Homol-D cis-regulatory elements mediate the transcriptional coregulation of box C/D snoRNA and RP genes in fission yeasts. (A) Homol-D box was overrepresented upstream of box C/D snoRNA and RP genes in S. pombe, S. cryophilus, S. octosporus, and S. japonicus. Z-scores were calculated as described in Materials and Methods. (B) Distribution of distances from the Homol-D sites to the box C/D snoRNA and RP genes. (C) The percentages of RP genes, snoRNA genes, and genome-wide protein genes with Homol-D box in promoter regions in four fission yeasts. (D) Schematic diagram of deletions in the cluster I promoter region. (E) Northern blot: (WT) wild type S. pombe; (ΔT) TATATAA-box deleted strain; (ΔH) Homol D-box deleted strain; (ΔT-H) TATATAA-Homol D-box deleted strain; (M) molecular weight markers. U2 was used as a control. (F) RRN7 gene relative expressions detected by real-time PCR. (WT) wild type; (RRN7) RRN7 up-regulated strain. (G) DDB1 gene relative expressions detected by real-time PCR; (Empty) the strain stably expressing HA tag vector; (OE-DDB1) DDB1 up-regulated strain stably expressing DDB1 protein-HA tag vector. (H) Western blot: (Empty) the strain stably expressing HA tag vector; (OE-DDB1) DDB1 up-regulated strain stably expressing DDB1 protein-HA tag vector. Anti-FLAG indicates FLAG-tagged DDB1 protein. The lower gel shows Coomassie blue-stained total proteins from samples. (I) Fold changes of random selected RP and snoRNA genes expression detected by real-time PCR when Homol-D binding proteins were changed. (RRN7/WT) RRN7 up-regulated strain compared to wild type; (Δrqh1/WT) rqh1 knockout strain compared to wild type; (ΔDDB1/WT) DDB1 knockout strain compared to wild type; (OE-DDB1/Empty) DDB1 overexpressed strain compared to empty vector strain. Error bars indicate SEM; two-sided student's t-test; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (J) A model for Homol-D box mediating the transcriptional coregulation of box C/D snoRNA and RP genes.
