The Imitation SWItch (ISWI) chromatin remodeling factors have been implicated in nucleosome positioning. In vitro, they can mobilize nucleosomes bi-directionally, making it difficult to envision how they can establish precise translational positioning of nucleosomes in vivo. It has been proposed that they require other cellular factors to do so, but none has been identified thus far. Here, we demonstrate that both ISW2 and TUP1 are required to position nucleosomes across the entire coding sequence of the DNA damage-inducible gene RNR3. The chromatin structure downstream of the URS is indistinguishable in Deltaisw2 and Deltatup1 mutants, and the crosslinking of Tup1 and Isw2 to RNR3 is independent of each other, indicating that both complexes are required to maintain repressive chromatin structure. Furthermore, Tup1 repressed RNR3 and blocked preinitiation complex formation in the Deltaisw2 mutant, even though nucleosome positioning was completely disrupted over the promoter and ORF. Our study has revealed a novel collaboration between two nucleosome-positioning activities in vivo, and suggests that disruption of nucleosome positioning is insufficient to cause a high level of transcription.