Structure and Function of a Novel Silencer in 5' Flanking Distal Region of Human beta Globin Gene.

A novel silencer fragment(310 bp) was recently discovered and identified to locate between -2 132 bp and -1 822 bp upstream from the cap site of beta-globin gene by gel retardation assay and luciferase reporter gene expression analysis. DNA footprinting assays were performed to determine the interaction between its DNA sequence and binding proteins from the nuclear extract of Hela cells. The results showed that there were two nuclear protein binding sites in thissilencer, one was the -2 017--2 011 bp sequence CTTCCGC" and the other was the -2 006--1 997 bp sequence "CACTTTATTT". Two sequences were mutated into "CTTAAGC" and "CACTTAAGTT", respectively by two mutagenic primer pairs, in order to construct two mutation types of the 310 bp fragment. The competitive gel retardation assays showed that two mutation types of the 310 bp fragment and their four smaller DNA fragments, which were formed respectively by the digestion of restriction enzyme BspTI, all lost their competitive ability against the wild type of 310 bp fragment probe for DNA-binding proteins without exception. Furthermore, the double-strand oligonucleotides, which contained both the sequences of "CTTCCGC" and "CACTTTATTT", were synthesized, and the competitive gel retardation assays showed that they competed ability against wild type 310 bp fragment probe for DNA-binding proteins. The results suggest that two binding sites of the nuclear proteins are involved or associated with a potential DNA-DNA interaction. Moreover, the specific DNA-binding proteins were purified from the nuclear extract of Hela cells by using "DNA-binding protein purification kit" for magnetic isolation. In order to identify the purified DNA-binding proteins, a SDS-PAGE was performed. By using the silver staining, the PAGE electrophoretogram showed that these two nuclear proteins specifically bound to these two sites of the silencer, appearing as two definite bands. The molecular weight of each protein was determined to be 37 kD or 81 kD, respectively.