LeuO protein relieves bacterial gene silencer AT8-mediated transcriptional repression as part of a promoter relay mechanism found in the ilvIH-leuO-leuABCD gene cluster. The gene silencing activity has recently been characterized as a nucleoprotein filament initiated at the gene silencer. In this gene locus, the nucleoprotein filament cis-spreads toward the target leuO promoter and results in the repression of the leuO gene. Although the cis-spreading nature of the transcriptionally repressive nucleoprotein filament has been revealed, the mechanism underlying LeuO-mediated gene silencing relief remains unknown. We have demonstrated here that LeuO functions analogously to the eukaryotic boundary element that delimits the transcriptionally active and repressive domains on the chromosome by blocking the cis-spreading pathway of the transcriptionally repressive heterochromatin. Given that one LeuO-binding site is positioned between the gene silencer and the target promoter, the simultaneous presence of a second LeuO-binding site synergistically enhances the blockade, resulting in a cooperative increase in LeuO-mediated gene silencing relief. A known DNA loop-forming protein, the lac repressor (LacI), was used to confirm that cooperative protein binding via DNA looping is responsible for the blocking synergy. Indeed, a distal LeuO site located downstream cooperates with the LeuO sites located upstream of the leuO gene, resulting in synergistic relief for the repressed leuO gene via looping out the intervening DNA between LeuO sites in the ilvIH-leuO-leuABCD gene cluster.