High-mobility-group protein I can modulate binding of transcription factors to the U5 region of the human immunodeficiency virus type 1 proviral promoter

Abstract

HMG I/Y appears to be a multifunctional protein that relies on in its ability to interact with DNA in a structure-specific manner and with DNA, binding transcriptional activators via distinct protein-protein interaction surfaces. To investigate the hypothesis that HMG I/Y may have a role in human immunodeficiency virus type 1 (HIV-1) expression, we have analyzed whether HMG I/Y interacts with the 5' long terminal repeat and whether this interaction can modulate transcription factor binding. Using purified recombinant HMG I, we have identified several high-affinity binding sites which overlap important transcription factor binding sites. One of these HMG I binding sites coincides with an important binding site for AP-1 located downstream of the transcriptional start site, in the 5' untranslated region at the boundary of a positioned nucleosome. HMG I binding to this composite site inhibits the binding of recombinant AP-1. Consistent with this observation, using nuclear extracts prepared from Jurkat T cells, we show that HMG I (but not HMG Y) is strongly induced upon phorbol myristate acetate stimulation and this induced HMG I appears to both selectively inhibit the binding of basal DNA-binding proteins and enhance the binding of an inducible AP-1 transcription factor to this AP-1 binding site. We also report the novel finding that a component present in this inducible AP-1 complex is ATF-3. Taken together, these results argue that HMG I may play a fundamental role in HIV-1 expression by determining the nature of transcription factor-promoter interactions.

FIG. 1

5′-UTR of the HIV-1 promoter contains multiple high-affinity HMG I binding sites. To determine the location of potential HMG I binding sites, DNase I footprinting reactions were carried out as described in the text. Analysis of three segments within the 5′-UTR is shown. These include regions −105 to +1 (A), +56 to +128 (B), and +112 to +194 (C). The numbering is relative to the start site of transcription at +1. Solid boxes represent high-affinity binding sites, whereas shaded boxes illustrate the position of low-affinity HMG I binding sites. Since HMG I contains three distinct individual DNA-binding domains, each capable of producing a small footprint, the combination of these individual footprints has been described as a footprint region. (D) SDS–15% polyacrylamide gel showing 2 μg of recombinant HMG I.

FIG. 2

Summary of the positions of HMG I binding sites within the 5′-LTR. Shown is the sequence of the 5′-LTR from −187 to +230. Previously identified transcription factor binding sites are also shown (2, 44). Solid and hatched rectangles below the sequence represent high- and low-affinity binding sites for HMG I, respectively. The shaded region (+10 to +155) indicates the approximate location of the positioned nucleosome identified by in vivo footprinting (43). The three AP-1 binding sites within this nucleosome are labeled AP1-1, AP1-2, and AP1-3. The two T residues that were mutated to G residues in footprint region 5 are highlighted with stars. Bracketed is the DNA probe used for gel shift analysis. R, start site of transcription.

FIG. 3

HMG I inhibits the binding of Fos-Jun to site AP1-3. (A) To examine the effect of HMG I on the binding of Fos-Jun to site AP1-3, a Fos-Jun titration (see Materials and Methods) was carried out in which either no protein, 25 ng of HMG I, or 25 ng of a mutant DNA-binding form of HMG I (mHMG I) was added to binding reactions containing a 25-bp probe. (B) The experiment in panel A was repeated except that the HMG I/Y binding site was mutated. Lanes 2, 5, 8, and 11 received 12 ng of HMG I, while lanes 3, 6, 9, and 12 received 25 ng of HMG I.

FIG. 4

Endogenous HMG I binds to site AP1-3. (A) DNA-binding reactions were carried out using a labeled probe containing site AP1-3 and increasing amounts of a nuclear extract (NE) prepared from PMA-induced Jurkat cells. As indicated, reactions received either poly(dI-dC) or poly(dG-dC) (2 μg). Lanes 2 and 7, 3 and 8, 4 and 9, and 5 and 10 received 1, 5, 10, and 20 μg of nuclear extract, respectively. A mobility gel shift assay was performed to identify assembled nucleoprotein complexes. (B) DNA-binding reactions, as indicated, were carried out using nuclear extracts prepared from uninduced and PMA-induced Jurkat cells. The nucleoprotein complexes generated are shown as complexes 1 to 4. The formation of HMG I-DNA complexes is also highlighted. Lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8 received 2, 5, 10, and 15 μg of nuclear extract, respectively. (C) Mobility gel shift assay was carried out using labeled DNA probes that contained either a mutated or an unmodified HMG I/Y binding site. Lanes 2 to 5 and lanes 7 to 10 received 15, 20, 25, and 30 μg of nuclear extract, respectively.

