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. 2001 Feb;75(4):1842-56.
doi: 10.1128/JVI.75.4.1842-1856.2001.

Interaction between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human immunodeficiency virus type 1 transcription in cells of the monocyte/macrophage lineage

H L Ross  1 , M R Nonnemacher, T H Hogan, S J Quiterio, A Henderson, J J McAllister, F C Krebs, B Wigdahl
Affiliations

Interaction between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human immunodeficiency virus type 1 transcription in cells of the monocyte/macrophage lineage

H L Ross et al. J Virol. 2001 Feb.

Abstract

Recent observations have shown two CCAAT/enhancer binding protein (C/EBP) binding sites to be critically important for efficient human immunodeficiency virus type 1 (HIV-1) replication within cells of the monocyte/macrophage lineage, a cell type likely involved in transport of the virus to the brain. Additionally, sequence variation at C/EBP site I, which lies immediately upstream of the distal nuclear factor kappa B site and immediately downstream of a binding site for activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB), has been shown to affect HIV-1 long terminal repeat (LTR) activity. Given that C/EBP proteins have been shown to interact with many other transcription factors including members of the ATF/CREB family, we proceeded to determine whether an adjacent ATF/CREB binding site could affect C/EBP protein binding to C/EBP site I. Electrophoretic mobility shift analyses indicated that selected ATF/CREB site variants assisted in the recruitment of C/EBP proteins to an adjacent, naturally occurring, low-affinity C/EBP site. This biophysical interaction appears to occur via at least two mechanisms. First, low amounts of CREB-1 and C/EBP appear to heterodimerize and bind to a site consisting of a half site from both the ATF/CREB and C/EBP binding sites. In addition, CREB-1 homodimers bind to the ATF/CREB site and recruit C/EBP dimers to their cognate weak binding sites. This interaction is reciprocal, since C/EBP dimer binding to a strong C/EBP site leads to enhanced CREB-1 recruitment to ATF/CREB sites that are weakly bound by CREB. Sequence variation at both C/EBP and ATF/CREB sites affects the molecular interactions involved in mediating both of these mechanisms. Most importantly, sequence variation at the ATF/CREB binding site affected basal LTR activity as well as LTR function following interleukin-6 stimulation, a treatment that leads to increases in C/EBP activation. Thus, HIV-1 LTR ATF/CREB binding site sequence variation may modulate cellular signaling at the viral promoter through the C/EBP pathway.

