The E2 protein encoded by human papillomaviruses (HPVs) inhibits expression of the viral E6 oncoprotein, which, in turn, regulates p53 target gene transcription. To identify cellular proteins involved in E2-mediated transcriptional repression, we isolated an E2 complex from human cells conditionally expressing HPV-11 E2. Surprisingly, the double bromodomain-containing protein Brd4, which is implicated in cell cycle control and viral genome segregation, was found associated with E2 and conferred on E2 the ability to inhibit AP-1-dependent HPV chromatin transcription in an E2-binding site- [Keywords: HPV; E2; AP-1; Brd4; chromatin transcription; gene silencing] Supplemental material is available at http://www.genesdev.org.

Transcription in human papillomaviruses (HPVs) is mainly regulated by cellular transcription factors and virus-encoded E2 proteins that act as sequence-specific DNA-binding proteins. Although the functions of E2 as a transcriptional activator and a repressor have been well documented, the role of cellular factors involved in E2-mediated regulation of the HPV promoters and the mechanism by which E2 modulates viral gene expression remain unclear. Using reconstituted cell-free transcription systems, we found that cellular enhancerbinding factors and general cofactors, such as TAF II s, TFIIA, Mediator, and PC4, are not required for E2-mediated repression. Unlike other transcriptional repressors that function through recruitment of histone deacetylase or corepressor complexes, HPV E2 is able to directly target components of the general transcription machinery to exert its repressor activity on the natural HPV E6 promoter. Interestingly, preincubation of TATA binding protein (TBP) or TFIID with HPV template is not sufficient to overcome E2-mediated repression, which can be alleviated only via formation of a minimal TBP (or TFIID)-TFIIB-RNA polymerase II-TFIIF preinitiation complex. Our data therefore indicate that E2 does not simply work by displacing TBP or TFIID from binding to the adjacent TATA box. Instead, E2 appears to function as an active repressor that directly inhibits HPV transcription at steps after TATA recognition by TBP or TFIID.Transcription in eukaryotes is often regulated by extracellular molecules that act through distinct signal transduction pathways to modulate specific gene expression via controlling the activity of gene-specific transcription factors. These genespecific transcription factors then work in conjunction with general transcription factors (GTFs) and cofactors to enhance or inhibit the level of transcription. Although many studies have been conducted to elucidate the mechanisms of transcriptional activation in eukaryotes, relatively little is known about the mechanisms of repression. In general, transcriptional repressors can work either passively to antagonize the activator function or actively to inhibit the activity of the general transcription machinery (30). Counteraction of the activator function by passive repressors can be achieved by direct competition of the same DNA-binding sites (36,37,41,54,55), interference of overlapping or neighboring activator-binding sites (21,24,38,58), modification of the DNA-binding property of the activators (60), titrating away limiting protein factors required for activator function (15, 31), or masking and/or altering the function of the activation domain or blocking the DNA-binding activity of the activators through protein-protein interactions (3,24,46,61). In contrast, active repressors are able to directly inhibit the activity or the assembly of the general transcription machinery, with or without the help of corepressors (2,23,27,29,40,43,45,51). The recruitment of histone deacetylase complexes by some repressors or corepressors represen...

