A TRANSCRIPTIONAL REGULATORY ELEMENT IS ASSOCIATED WITH A NUCLEASE-HYPERSENSITIVE SITE IN THE POL GENE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Analysis of the chromatin organization of the integrated human immunod eficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high -resolution mapping of this sits with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four d istinct nuclear protein complexes: a ubiquitous factor, a T-cell-speci fic factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolat es in which this PU box was not conserved, it was replaced by a bindin g site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors bin ding to site B, except for PU.1/Spi-1 A GC box containing a binding si te for Sp1 was identified (nt 4623 to 4631), Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results iden tify a transcriptional regulatory element associated with a nuclease-h ypersensitive site in the pol gene of HIV-1 and suggest that its activ ity mag. be controlled by a complex interplay of cis-regulatory elemen ts.