NF-Y influences directionality of transcription from the bidirectional Mrps12/Sarsm promoter in both mouse and human cells.

The bidirectional mammalian promoter for mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm) contains an array of four CCAAT boxes separated by 34-49 bp. In mouse, these elements were shown previously to interact with transcription factor NF-Y and to be required for efficient transcription. Here we show that the CCAAT boxes of the human promoter also influence relative transcriptional activities in the two directions, although they are not absolutely required for transcription. By mutating CCAAT boxes in all possible permutations, we demonstrate that their function is combinatorial, although not simply additive. EMSA indicated that NF-Y interacts with the array in two alternate ways related to the directional selectivity of transcription. Inversion and/or exchange of individual CCAAT boxes had minimal effects on directional selectivity. Over-expression of wild-type or dominant-negative NF-Y affected transcription in the Sarsm direction only, but in human cells, concomitant expression of dominant-negative constructs for other factors was needed to reveal such effects. We propose that the array of NF-Y type CCAAT boxes maintains bidirectional transcription with an appropriate directional selectivity. Computational analysis confirmed that NF-Y type CCAAT boxes are found preferentially in bidirectional promoters, but many such promoters lack them and must be regulated in another way.