Article Figures & Data
Figures
- FIG. 1.
PU.1 interactions with EIAV ets motifs. (A) Model of the interaction of the PU.1 binding domain with the three EIAV binding motifs. The binding domain of PU.1, as determined by Kodandapani et al. (24), was modeled onto B-form DNA. The second helix of a helix-turn-helix motif of PU.1 binds in the major groove of the helix through interactions with the core ets motif (GGAA) that is present on the antisense strand of the EIAV enhancer. (B) Schematic of the EIAV LTR and the nucleotide sequences of the EIAV LTR enhancer ets or PU.1 sites. The Oct motif is identified below the construct because the site physically overlaps both the 5′ and middle PU.1 sites. The empty blocks within the enhancer represent 3- to 5-bp blocks of DNA that are not believed to be involved with transcription factor binding. (C) DNase I protection of the EIAV enhancer region complexed with recombinant PU.1 protein. Lanes 1 to 3, increasing concentrations of DNase I in the absence of PU.1; lane 4, probe that was not treated with DNase; lanes 5 to 7, increasing concentrations of DNase I in the presence of recombinant PU.1. The 5′ to 3′ EIAV LTR enhancer nucleotide sequence is shown in the center of the figure. Hypersensitive regions (circles) as well as PU.1-protected nucleotides (bars) at each of the three PU.1 motifs are indicated.
- FIG. 2.
PU.1 binding motifs in the EIAV enhancer differentially impact LTR activity in the canine macrophage cell line DH82. (A) Enhancer sequences tested for activity within the context of LTR/CAT constructs. (B) Basal levels of LTR activity. (C) Tat-transactivated levels of LTR activity of constructs containing the promoter-proximal (3′) PU.1 binding site (left panel) and constructs that do not contain the promoter-proximal (3′) PU.1 binding site (right panel).
- FIG. 3.
Scatchard analysis of recombinant PU.1 binding to the three PU.1 binding motifs in the EIAV LTR. (A) Representative Scatchard plot of PU.1 binding to the 5′ PU.1 binding motif. The average disassociation constant was determined to be 4.123 nM. (B) Representative Scatchard plot of PU.1 binding to the middle PU.1 binding motif. The average disassociation constant was determined to be 3.269 nM. (C) Representative Scatchard plot of PU.1 binding to the 3′ PU.1 binding motif. The average disassociation constant was determined to be 2.609 nM. (D) Average disassociation constants of PU.1 for the three motifs within the EIAV LTR. Values represent means and standard errors of three independent experiments. The inset graphs in panels A to C demonstrate the saturation of the oligonucleotides with PU.1.
- FIG. 4.
Binding curves of DH82 NE to an oligonucleotide containing the 5′ and middle PU.1 sites (5′ + mid oligonucleotide; squares) or an oligonucleotide containing the middle and 3′ PU.1 sites (mid + 3′ oligonucleotide; diamonds). All other transcription factor binding motifs that are present in that region of the EIAV LTR enhancer were altered in the oligonucleotides by the introduction of point mutations in the appropriate locations. Lines through the data were simulated by using the association constants resolved from a fit of averaged data.
- FIG. 5.
Promoter-proximal location of the PU.1 site is critical for optimal Tat-transactivated expression of the LTR. (A) Constructs tested with transient transfections performed in DH82 cells. The 3′ PU.1 binding motif was substituted for both the 5′ and middle PU.1 motifs in 3′ UP. (B) Basal levels of CAT activity in DH82 cells. (C) Tat-transactivated levels of CAT activity.
- FIG. 6.
HIV enhancer-promoter elements substitute for the EIAV elements in macrophages. (A) Constructs tested in transient transfections performed in DH82 cells. pEIA P4 (30) contains the HIV enhancer-promoter region within the context of the EIAV LTR. (B) Basal levels of expression. (C) EIAV Tat-transactivated levels of expression.
- FIG. 7.
Three PU.1 sites are not sufficient to support EIAV replication in equine MDMs. (A) Enhancer sequences of the molecular clones tested for infectivity. (B) RT activity of culture supernatants from EIAV-infected MDMs.
Tables
- TABLE 1.
Oligonucleotides used for PU.1 binding studiesa
Oligonucleotide Sequence (5′→3′) 5′PU.1 ATA CTG TAG TTC CTC AAT ATA G 5′PU.1 C′ TTC ACT ATA TTG AGG AAC TAC A MidPU.1 TCA ATA TAG TTC CGC ATT TGT G MidPU.1 C′ ACG TCA CAA ATG CGG AAC TAT A 3′PU.1 CGT GTT AAG TTC CTG TTT TTA C 3′PU.1 C′ TAC TGT AAA AAC AGG AAC TTA A 5′ + mid PU.1 TCC TGT AGT TCC TCA ATA TAG TTC CGC A 5′ + mid PU.1 C′ CAA ATG CGG AAC TAT ATT GAG GAA CTA CAG G Mid + 3′ PU.1 TCA ATA TAG TTC CGC ATT TGC TAC GCG TTA AGT TCC TG Mid + 3′ PU.1 C′ ATA CTG TAA AAA CAG GAA CTT AAC GCG TAG CAA ATG CGG AAC ↵ a Double-stranded oligonucleotides were made by annealing positive-strand oligonucleotides to their complementary (C′) partners. The core of each PU.1 binding motif is underlined. Other known transcription factor binding motifs (Oct and CRE) were eliminated from the oligonucleotides by point mutations.
