DNA elements which negatively regulate transcription have been identified in many genes. One kind of these DNA elements which inhibits transcription in an position - and orientation - independent manner has been termed silencer. Silencers have been found in the rat insulin I gene, c-myc gene, mouse beta major globin gene, chicken lysozyme gene and in yeast, and appear to play an important role in the control of gene activity. We have studied the upstream control region of the human epsilon-globin gene. By deletion analysis, we found that a number of upstream regions have regulatory functions. Particularly, two negative regulatory regions located between -177 bp and -392 bp, and -535 bp and -1400 bp, and one positive regulatory region between -392 and -535 bp have been identified. As a first step to characterize these regulatory elements, we have concentrated on the region between - 177 and -392 bp. This 220 bp fragment has been cloned in CAT expression plasmid containing 177 bp upstream sequences of the epsilon gene in both 5' and 3' positions and in both sense and antisense orientations. Upon introduction into the erythroleukemia K562 and Hela cells, the plasmids containing the 220 bp DNA fragment showed significantly lower CAT activity compared with the plasmid without tne fragment. This indicates that this fragment can act as a transcriptional silencer. To test whether this silencer functions in a heterologous system, we placed this fragment in the 5' side of the tK promoter. When transfected into HeLa cells, this silencer also inhibits tKCAT expression. Thus, the silencer not only functions on the epsilon-globin gene, it also functions on other promoters.