The full-length E2 protein, encoded by human papillomaviruses (HPVs), is a sequence-specific transcription factor found in all HPVs, including cancer-causing high risk HPV types 16 and 18 and wart-inducing low risk HPV types 6 and 11. To investigate whether E2 proteins encoded by high risk HPVs may function differentially from E2 proteins encoded by low risk HPVs and animal papillomaviruses, we conducted comparative DNAbinding and transcription studies using electrophoretic mobility shift assays and cell-free transcription systems reconstituted with purified general transcription factors, cofactor, RNA polymerase II, and with E2 proteins encoded by HPV-16, HPV-18, HPV-11, and bovine papillomavirus type 1 (BPV-1). We found that although different types of E2 proteins all exhibited transactivation and repression activities, depending on the sequence context of the E2-binding sites, HPV-16 E2 shows stronger transcription activity and greater DNA-binding affinity than those displayed by the other E2 proteins. Surprisingly, HPV-18 E2 behaves more similarly to BPV-1 E2 than HPV-16 E2 in its functional properties. Our studies thus categorize HPV-18 E2 and BPV-1 E2 in the same protein family, a finding consistent with the available E2 structural data that separate the closely related HPV-16 and HPV-18 E2 proteins but classify together the more divergent BPV-1 and HPV-18 E2 proteins.Human papillomaviruses (HPVs) 1 are a family of small DNA viruses that cause a wide variety of human diseases ranging from benign epithelial lesions, such as warts, to invasive cancers, such as cervical carcinoma. So far, more than 100 HPV types have been identified and fully sequenced, whereas more than 120 putative novel types have been partially characterized (1, 2). HPV types frequently found in invasive cancers include These are classified as high risk HPVs. In contrast, HPV types that are rarely found in cancers but are associated with genital warts, such as HPV-6 and HPV-11, are considered low risk HPVs (1-3). Because the genomic structures of HPVs are highly conserved, it is important to determine the functional differences among individual HPV gene products which lead to etiologically high and low risk phenotypes. Previous comparative studies have primarily focused on HPV-encoded E6-and E7-transforming proteins. These studies found that E6 and E7, from high risk HPVs, lead to cellular transformation much more readily than low risk E6 and E7 proteins (for review, see Refs. 4 and 5). In both high and low risk HPVs, expression of E6 and E7 is transcriptionally regulated via the E6 promoter by many cellular and viral proteins. The full-length viral E2 protein is a sequence-specific transcription factor that functions as an activator or repressor to regulate tightly the E6 promoter through four consensus E2-binding sites (E2-BSs), ACCGN 4 CGGT (6, 7), whose locations within the upstream regulatory region (URR) are highly conserved among genital HPVs. Efficient activation of the E6 promoter requires binding of E2 protein to the promoter...

TFIID is a general transcription factor required for the assembly of the transcription machinery on most eukaryotic promoters transcribed by RNA polymerase II. Although the TATA-binding subunit (TBP) of TFIID is able to support core promoter and activator-dependent transcription under some circumstances, the roles of TBP-associated factors (TAF II s) in TFIID-mediated activation remain unclear. To define the evolutionarily conserved function of TFIID and to elucidate the roles of TAF II s in gene activation, we have cloned the mouse TAF II 55 subunit of TFIID and further isolated mouse TFIID from a murine FM3A-derived cell line that constitutively expresses FLAG-tagged mouse TAF II 55. Both mouse and human TFIIDs are capable of mediating transcriptional activation by Gal4 fusions containing different activation domains in a highly purified human cellfree transcription system devoid of TFIIA and Mediator. Although TAF II -independent activation by Gal4-VP16 can also be observed in this highly purified human transcription system with either mouse or yeast TBP, TAF II s are strictly required for estrogen receptor-mediated activation independently of the core promoter sequence. In addition, TAF II s are necessary for transcription from a preassembled chromatin template. These findings clearly demonstrate an essential role of TAF II s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.Regulation of eukaryotic transcription by gene-specific transcription factors often requires protein cofactors, in addition to the general transcription machinery. Currently, there are three classes of general cofactors commonly thought to be essential for activator-dependent transcription. The first class is RNA polymerase II-specific TBP-associated factors (TAF II s) 1 initially defined as components of TFIID (1-6). TAF II s are highly conserved through evolution and exhibit many properties accounting for the functional activities of TFIID. In general, TFIID is a core promoter-binding factor that has intrinsic activity to recognize the TATA box, initiator and downstream promoter elements, and initiates preinitiation complex assembly on both TATA-containing and TATA-less promoters (1,7,8). The nucleation pathway for preinitiation complex formation usually begins with TFIID binding to the core promoter region, followed either by sequential assembly of other general transcription factors (GTFs) and RNA polymerase II (pol II) or by recruitment of a preassembled pol II holoenzyme complex (9 -11). In addition to the core promoter-binding activity, TFIID has also been implicated as a general coactivator or corepressor in transducing the regulatory signals to the general transcription machinery, as exemplified by many protein-protein interactions occurring between gene-specific regulatory factors and components of TFIID (1)(2)(3)(12)(13)(14). A universal coactivator function of TFIID has recently been challenged by both in vivo yeast studies (15)(16)(17) and in vitro mammalian cell-free transcription assays ...

