Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter

Transcriptional repression within a complex modular promoter may play a key role in determining the action of enhancer elements. In human cytomegalovi rus? the major immediate-early promoter (MIEP) locus contains a highly pote nt and complex modular enhancer. Evidence is presented suggesting that sequ ences of the MIEP between nucleotide positions -556 and -673 function to pr event transcription activation by enhancer elements from the UL127 open rea ding frame divergent promoter. Transient transfection assays of reporter pl asmids revealed repressor sequences located between nucleotides -556 and -6 38. The ability of these sequences to confer repression in the context of a n infection was shown using recombinant viruses generated from a bacterial artificial chromosome containing an infectious human cytomegalovirus genome , In addition to repressor sequences between -556 and -638, infection exper iments using recombinant virus mutants indicated that sequences between -63 8 and -673 also contribute to repression of the UL127 promoter. On the basi s of in vitro transcription and transient transfection assays, we further s how that interposed viral repressor sequences completely inhibit enhancer-m ediated activation of not only the homologous but also heterologous promote rs. These and other experiments suggest that repression involves an interac tion of host-encoded regulatory factors with defined promoter sequences tha t have the property of proximally interfering with upstream enhancer elemen ts in a chromatin-independent manner. Altogether, our findings establish th e presence of a boundary domain that efficiently blocks enhancer-promoter i nteractions, thus explaining how the enhancer can work to selectively activ ate the MIEP.