IDENTIFICATION AND CHARACTERIZATION OF A NOVEL TRANSCRIPTIONAL SILENCER IN THE HUMAN COLLAGEN TYPE-IV GENE COL4A2

Collagen type IV [alpha 1(IV)(2) alpha 2(IV)] is the basic structural component of all basement membranes. The two subunit genes COL4A1 and COL4A2 are found closely linked in the human and murine genomes and are transcribed divergently from a common promoter, Previously, activating elements had been detected within both genes which are indispensable for efficient transcription. An additional negative regulatory element has now been identified within the third intron of the COL4A2 gene which is able to inhibit transcription of both COL4 genes from their shared promoter, as well as the nonrelated herpes simplex virus thymidine kinase promoter. The element exerts its inhibitory effect largely independently from its relative orientation and distance from the initiation site of transcription. Therefore, the element represents a silencer, which is named the ''COL4 silencer.'' The minimal functional silencer could be narrowed down by deletion mapping to a sequence element located within intron 3 of the COL4A2 gene. This motif is specifically recognized by a nuclear protein, named ''SILBF,'' and the binding site of which was determined by footprinting assays. Mutation studies and deletion analysis proved that the presence of this sequence element and its interaction with SILBF is not only essential but also sufficient for the silencing function. We assume that the COL4 silencer plays an important role in the control of overall expression and the balance of divergent transcription of both COL4 genes.