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Figures

FIG. 1
FIG. 1
A C/EBP binding site I variant exhibits very low reactivity for members of the C/EBP transcription factor family. (A) The U3 region of the HIV-1 LTR contains many cis-acting promoter elements that control viral transcription. Included among the many transcription factor binding sites which regulate viral replication are adjacent ATF/CREB and C/EBP binding sites that lie immediately upstream of the tandem NF-κB sites. (B) Double-stranded radiolabeled oligonucleotide probes spanning either the 6G or 3T C/EBP binding site I sequence variants were reacted with IL-6-stimulated U-937 nuclear extract. These reactions were conducted in the absence or presence of antibody directed against C/EBP α (lanes 3 and 7) or C/EBP β (lanes 4 and 8). Control rabbit immunoglobulin (CS) was added (lanes 2 and 6) to illustrate the specific nature of supershifted complexes. Arrows to the right indicate supershifted C/EBP complexes, and the bracket to the left identifies the DNA-protein complexes. The EMS reactions were performed in probe excess, and the unreacted free probe is visible at the bottom. The free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion.
FIG. 2
FIG. 2
ATF/CREB binding site sequence variation results in different binding reactivities with respect to CREB-1. (A) Four HIV-1 ATF/CREB sequence variants that differ from the HIV-1 consensus clade B sequence were selected. The ATF/CREB probes (15 bp) were designated as ATF/CREB Var1 to Var4 (divergent nucleotides are underlined). Probes spanning 27 nucleotides that included an ATF/CREB variant and the 3T C/EBP site I sequence were used in subsequent experimentation. The four ATF/CREB probes were used in EMS analyses in which radiolabeled oligonucleotides spanning the ATF/CREB site were reacted with nuclear extract prepared from a baculovirus-infected insect SF9 cell line which overexpressed CREB-1 (B) or the human monocytic U-937 nuclear extract (C). CREB-1 antisera was added (lanes 3, 6, 9, and 12) to demonstrate the presence of CREB in the DNA-protein complexes. Control rabbit immunoglobulin (CS) was added (lanes 2, 5, 8, and 11) to demonstrate the specific nature of supershifted complexes. The asterisk indicates potential ATF-related complexes. Arrows to the right indicate supershifted CREB-1 complexes, and the bracket to the left identifies the DNA-protein complexes. The EMS reactions were performed in probe excess, and the free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion (data not shown).
FIG. 3
FIG. 3
Probes containing ATF/CREB binding site sequence variants adjacent to a weakly reactive C/EBP binding site can differentially enhance binding of C/EBP nuclear factors derived from monocytic cell lines. (A) Chimeric probes containing the Var1, Var2, Var3, and Var4 ATF/CREB binding sites adjacent to the 3T C/EBP site I were reacted with U-937 (lanes 1 to 4), IL-6-induced U-937 (lanes 5 to 8), and THP-1 (lanes 9 to 12) nuclear extracts. Brackets to the left identify the DNA-protein complexes. (B) Probes containing the Var1 and Var2 ATF/CREB binding sites adjacent to the weakly reactive 3T C/EBP binding site were reacted with nuclear extract from IL-6-induced U-937 cells in EMS analyses. Antisera to C/EBP α (lanes 3, 7, 11, 15, 19, and 23) and C/EBP β (lanes 4, 8, 12, 16, 20, and 24) were added to identify the quantities of these proteins recruited to the DNA-protein complexes. A probe containing the single 3T C/EBP binding site was included (lanes 1 to 4 and 13 to 16) to demonstrate the level of C/EBP recruited to the weakly reactive site alone. Control rabbit immunoglobulin (CS) was added (lanes 2, 6, 10, 14, 18, and 22) to demonstrate the specific nature of supershifted complexes. Arrows to the right indicate supershifted C/EBP complexes, and brackets to the left identify the DNA-protein complexes. The EMS reactions were performed in probe excess, and the free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion (data not shown).
FIG. 4
FIG. 4
ATF/CREB variants adjacent to the weakly reactive 3T C/EBP binding site exhibit differential abilities to compete for C/EBP proteins derived from U-937 monocytic nuclear extract. Radiolabeled Var1/3T probe was reacted with U-937 nuclear extract in the presence of unlabeled competitor oligonucleotide Var1/3T (A), Var2/3T (B), Var3/3T (C), or Var 4/3T (D) in EMS analyses. The amount of remaining radioactive C/EBP complex was quantitated by phosphorimaging analyses. The fold amount of competitor required to decrease C/EBP binding by 50% is illustrated with a dashed line. Each graph is the summary of four independent experiments. The EMS reactions were performed in probe excess, and the free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion (data not shown).
FIG. 5
FIG. 5
