IN-VIVO FOOTPRINTING OF THE ENHANCER SEQUENCES IN THE UPSTREAM LONG TERMINAL REPEAT OF MOLONEY MURINE LEUKEMIA-VIRUS - DIFFERENTIAL BINDINGOF NUCLEAR FACTORS IN DIFFERENT CELL-TYPES

The enhancer sequences in the Moloney murine leukemia virus (M-MuLV) l ong terminal repeat (LTR) are of considerable interest since they are crucial for virus replication and the ability of the virus to induce T lymphomas, While extensive studies have identified numerous nuclear f actors that can potentially bind to M-MuLV enhancer DNA in vitro, it h as not been made clear which of these factors are bound in vivo. To ad dress this problem, we carried out in vivo footprinting of the M-MuLV enhancer in infected cells by in vivo treatment with dimethyl sulfate (DMS) followed by visualization through ligation-mediated PCR (LMPCR) and gel electrophoresis. In vivo DMS-LMPCR footprinting of the upstrea m LTR revealed evidence for factor binding at several previously chara cterized motifs, In particular, protection of guanines in the central LVb/Ets and Core sites within the 75-bp repeats was detected in infect ed NIH 3T3 fibroblasts, Ti-6 lymphoid cells, and thymic tumor cells, I n contrast, factor binding at the NF-1 sites was found in infected fib roblasts but not in T-lymphoid cells, These results are consistent wit h the results of previous experiments indicating the importance of the LVb/Ets and Core sequences for many retroviruses and the biological i mportance especially of the NF-1 sites in fibroblasts and T-lymphoid c ells, No evidence for factor binding to the glucocorticoid responsive element and LVa sites was found, Additional sites of protein binding i ncluded a region in the GC-rich sequences downstream of the 75-bp repe ats (only in fibroblasts), a hypersensitive guanine on the minus stran d in the LVc site (only in T-lymphoid cells), and a region upstream of the 75-bp repeats. These experiments provide concrete evidence for th e differential in vive binding of nuclear factors to the M-MuLV enhanc ers in different cell types.