Nucleosome-depleted regions (NDRs) are present immediately adjacent to the transcription start site in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR， creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator， SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance， probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators first to URS1， second to the left-half of URS2 (URS2-L)， and finally to the right-half of URS2 (URS2-R)， with each region representing distinct gates that must be unlocked to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates， as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore， nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.