INCREASING BINDING OF A TRANSCRIPTION FACTOR IMMEDIATELY DOWNSTREAM OF THE CAP SITE OF A CYTOMEGALOVIRUS GENE REPRESSES EXPRESSION

A closely related family of ubiquitous DNA binding proteins, called MD BP, binds with high affinity to two 14 base pair (bp) sites within the human cytomegalovirus immediate early gene 1 (CMV IE1) enhancer and w ith low affinity to one site beginning 5 bp downstream of the CMV IE1 transcription start point (+5 site). Unlike several cap position downs tream MDBP sites in mammalian genes, these MDBP sites do not require c ytosine methylation for optimal binding, Mutation of one of the enhanc er MDBP sites to prevent MDBP recognition modestly increased the funct ion of a neighboring CREB binding site in a transient transfection ass ay in the context of one promoter construct, A much larger effect on r eporter gene expression (a 10-fold reduction) was seen when the low af finity MDBP recognition sequence at position +5 was converted to a hig h affinity site in a plasmid containing the CMV IE1 promoter upstream of the reporter gene, Evidence that the increased binding of MDBP at t he mutant site is largely responsible for the observed results was pro vided by transfection experiments with this high affinity MDBP +5 site re-mutated to a non-binding site and by in vitro transcription assays .