Recruitment of different levels of CREB-C/EBP heterodimers and C/EBP homodimers in EMS analyses is dependent on ATF/CREB sequence variation at hybrid binding sites containing adjacent half sites of the ATF/CREB and C/EBP binding sites. (A) Several mechanisms may explain CREB enhancement of C/EBP recruitment. CREB/C/EBP heterodimers may bind to the ATF/CREB binding site (model 1), or CREB/C/EBP heterodimers may bind to a chimeric binding site created by half sites from the ATF/CREB and C/EBP binding sites (model 2). CREB homodimers may bind to the ATF/CREB site and recruit C/EBP dimers to their cognate sequence (model 3). (B) The Var1/3T hybrid half-site probe was reacted with U-937 (lanes 1 to 5) and IL-6-induced U-937 (lanes 6 to 10) nuclear extracts. Monoclonal antisera directed against C/EBP proteins (lanes 3, 4, 8, and 9) and CREB-1 (lanes 5 and 10) were added to the EMS reactions to identify the proteins binding to the hybrid half sites. Control rabbit immunoglobulin (CS) was added to lanes 2 and 7 to demonstrate the specific nature of supershifted complexes. (C) The Var4/3T hybrid half site was reacted with IL-6-stimulated U-937 nuclear extract. Antisera to C/EBP proteins were added to lanes 3, 4, 7, and 8. Control rabbit immunoglobulin (CS) was also added (lanes 2 and 6) to demonstrate the specific nature of supershifted complexes. Arrows to the right indicate supershifted C/EBP complexes (filled) and CREB-1 complexes (open), and brackets to the left identify the DNA-protein complexes. The EMS reactions were performed in probe excess, and the free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion (data not shown). (D) DNase I footprinting analysis was conducted using two 204-bp probes which contained either the Var4 ATF/CREB and 3T C/EBP binding sites within the context of the HIV-1 strain LAI backbone (lanes 1 and 2) or the Var1 ATF/CREB and highly reactive 6G C/EBP binding sites within the LAI backbone (lanes 3 and 4). The probes were reacted with purified six-histidine-tagged C/EBP β (lanes 2 and 4), and regions of DNase I footprinting were compared with probe incubated with DNase I only (lanes 1 and 3). Sequencing ladders of the probes were also included during electrophoresis (data not shown) to confirm the regions of DNase I footprint, and the confirmed binding sites are illustrated to the left.
FIG. 6
FIG. 6
EMS analyses with a Var1/linker/3T probe indicate that the majority of enhanced C/EBP binding to Var1/3T depends on CREB dimers binding to their cognate sequences. The Var1/3T (lanes 1 to 5) and Var1/linker/3T (lanes 6 to 10) probes were reacted with IL-6-stimulated U-937 nuclear extract. Antisera directed against C/EBP α (lanes 2 and 7) and C/EBP β (lanes 3 and 8) as well as CREB-1 (lanes 4 and 9) were all used to identify these proteins in the DNA-protein complexes. Control rabbit immunoglobulin (CS) was added to lanes 5 and 10 to demonstrate the specific nature of supershifted complexes. Arrows to the right indicate the supershifted C/EBP complexes (filled) and supershifted CREB-1 complexes (open), and brackets to the left identify the DNA-protein complexes. The EMS reactions were performed in probe excess, and the free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion (data not shown).
FIG. 7
FIG. 7
ATF/CREB sequence variation affects basal and IL-6-induced activity of the HIV-1 LTR with the weakly reactive C/EBP site I. Chimeric LTRs were constructed to contain the ATF/CREB variants adjacent to a weakly reactive 3T binding site within the context of the LAI LTR backbone. The parental LAI LTR (containing the clade B consensus sequence at the ATF/CREB site and the 6G C/EBP binding site) was also included in the analyses. The LTRs were transiently transfected into the U-937 cell line, and the cultures were treated with IL-6 for a 24-h interval. Firefly luminescence was normalized to the Renilla luminescence to control for variations in transfection efficiency. The luciferase activity of each of the chimeric LTRs was normalized to an arbitrary activity level of 1.0 for the parental LAI LTR. Basal (A) activity and IL-6-induced (B) activities were plotted separately. Each transient transfection was done in duplicate, with three independent experiments. The average luciferase count for the LAI LTR was 3,265 ± 1,075, and the average background count was 66 ± 16.
FIG. 8
FIG. 8
Highly reactive C/EBP binding sites can enhance binding of CREB-1 to an adjacent weakly reactive ATF/CREB site. Chimeric oligonucleotides containing the Var1 (A, lanes 1 to 10), Var2 (A, lanes 11 to 20), Var3 (B, lanes 1 to 10), and Var4 (B, lanes 11 to 20) ATF/CREB binding sites adjacent to either a weak 3T C/EBP site (A and B, lanes 1 to 5 and 11 to 15) or a strong 6G C/EBP binding site (A and B, lanes 6 to 10 and 16 to 20) were reacted with IL-6-induced U-937 nuclear extract in EMS analyses. Antisera specific for C/EBP α (lanes 3, 8, 13, and 18) and C/EBP β (lanes 4, 9, 14, and 19) were added to the indicated reactions. Control rabbit immunoglobulin (CS) was added (lanes 2, 7, 12, and 17) to demonstrate the specific nature of any supershifted complexes. Arrows to the right indicate supershifted C/EBP complexes (filled) and supershifted CREB-1 complexes (open) and brackets to the left identify the DNA-protein complexes. The EMS reactions were performed in probe excess, and the free probe accounted for approximately 75 to 90% of the total probe in each reaction at completion (data not shown).
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