Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein - PubMed (original) (raw)

Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein

Miriam Denne et al. J Virol. 2007 Jun.

Abstract

Only few of the human endogenous retrovirus (HERV) sequences in the human genome can produce proteins. We have previously reported that (i) patients with germ cell tumors often make antibodies against proteins encoded by HERV-K elements, (ii) expression of the HERV-K rec gene in transgenic mice can interfere with germ cell development and induce carcinoma in situ, and (iii) HERV-K np9 transcript is overproduced in many tumors including breast cancers. Here we document that both Np9 and Rec physically and functionally interact with the promyelocytic leukemia zinc finger (PLZF) tumor suppressor, a transcriptional repressor and chromatin remodeler implicated in cancer and the self-renewal of spermatogonial stem cells. Interaction is mediated via two different central and C-terminal domains of Np9 and Rec and the C-terminal zinc fingers of PLZF. One major target of PLZF is the c-myc proto-oncogene. Coexpression of Np9 and Rec with PLZF abrogates the transcriptional repression of the c-myc gene promoter by PLZF and results in c-Myc overproduction, altered expression of c-Myc-regulated genes, and corresponding effects on cell proliferation and survival. Thus, the human endogenous retrovirus proteins Np9 and Rec may act oncogenically by derepressing c-myc through the inhibition of PLZF.

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Figures

FIG. 1.

The origins of the endogenous retrovirus proteins Np9 and Rec. (A) Schematic presentation of the HERV-K type 1 and 2 proviral sequences. The indicated open reading frames encode the proteins Gag (group-specific antigens and the viral capsid proteins), Prt (protease), Pol (reverse transcriptase/RNase H) and Env (envelope proteins). A 292-bp deletion in the env reading frame of HERV-K type 1 gives rise to the alternatively spliced np9 transcript; the rec transcript is spliced from the full-length env reading frame in the type 2 sequence. Np9 and Rec share the N-terminal 14 amino acid residues (hatched boxes). NES, nuclear export signal; LTR, long terminal repeat. (B) Results of reverse transcription-PCR amplification of the rec and np9 transcripts from Tera-1 cells are shown in the top panel. M, marker; RT, reverse transcriptase. The bottom panel shows the expression of Rec and Np9 in Tera-1 cells. Western blot analysis was performed on 15 μg total protein with the rabbit polyclonal anti-Np9 antibody K82 and anti-Rec antibody K3086 at a 1:100 dilution.

FIG. 2.

Mapping of the interaction of Np9 and Rec with PLZF by GST pull-down assays. (A) GST-Np9 and the indicated GST-Np9 mutants but not GST alone precipitate full-length in vitro translated 35S-labeled PLZF. An outline of the Np9 variants and the PLZF interaction is also shown at bottom. Np9-55mut1 harbors a mutation that incapacitates the NLS1. (B) GST-Rec and the indicated variants bind to radiolabeled PLZF. A scheme summarizing the interactions is given below. NES, nuclear export signal. (C) GST-Np9 but not GST alone precipitates the indicated full-length or truncated variants of in vitro translated 35S-labeled PLZF. (D) GST-Rec precipitates the indicated deletion mutants of radiolabeled PLZF. (E) In vitro coimmunoprecipitation of Np9 and PLZF. The GST-Np9 and PLZF proteins as well as the Np9 binding-defective PLZF(245/543) were transcribed and translated in vitro and coprecipitated with the anti-GST monoclonal antibody 6G9. Note that GST-Np9 fails to bring down PLZF(245/543).

FIG. 3.

Schematic summary of the interaction between Np9 and Rec with PLZF and comparison of the Np9 and Rec domains that interact with PLZF. (A) The NLS1 region between amino acid residues 23 and 29 on Np9 associates with the four C-terminal zinc fingers of PLZF. On Rec, a region between amino acid residues 21 and 47 situated downstream of the NLS and the C-terminal 16 amino acid residues make contact with the PLZF zinc fingers. NES, nuclear export signal. (B) Alignment and comparison of the PLZF-interacting domains on Np9 and Rec (highlighted). NES, nuclear export signal. Note the differences between the PLZF-interacting NLS1 of Np9 and the noninteracting NLS of Rec.

FIG. 4.

Intracellular localization of Np9, Rec, and PLZF. (A) Cos-1 cells were transiently transfected with plasmids producing the indicated fusion proteins involving EGFP or the red fluorescent protein (Dsred). (B) Transient transfection of Cos-1 cells with plasmids producing the indicated fusion proteins revealed a partial colocalization of Np9 and Rec with PLZF in dot-like subnuclear structures. Some of these were identical with the nucleoli spared in the DAPI-stained nuclei. (C) Transient transfection of Tera-1 cells with the indicated plasmids.

FIG. 5.

Effect of Np9 and Rec on the repression of the c-myc promoter by PLZF. (A) 293T cells were transiently cotransfected with the reporter plasmids cmyc2.5 or cmyc2.5ΔPLZF defective for PLZF binding and with either empty vector or an effector plasmid producing PLZF. The relative luciferase activity was calculated as the percentage of the controls. Error bars indicate the standard deviations from at least three experiments. (B) Transient cotransfection of the cmyc2.5 reporter plasmid with empty vector alone, with empty vector plus Np9- or Rec-producing effector plasmids, and with the PLZF expression plasmid. Note that Np9 and Rec alone do not affect luciferase expression in these PLZF-negative cells. In contrast, PLZF represses luciferase production, and Np9 and Rec can overcome this effect. Luc, luciferase.

FIG. 6.

Effect of Rec on the regulation of the endogenous c-myc gene by PLZF. (A) U937T cells that conditionally produce PLZF and a derived cell line which, in addition, expresses Rec from the CMV promoter/enhancer, give rise to equal levels of PLZF upon exposure to low levels of Tet. Western immunoblottings were performed on the indicated quantities of total cell protein with the anti-PLZF monoclonal antibody at a dilution of 1:50, the anti-β actin monoclonal antibody at a dilution of 1:2,500, and the anti-Rec antiserum K3086 at a dilution of 1:100. (B) Western blot analysis shows that Rec expression causes overproduction of endogenous c-Myc despite equally high levels of PLZF. The monoclonal c-Myc antibody 9E10 was used at a dilution of 1:500. The bar diagram indicates the relative signal intensities of the PLZF and c-Myc bands, normalized to β-actin and measured by laser densitometry. The standard deviations of the levels of c-Myc depending on Rec were determined from three blots. (C) Western blots documenting the elevated expression of c-Myc and of known c-Myc-responsive genes in the presence of Rec. The anti-p53 monoclonal antibody DO-1 was used at 1:2,000, the anti-PCNA monoclonal antibody SC56 was used at 1:200, the anti-IκBα antibody was used at 1:500, and the anti-actin antibody was used at 1:2,500. The bar diagrams show the signal intensities.

FIG. 7.

Cell proliferation and apoptosis in dependence of Rec expression. (A) U937 cells that conditionally produce PLZF and a derived cell line which, in addition, expresses Rec from the CMV promoter/enhancer, were exposed to the indicated doses of Tet for 24 h. Cell proliferation was determined by measuring absorbance in a standard MTT assay. Error bars denote standard deviations from three experiments. (B) Flow cytometry was employed to determine the number of cells with a sub-2N DNA content indicative of apoptosis, after 24 h of weak (0.005 μg/ml Tet) or strong (no Tet) expression of PLZF. Error bars show standard deviations from three experiments.

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Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein - PubMed (original) (raw)