COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

Abstract

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415-5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1alpha, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region.

Figure 1

CTIP1 and CTIP2 proteins repress HIV-1 gene transcription via the proximal LTR region. Microglial cells were transfected with 3 μg of pLTR-CAT or 3 μg of pLTR-CAT (287–535) in the presence or absence of 1 μg of HA-CTIP1 or Flag-CTIP2. Two days post-transfection, CAT activities were measured and expressed relative to the CAT activity obtained with pLTR-CAT alone with the standard deviations indicated (values correspond to an average of at least three independent experiments performed in duplicate).

Figure 2

CTIP1 and CTIP2 inhibit Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and related viral replication. Microglial cells were cotransfected with 3 μg of pLTR-CAT (A and B) or 3 μg of pNL4-3 (C and D), 1 μg of RSV-COUP-TF (A and C) or CMV-Sp1 (B and D) expression vectors and increasing amounts of HA-CTIP1 or Flag-CTIP2 (0.1, 0.5 or 1 μg). (A and B) Two days post-transfection, CAT activities were measured and expressed relative to the value obtained with pLTR-CAT alone with the standard deviation indicated (values correspond to an average of at least three independent experiments performed in duplicate). Western blot experiments were performed on nuclear extracts with antibodies directed against COUP-TF or Sp1 proteins as indicated. (C and D) Two days post-transfection, culture supernatants were analyzed for p24 Gag contents and expressed relative to the value obtained with pNL4-3 alone taken as 1. Depending on the cell confluency, this value varied between 500 and 5000 pg/ml. Values correspond to an average of at least three independent experiments performed in duplicate.

Figure 3

CTIP2 interacts in vitro with COUP-TF and Sp1 by two interfaces. (A) Upper panels: schematic representation of the COUP-TF and Sp1 proteins; lower panels: GST pull-down assays were performed with ³⁵S-labeled CTIP2 incubated with GST (lanes 2 and 7) or GST fusion proteins of the indicated COUP-TF domains (lanes 3–5) or Sp1 domains (lanes 8–11). Approximately 1% of the total ³⁵S-labeled CTIP2 obtained was loaded as input control (lanes 1 and 6). (B) GST pull-down assays were performed with ³⁵S-labeled full-length or truncated CTIP2 proteins incubated with GST (lanes 2 and 5), GST-COUP-TF (lane 6) or GST-Sp1 (lane 3) fusion proteins. Approximately 1% of the total ³⁵S-labeled proteins used were loaded as input control (lanes 1 and 4). Representative Coomassie stainings of GST, GST-Sp1 and GST-COUP-TF proteins were presented (B lower panels).

Figure 4

CTIP2 colocalizes with Sp1 and COUP-TF within Hp1α-associated structures. (A) Microglial cells were transfected or not with Flag-CTIP2 as indicated. After being treated, endogenous Sp1, COUP-TF and Hp1α proteins were immunodetected with primary anti-COUP-TF (Santa Cruz Biotechnology) (images 1 and 2), anti-Sp1 (images 6 and 7) and anti-Hp1α antibodies (images 11 and 12). Overexpressed Flag-CTIP2 was detected with antibodies directed against the Flag epitope (images 3, 8 and 13). The primary immunocomplexes were revealed by CY2- or CY3-labeled anti-species secondary antibodies (green or red staining). Mask column (images 5, 10 and 15) shows the colocalized CY2 and CY3 stainings. (B) Microglial cells expressing RFP-CTIP2 (image 1) and GFP-Sp1 (image 3) were subjected to endogenous COUP-TF immunodetection with anti-COUP-TF antibodies (kindly provided by J. E. Mertz). COUP-TF immunocomplexes were stained by CY5- (blue staining) labeled anti-species secondary antibodies (image 2). Pattern of RFP-CTIP2 and GFP-Sp1 expressed alone are presented on images 5 and 6, respectively. (A and B) Coverslips were subjected to confocal microscopy analysis. Bar, 10 μm.

Figure 5

CTIP2 interacts with COUP-TF, Sp1 and Hp1α in microglial cells. (A) Nuclear extracts of microglial cells expressing (lanes 2, 4, 5, 7, 8 and 10) or not (lanes 3, 6 and 9) Flag-CTIP2 were immunoprecipitated with antibodies directed against COUP-TF (lanes 3 and 4), Sp1 (lanes 6 and 7) or Hp1α (lanes 9 and 10) proteins. Proteins were separated by SDS–PAGE and western blot analysis with anti-Flag antibodies were performed. (B) GST pull-down competition assays were performed with equal amounts of ³⁵S-labeled 145–434 and 717–813 CTIP2 proteins and increasing amounts of ³⁵S-labeled Hp1α. Proteins were incubated with GST-COUP-TF or GST-Sp1 fusion proteins as indicated. (C) GST pull-down assays were performed with equal amounts of ³⁵S-labeled Sp1 and COUP-TF proteins and GST or GST-CTIP2 fusion proteins as indicated. (D) GST pull-down competition assays were performed with the indicated amounts of ³⁵S-labeled 145–434 CTIP2 and COUP-TF proteins and GST-Sp1. (A–D) Approximately 1% of the total ³⁵S-labeled proteins used were loaded as input control.

Figure 6

CTIP2 is anchored to the HIV-1 LTR by direct interactions with Sp1 bound to the LTR proximal region. (A and B) EMSA experiments were performed using purified Sp1, GST, GST-COUP-TF and GST-CTIP2 fusion proteins. Proteins were incubated with a ³²P-labeled probe corresponding to the three Sp1 binding sites located downstream of the LTR (LAI) TATAA sequence. (A) Increasing amounts of GST-CTIP2 proteins correspond to 5 μl (lane 9) and 10 μl (lane 10) of the used GST-CTIP2 preparation. Supershift experiments performed in (B) were carried out with non-immune serum (lane 5) and with antibodies directed against CTIP2 (lane 2), COUP-TF (lane 3), and Sp1 (lane 4). The specific shifted and supershifted complexes are presented. (C) The TZM-bl cells, which contain a stably integrated LTR, were transfected or not with 30 μg of the indicated Flag-CTIP2 expression vector. (D) HEK 293T cells were transfected with 5 μg of the indicated pLTR (left panel) or pLTR mutGC (right panel) constructs and 10 μg of Flag-CTIP2 expression vector if indicated. (C and D) Input lanes correspond to positive controls conduced with a fraction of the lysates used for the immunoprecipitation. Control lanes correspond to negative controls, in which immunoprecipitation reactions were performed without antibodies. Anti-Flag, anti-Sp1, anti-COUP-TF and anti-Hp1α lanes represent amplification reactions from samples immunoprecipitated with the indicated antibodies. Results are representative of three independent experiments.

Figure 7

Sp1 binding sites are dominant and sufficient but not absolutely necessary for transcriptional repression mediated by CTIP2. (A) Microglial cells were transfected with 3 μg of pGC-WAP-CAT reporter vector and 1 μg of expression vectors as indicated. (B) Microglial cells were transfected with 3 μg of pLTR-CAT, pLTR-CAT mutGC or pLTR-CAT ΔGC and 1 μg of the indicated Flag-CTIP2 vector. Two days post-transfection, CAT activities were measured and expressed relative to the value obtained with the reporter plasmids pGC-WAP-CAT or pLTR-CAT alone with the standard deviations indicated (values correspond to an average of at least three independent experiments carried out in duplicate).