Intracellular forms of human NOTCH1 functionally activate essential Epstein-Barr virus major latent promoters in the Burkitt's lymphoma BJAB cell line but repress these promoters in Jurkat cells - PubMed (original) (raw)

Intracellular forms of human NOTCH1 functionally activate essential Epstein-Barr virus major latent promoters in the Burkitt's lymphoma BJAB cell line but repress these promoters in Jurkat cells

M Cotter et al. J Virol. 2000 Feb.

Abstract

We have demonstrated that intracellular forms of NOTCH1 transactivate two major Epstein-Barr virus (EBV) latent promoters, the LMP1 and Cp1 promoters in an EBV-negative B-cell line, BJAB. Truncated intracellular NOTCH1 associated with the nuclear membrane (DeltaE) transactivates the LMP1 promoter fivefold; however, the intranucleus localized form of NOTCH1 (ICN) transactivates this promoter approximately twofold in chloroamphenicol acetyltransferase (CAT) reporter assays in BJAB cells. Additionally, DeltaE activated the major Cp1 promoter 12-fold, whereas the ICN form of NOTCH1 activates at only about half that level when compared to that of DeltaE membrane-bound NOTCH1. This result differs from previously observed data, where intracellular NOTCH1 bound to the nuclear membrane, DeltaE, and nucleus-localized NOTCH1, ICN, all had similar levels of activation in 293 cells. This suggests distinct transcriptional activities in different cell types. Moreover, in Jurkat cells, a T-cell line, intranucleus localized NOTCH1 molecules demonstrated a repressive activity against the two EBV major latent promoters. Only DeltaE activated the Cp1 and LMP1 promoters at a level slightly above background, whereas intranucleus localized NOTCH1 ICN, or the form of NOTCH1 lacking the ankyrin repeats, DeltaE(TAR), surprisingly resulted in the repression of these promoters in Jurkat cells. Similarly, another truncated form of NOTCH1, referred to as ICNW, which contains the tryptophan residue W(1767) within one of the RBP-Jkappa interacting domains, repressed the LMP1 promoter approximately twofold. Further analysis of the truncated NOTCH1 molecules on the LMP1 promoter element, lacking the two RBP-Jkappa binding sites, suggests that repression in Jurkat cells may be affected by the presence of the two RBP-Jkappa binding sites. These studies indicate that intracellular NOTCH1 can activate the EBV major latent promoters in BJAB cells. However, in Jurkat cells, intracellular truncated forms of NOTCH1 lacking the RBP-Jkappa binding sites repress these EBV latent promoters. Only the membrane-bound form of NOTCH1, DeltaE, activated the EBV major latent promoters in Jurkat cells, albeit at a lower level than that seen in BJAB cells. Our data suggest that EBNA2 and truncated intracellular nuclear localized forms of NOTCH1 may be functionally similar in their interactions with RBP-Jkappa; however, these molecules may have distinctly different transcriptional partners in BJAB and Jurkat cells. Moreover, these truncated NOTCH1 molecules may not represent the normal processed forms of NOTCH1 in cells and may exhibit dominant negative phenotypes in the absence of the required posttranslational modifications. Further investigations are necessary to determine the similarity and differences occurring with intracellular NOTCH1 in other B- and T-cell lines.

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Figures

FIG. 1

Schematic diagram showing the full-length NOTCH1 protein with the identified extracellular and intracellular domains. The epidermal growth factor-like repeats and the lin-12-like repeats are positioned extracellular to the transmembrane domain. Two cysteine repeats are potentially involved in the formation of disulfide bridges. The cdc10 (ankyrin) repeats, the two nuclear localization signals (N1 and N2), and the PEST (P) and OPA (O) sequences are intracellular domains (6). The ΔE construct contains the leader peptide, the transmembrane domain, and the intracellular region of NOTCH1 and localizes to the nuclear membrane. The ICN construct lacks the leader peptide and the transmembrane region and localizes to the nucleus. The ΔETAR construct contains the same sequence as ΔE but lacks the ankyrin repeats (3). ICNW contains the entire intracellular domain, including W1767 that is crucial for RBP-Jκ association (3).

