Mutations in the Mbp1/Swi4 consensus binding sites (MCB/SCB) affect TLC1 transcription levels. (A) Schematic representation of the TLC1 chromosomal locus and the flanking upstream gene. The 5′-ends of the mature snR161 and TLC1 RNAs are represented by arrows indicating the transcription direction. The identity of the conserved sequence motifs is indicated by letters. The region containing the putative MCB/SCB motifs is magnified at bottom. The sequence of SCB1 is in bold, and the sequences of MCB1 and MCB2 are indicated by a line. The point mutations are indicated beneath. The mutant mcb1/2a was used in all the experiments except in Figure 4B, where it was mutant mcb1/2b. (B) Northern blot analysis of TLC1 RNA from CSHY76 (tlc1Δ) or CSHY76 carrying TLC1 (WT) or tlc1-mcb1 (mcb1a) or tlc1-mcb1/2a (mcb1/2a). Total RNA was extracted from log-phase cells, separated on an Agarose gel, and visualized using radioactive probes complementary to TLC1 and the U1 snRNA as a loading control. (C) The same RNA samples used in B were analyzed by qRT-PCR. The TLC1 RNA levels were measured and normalized against Act1. The WT value is arbitrarily set to one. (D) ChIP analysis of Mbp1-myc (Z1372) and Swi4-myc (Z1335) on the genomic TLC1 promoter. Enrichment levels were determined by qPCR and are represented as the fold increase over the untagged strain (Z1256). Average value and error bars are derived from biological duplicates and experimental duplicates. (E) ChIP analysis of Swi4-myc on the genomic WT TLC1 promoter (IDY1014-3a) or the mutated tlc1-mcb1/2 promoter (IDY1014-6c). Enrichment levels were determined by qPCR and are represented as the fold increase over the untagged strain (Z1256). Average value and error bars are derived from biological duplicates.
