NF-Y influences directionality of transcription from the bidirectional Mrps12/Sarsm promoter in both mouse and human cells Ernesto Zanotto a , Antti Häkkinen b , Gabriel Teku a , Bairong Shen a , Andre S. Ribeiro b , Howard T. Jacobs a, ⁎ a Institute of Medical Technology and Tampere University Hospital, University of Tampere, Finland b Institute of Signal Processing, Tampere University of Technology, Tampere, Finland abstract article info Article history: Received 11 March 2009 Received in revised form 30 April 2009 Accepted 4 May 2009 Available online 9 May 2009 Keywords: Transcription factor Bidirectional promoter CCAAT box NF-Y 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-Yaffected 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. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Many genes in mammalian DNA are organized in head-to-head configuration, with transcription start sites separated by less than 1 kb of intervening promoter DNA. These bidirectional promoters present a number of inherent problems for the transcriptional apparatus. Firstly, all cis-acting information for efficient transcription in opposite directions, and its regulation in different physiological or develop- mental contexts, must be contained within a relatively short stretch of DNA. One solution to this problem would require that the two genes share regulatory information and are effectively co-expressed and co- regulated, though not necessarily at the same level as each other. Shared enhancer elements fulfilling such functions need, never- theless, to be able to interact with the basal transcriptional machinery in either of two different configurations, which may be mutually exclusive. In a previous study [1], using a dual-reporter strategy in combination with EMSA and ChIP assays, we characterized the bidirectional promoter of two mouse nuclear genes encoding components of the mitochondrial translational apparatus, mitoribo- somal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm). The transcription start sites for these genes are located less than 200 bp apart (Fig. 1). In the intervening DNA we identified an array of four CCAAT boxes, interacting both in vitro and in vivo with the transcription factor NF-Y. These elements were shown to be required for efficient, bidirectional transcription, with the terminal CCAAT boxes of the array playing key roles in enforcing directionality towards the opposite end of the promoter. Subsequently, a partial mutational analysis of the homo- logous human promoter [2] indicated that it may function in a similar manner. The transcription factor NF-Y is unusual in that it can activate transcription in both orientations with respect to the transcription start site, although the mechanism by which it acts thus is not known. A recent study [3] found that NF-Y binding is strongly correlated with the local deposition of histone methylation marks that can confer either positive or negative effects on transcription. By recruiting the methylation (MLL) complex, NF-Y was proposed to induce changes in chromatin structure which favor the further binding of other transcription factors and the activation, or in some cases repression, of transcription. This action should be independent of the orientation of the NF-Y binding site, allowing it to function bidirectionally. Whatever the mechanism which underlies this bidirectional property of NF-Y, it may be predicted that inversion of an NF-Y-dependent CCAAT box should have little impact on its transcriptional activity. The Biochimica et Biophysica Acta 1789 (2009) 432–442 ⁎ Corresponding author. Institute of Medical Technology, FI-33014 University of Tampere, Finland. Tel.: +358 3 35517731; fax: +358 3 35517710. E-mail address: howard.t.jacobs@uta.fi (H.T. Jacobs). 1874-9399/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2009.05.001 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm