at least three independent experiments and represent percent recovery relative to the total input DNA. Error bars indicate standard deviation. (B) Level of de novo 18S rRNA synthesis under the light/dark cycle. C. merolae cells were grown under white light until OD₇₅₀=0.5 and incubated in complete darkness for 18 h. Lights were turned on for 6 h and then turned off again. Cells were sequentially harvested at the time (h) shown at the top, and the newly synthesized 18S rRNA was detected by run-on analysis. Signals obtained with rDNA (18S rRNA) or pDEST-HIS (negative control) as probe DNAs are shown in the middle. Signal intensities of de novo synthesized 18S rRNA from three independent experiments were quantified and values (L₀ as 100%) are presented (meanss.d.) as relative levels at the bottom. (C) Protein level of CmpBrp in the light/dark cycle. Aliquots of total protein (15 g) from the cell lysate were subjected to immunoblot analysis with CmpBrp antibody. Total protein (around 55 kDa) stained with Coomassie Brilliant Blue (CBB) is shown as loading control (lower panel). (D) CmpBrp association on the rDNA promoter region in the light/dark cycle. ChIP analysis was carried out in the same way as in panel (A) but with sample prepared similar to panel (B). Occupancy of CmpBrp at promoter regions of rDNA (rDNA no. 1) and CMK028C (K028) was analysed. (E, F) ChIP analysis with CmTFIIB (E) and CmBRF (F) antibodies. ChIP analyses were carried out with sample prepared at L₂. Occupancies of relevant factors were analysed at the indicated promoter regions. Others are the same as in panel (A).