STEM-CELL FACTOR-BINDING TO RETROVIRUS PRIMER BINDING-SITE SILENCERS

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and o verlaps the Moloney murine leukemia virus (M-MuLV) proline primer bind ing site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silenc er. Factor A bound specifically to the,wild-type silencer element at r oom temperature and 30 degrees C, but not at 4 degrees C, and bound 10 -fold better to the full-length silencer than to a minimal silencer co re element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiate d cells in which the silencer is nonfunctional. Salt and ion requireme nts for factor A binding were investigated, and partial purification s teps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silenc er and PBS activities, sequences representing phenylalanine, isoleucin e, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell specific repression of M-MuLV expres sion and in vitro in DNA binding assays. Of these PBS elements, only t he lysine-1,2 PBS DNA fragment showed consistently high levels of repr ession. Interestingly, the lysine-1,2 PBS DNA fragment also formed a c omplex,vith an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding s tudies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna v irus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited i n undifferentiated stem cells.