FIG. 5

PMA induction of Jurkat T cells induces HMG I. (A) Nuclear extract (NE) proteins prepared from uninduced and PMA-induced Jurkat T cells were precipitated using 60% (NH₄)₂SO₄ (see Materials and Methods). Western blot analysis of precipitated and supernatant fractions was carried out using affinity-purified polyclonal antibodies raised against HMG I/Y. Lanes 2 to 5 and 6 and 7 received 40 and 10 μg of total protein, respectively. (B) Mobility gel shift assay carried out using site AP1-3 and (NH₄)₂SO₄-precipitated (ppt) and supernatant (supern.) fractions derived from uninduced and induced nuclear extracts. Lanes 2 to 6 and 7 to 11, received 1, 2, 5, 10, and 15 μg of total protein, respectively. Lanes 12 to 15 and 16 to 19 received 2, 5, 10, and 15 μg of total protein, respectively.

FIG. 6

HMG I can both selectively inhibit and promote endogenous factor binding to site AP1-3. (A) The 60% (NH₄)₂SO₄ supernatant fraction, derived from PMA-induced nuclear extracts (NE), was mock immunodepleted (lanes 2 to 6) or immunodepleted with affinity-purified antibodies against HMG I/Y (lanes 7 to 11) or H2A (control, lanes 12 to 16). A mobility gel shift assay, using site AP1-3, was carried out using these immunodepleted supernatant fractions. Lanes 2 to 6, 7 to 11, and 12 to 16 received 2, 5, 10, 15, and 20 μg of the supernatant fraction, respectively. (B) PMA-induced supernatant fractions that were immunodepleted with either control (H2A) or HMG I affinity-purified antibodies were titrated back to a PMA-induced nuclear precipitated fraction. Lanes 2 to 12 received 7 μg of nuclear (NH₄)₂SO₄-precipitated proteins. Lanes 3 to 7 and lanes 8 to 12 received 5, 10, 20, 30, and 40 μg of H2A- or HMG I/Y-depleted supernatant fractions, respectively. (C) Increasing amounts of recombinant HMG I were added to DNA-binding reactions that received 10 μg of uninduced nuclear precipitated extract and a labeled oligonucleotide that contained site AP1-3. Lanes 2 to 6 received 0, 25, 50, 100, and 200 ng of HMG I, respectively.

FIG. 6

HMG I can both selectively inhibit and promote endogenous factor binding to site AP1-3. (A) The 60% (NH₄)₂SO₄ supernatant fraction, derived from PMA-induced nuclear extracts (NE), was mock immunodepleted (lanes 2 to 6) or immunodepleted with affinity-purified antibodies against HMG I/Y (lanes 7 to 11) or H2A (control, lanes 12 to 16). A mobility gel shift assay, using site AP1-3, was carried out using these immunodepleted supernatant fractions. Lanes 2 to 6, 7 to 11, and 12 to 16 received 2, 5, 10, 15, and 20 μg of the supernatant fraction, respectively. (B) PMA-induced supernatant fractions that were immunodepleted with either control (H2A) or HMG I affinity-purified antibodies were titrated back to a PMA-induced nuclear precipitated fraction. Lanes 2 to 12 received 7 μg of nuclear (NH₄)₂SO₄-precipitated proteins. Lanes 3 to 7 and lanes 8 to 12 received 5, 10, 20, 30, and 40 μg of H2A- or HMG I/Y-depleted supernatant fractions, respectively. (C) Increasing amounts of recombinant HMG I were added to DNA-binding reactions that received 10 μg of uninduced nuclear precipitated extract and a labeled oligonucleotide that contained site AP1-3. Lanes 2 to 6 received 0, 25, 50, 100, and 200 ng of HMG I, respectively.

FIG. 7

Inducible AP-1 complex 3 contains ATF-3. Biotinylated site AP1-3 was incubated with induced or uninduced nuclear extract (NE) (250 μg of total protein), and the DNA-bound AP-1 factors were isolated as described in Materials and Methods. The isolated proteins were run on an SDS–12% polyacrylamide gel and either probed with commercial antibodies raised against different AP-1/CREB family members (A) or stained with silver (B). To determine which AP-1/CREB family members are present initially in uninduced and induced nuclear extracts, 15 μg of unpurified nuclear extract was also probed with the different antibodies (A) and stained with silver (B). NE, uninduced Jurkat T cells; PMA-NE, Jurkat T cells stimulated with PMA.