BackgroundCD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies.MethodsGBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401.ResultsGBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization.ConclusionThese results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

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Background: Antibody strategies are of huge importance in targeted therapies drug development. However, most of the time, antigen is specific of human species with bad cross-reactivity with mouse antigen, leading to difficulties to apply regular mouse models. The “gold” model for the preclinical evaluation of antibody strategy should have cells expressing human antigen, to analyze Fab binding activity but should also contain human immune effector cells in order to be able to test the antibody Fc mediated activity (Antibody Dependent Cell Cytotoxicity, ADCC). We here propose the use of CB17-SCID mouse strain having the ability to exhibit complement activation, useful to test the CDC (Complement Dependent Cytotoxicity) activity of antibody. This strain of mice supports the engraftment of human peripheral blood mononuclear cells (hPBMCs), containing human effector cells as NK cells and moncoytes, responsible of ADCC activity. In our case, the target of interest is expressed on human B lymphocytes, whatever the tumoral or healthy origin. The engrafted hPBMCs contained both the NKs as effector cells but also the human B cells as target cells. Methods: Whole body irradiated (D-3) female CB17-SCID mice were treated twice (Q7D×2) (starting at D-2) with subcutaneous injections of mouse NK cell-depleting antibody TM-Beta 1 and received an intraperitoneal injection of freshly prepared hPBMCs at D0. At D11 mice received a single intravenous injection of trastuzumab used as negative control, of new B cell targeting antibody or rituximab used as positive control (10 mice per group). At D15, mice were sacrificed to collect spleen. Single cell suspensions were prepared from each collected spleen, labeled with mCD45, hCD45, hCD19 and hCD20 antibodies before FACS analysis. Absolute cell number as well as percentage of human B cells were calculated. Statistical comparisons were performed using Mann-Whitney U test after having discarded outlier values by Dixon Q test. Results: All mice injected with hPBMCs were found humanized at the time of sacrifice. Percentage of human leukocytes in mouse spleen ranged from 0.6 to 59.4% with a mean value of 12.9 ± 12.5 % and a median value of 8.3 %. Considering the human B-cell percentage, only 3 out of 80 values were considered as outliers. In the trastuzumab treated group (10 mg/kg), the spleen contained about 6 % of human B cells (within the human leukocytes, i.e. hCD45+). When the mice were treated with rituximab at 2 mg/kg, a significant decrease in the percentage of human B cells was evidenced in the mouse spleen, with about 1.5% of human B cells. A dose-dependent decrease in human B cells was also observed in mouse spleen when mice were treated with the new B-cell targeting antibody (1.4 ± 1.1 % and 3.8 ± 2.2 % of human B cells for 10 mg/kg and 0.02 mg/kg respectively). The decrease starts to be significant from 0.1 mg/kg of the new B-cell targeting antibody. Conclusions: These results show that this humanized mouse model is suitable to evaluate B-cell targeting therapies. Humanization level as well as sensitivity to treatment are compatible with dose-response analysis and then offer the possibility to use this model for benchmarking of human B-cell targeting therapy tested on human target in the presence human effectors. Moreover, this model could be applied to other targets expressed on human immune cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B122.

GBR 401 is a humanized monoclonal antibody directed against domain 2 of human B-lymphocyte antigen CD19 and displays a number of mechanisms which can specifically target and kill B cells. Glenmark is developing this product as a B-cell depleting agent for B cell malignancies. CD19 is the archetypal B cell marker and is used in the diagnosis of B cell lymphomas/ leukemias by the identification of abnormal B cell numbers and/or populations. Expression of CD19 is found in the majority of B cell lineage malignancies including, but not limited to non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Given its role as a signaling molecule, an antibody directed against CD19 might be predicted to affect the progression of lymphomas and leukemias. GBR 401 is a humanized IgG1 which can specifically kill CD19 bearing cells with very efficient in vitro antibody-dependent cellular cytotoxicity (ADCC) and rapid-onset apoptosis-inducing abilities. It also can inhibit the proliferation of CD19+ tumor cells but has little to no complement dependent cytotoxicity (CDC), at least, in vitro. The in vivo anti-B cell activity of GBR 401 was evaluated using human peripheral blood mononuclear cell (huPBMC) graft models in SCID mice. GBR 401 significantly depleted human B cells in grafted mice at levels down to 0.1mg/kg compared to an isotype control. GBR 401 also produced approximately 50% depletion or EC50 at 0.02mg/kg. These in vitro and in vivo pharmacology data provide a rationale for the clinical testing of GBR 401 in patients with CD19+hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C164.