Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method

Our goal was to develop a system to study proteins that associate in vivo w ith the Moloney murine leukemia virus (M-MuLV) enhancer elements by the iso lation of intact proviral chromatin, The M-MuLV long terminal repeats (LTRs ) contain tandemly repeated transcriptional enhancer sequences consisting o f smaller motifs that bind cellular DNA-binding proteins implicated in tran scriptional regulation. The M-MuLV enhancers are also important for disease specificity and latency of disease induction. To enrich for proviral chrom atin containing M-MuLV LTR sequences, an affinity purification scheme was e mployed that relies on the affinity of bacterial Lac repressor protein for Lac operator (LacO) DNA sequences. An infectious M-MuLV recombinant was con structed that contains bacterial LacO sequences inserted into a nonessentia l region downstream from the 5 ' LTR of the virus (M-MuLV-LacO), Nuclei fro m M-MuLV-LacO-infected cells were digested with Pvull (which will liberate an LTR fragment containing LacO sequences), and digested chromatin was leac hed from the nuclei in hypotonic buffer. M-MuLV-LacO chromatin was then rec overed by binding to an affinity matrix consisting of a beta -galactosidase -Lac repressor fusion protein anchored to acrylamide beads by an anti-P-gal actosidase monoclonal antibody [7], Specifically bound chromatin was eluted under physiological conditions by incubation with the galactose analog iso propyl-beta -D-thiogalactopyranoside. Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin by this method. Copyright (C) 2001 National Science Council, ROC and S. Karger AG, Basel.