FIG. 2

Intracellular forms of NOTCH1 activate the major latent EBV promoters in BJAB cells. The CAT gene was used as the reporter for activity on the Cp1 (A) and −512/+40 LMP1 (B) promoter regions. Ten micrograms of each reporter construct and 20 μg (each) of the constructs cloned into pCDNA3.1 were transfected into BJAB cells to measure the level of transactivation of each reporter construct. Levels labelled LMP1 and Cp1 indicate transfection with reporter alone for basal activity. PCDNA3.1 vector DNA was used to normalize the amount of DNA in each transfection, and 2.5 μg of an expression plasmid containing the β-galactosidase gene was used as an internal control for transfection. Activity was counted on a Molecular Dynamics PhosphorImager and values are expressed as arbitrary units.

FIG. 3

Intracellular nucleus-localized forms of NOTCH1 repress the major latent EBV promoter elements in the Jurkat cells. Transient CAT reporter assays with the EBV Cp1 promoter (A) and transient transactivation activity on the EBV −512/+40 LMP1 promoter (B) are shown. Cells were transfected with equivalent amounts of DNA, including 10 μg of each reporter plasmid and 20 μg (each) of the various NOTCH1 constructs. The total amount of DNA was normalized by adding vector DNA. Levels labelled LMP1 and Cp1 represent transfection with reporter alone to determine basal levels of activation. The transfected cells were harvested after 22 h and CAT assays were done as described previously (3, 22). Data were collected on a Molecular Dynamics PhosphorImager and values are expressed as arbitrary units.

FIG. 4

Intracellular forms of NOTCH1 repress the larger −2350 LMP1 promoter element lacking the RBP-Jκ binding sites in Jurkat cells. Activity on the larger wild-type −2350 LMP1 promoter element (A) and activity of intracellular NOTCH1 on the −2350 (lacking the RBP-Jκ binding site) promoter construct (B) are shown. The effect of ICNW and EBNA2 on the −2350 LMP1 element lacking the RBP-Jκ binding sites (C) is shown. Ten million Jurkat cells were transfected with the appropriate amounts of DNA, and the cells were harvested after 22 h. Lysates were then used for CAT reporter assays, and the results were obtained with a PhosphorImager from Molecular Dynamics. Only ΔE transactivated this LMP1 promoter, lacking RBP-Jκ binding sites, slightly above background. All other forms of NOTCH1 repressed this promoter in this assay.

FIG. 5

Immunolocalization of activated forms of NOTCH1 in BJAB and Jurkat cells. As expected, ΔE constructs localize predominantly to the nuclear membrane in Jurkat and BJAB cells, whereas ICNW and ICN localize mostly to the nucleus (A). The upper section of panel A shows the immunofluorescence of transfected BJAB cells with the different intracellular NOTCH1 constructs. Note the preferential membrane localization of the ΔE constructs compared to the ICN construct, which predominantly localizes to the nucleus. The lower section shows the transfected Jurkat cells with the forms of activated NOTCH1 and shows similar localization of ΔE to the membrane and ICNW and ICN to the nucleus. A Western blot analysis of the transfected BJAB and Jurkat cells with the different NOTCH1 constructs is shown (B). Transfected cells were harvested, and 500,000 cells were then collected, washed in PBS, lysed, and fractionated by electrophoresis on a sodium dodecyl sulfate–6% polyacrylamide gel. The fractionated proteins were then transferred to nitrocellulose and then probed with rabbit polyclonal antibody against the cytoplasmic domain of NOTCH1. Note the expression of the different forms of NOTCH1 in all lanes.

FIG. 6

Schema of the potential interactions of RBP-Jκ and endogenous and exogenous forms of activated NOTCH1. In BJAB cells, RBP-Jκ is available for interaction with the transfected forms of NOTCH1, therefore activating the promoters. In Jurkat cells, the available RBP-Jκ is sequestered with endogenous activated forms of NOTCH1 and competes with the transfected NOTCH1 molecules, resulting in a dominant negative phenotype that is seen by repression of the EBV latent promoters.

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Intracellular forms of human NOTCH1 functionally activate essential Epstein-Barr virus major latent promoters in the Burkitt's lymphoma BJAB cell line but repress these promoters in Jurkat cells - PubMed (original) (raw)