ACTIVATION OF THE HIV-1 ENHANCER BY THE LEE-1 HMG PROTEIN ON NUCLEOSOME-ASSEMBLED DNA IN-VITRO

Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobili ty group (HMG) protein that activates the T cell receptor alpha (TCR a lpha) enhancer in a context-restricted manner in T cells. In this pape r we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 a nd Ets-1, also provides a functional context for activation by LEE-1. First, we show that mutations in the LEF-1-binding site inhibit the ac tivity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80 - to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nu cleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HTV-1 transcription and that repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets- 1 sites, further augments transcription in this system. Individually o r collectively, none of the three enhancer-binding proteins (LEF-1, Et s-1, and TFE-3) could activate transcription in the absence of Sp1. A truncation mutant of LEF-1 (HMG-88), which contains the HMG box but la cks the trans-activation domain, did not activate transcription from n ucleosomal DNA, indicating that bending of DNA by the HMG domain is no t sufficient to activate transcription in vitro. We conclude that tran scription activation by LEF-1 in vitro is a chromatin-dependent proces s that requires a functional trans-activation domain in addition to th e HMG domain.