Chromatin insulators or boundary elements protect genes from regulatory activities fromneighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, thedeletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gainof function phenotypes, presumably due to the loss of chromatin barriers. Homologous chro-mosomes are paired in Drosophila, creating a number of pairing dependent phenomenaincluding transvection, and whether transvection may affect the function of Polycombresponse elements (PREs) and thus contribute to the phenotypes are not known. Here, westudied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of thetransgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on theDsRed reporter gene in a CTCF binding sites dependent manner. However, the blockingalso depends on the zygosity of the transgene in that the barrier activity is present when thetransgene is homozygous, but absent when the transgene is heterozygous. To analyze thiseffect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR)experiments on homozygous transgenic embryos, and found that H3K27me3 andH3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRedgene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, themutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently,DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent thespread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting aninsulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator inone copy of the chromosome could lead to the loss of insulator activity on the homologouschromosome, and in other loci where chromosomal deletion created hemizygous regions ofthe genome, the chromatin barrier could be compromised. This study highlights a role ofhomologous chromosome pairing in the regulation of gene expression in the Drosophilagenome.