Bcl-2 P2 Promoter Research Articles

Oct transcription factors mediate t(14;18) lymphoma cell survival by directly regulating bcl-2 expression

Oct-1 and Oct-2 are members of the POU homeodomain family of transcriptional regulators and are critical for normal embryonic development. Gene-targeting studies showed that Oct-1 and Oct-2 are largely dispensable for B-cell development and immunoglobulin production, although both Oct-2 and Bob-1 are required for a proper immune response and germinal center formation. In these studies, we investigated the role of Oct factors in B-cell lymphomas. Recent investigations have shown increased expression of Oct-2 and Bob-1 in lymphomas, and we observed greatly increased levels of Oct-2 in lymphoma cells with the t(14;18) translocation. Decreased expression of Oct-1, Oct-2, or Bob-1 by RNA interference resulted in apoptosis and down-regulation of bcl-2 expression. Furthermore, Oct-2 induced bcl-2 promoter activity and mediated this effect through three regions in the bcl-2 P2 promoter. Although these regions did not contain canonical octamer motifs, we observed the direct interaction of Oct-2 with all three sites both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. Moreover, by mutation analysis we found that the ability of Oct-2 to activate bcl-2 required C/EBP, Cdx, and TATA-binding sites. Oct-2, therefore, acts as a cell survival factor in t(14;18) lymphoma cells by directly activating the antiapoptotic gene bcl-2.

C/EBPα and C/EBPα Myeloid Oncoproteins Inhibit Apoptosis and Induce Bcl-2 Via DNA-Binding Dependent and Independent Mechanisms.

C/EBPα dimerizes via its leucine zipper domain and then binds DNA via the adjacent basic region. One or both copies of the C/EBPα gene harbors point mutations in 5–8% of human AMLs. One common group of mutations leads to expression of C/EBPαp30, lacking an N-terminal transactivation domain. In addition, insertions and deletions commonly occur in the first α-helix of the leucine zipper, preventing DNA-binding - these alterations are typically in-frame, suggesting that the C/EBPαLZ mutants retain a role in transformation. We have now found that C/EBPα, C/EBPαp30, and four C/EBPαLZ mutants inhibit the apoptosis which occurs when the 32Dcl3 or HF1 myeloid or Ba/F3 or FL5.12 lymphoid cell lines are withdrawn from IL-3; for example, 30% of Ba/F3-αER cells survived at 24 hrs and 0–2% at 48 hrs in the absence of estradiol, and 180%, 80%, and 40% survived at 1, 2, or 3 days in its presence. However, TNF death receptor-mediated apoptosis was not affected. C/EBPα-ER did not alter expression of STAT3, P-STAT3, STAT5, P-STAT5, MAPK, P-MAPK, PI3K, AKT, c-Jun, JunB, bcl-xL, or bim in Ba/F3 cells. However, bcl-2 protein levels, which diminish several-fold after IL-3 withdrawal, were elevated 5-fold at 16 hours by C/EBPα-ER, but not in vector-transfected cells exposed to estradiol. Northern blotting demonstrated induction of transcription from the bcl-2 P2 promoter. Induction of bcl-2 by C/EBPα was previously seen in t(14;18) lymphoma lines, in which the bcl-2 gene is fused to the immunoglobulin heavy chain promoter, and C/EBPα was shown to transactivate P2-luciferase, in part via interaction with a Cdx:A-Myb complex. We have now also identified a consensus C/EBP-binding site, using EMSA, in the P2 promoter. C/EBPα-ER also induced endogenous bcl-2 in NIH 3T3 cells, in transduced normal murine myeloid progenitors, and in the splenocytes of H2K-Eμ-C/EBPα transgenic mice. C/EBPα enhanced survival of splenocytes exposed to low-dose ionizing radiation. The ability of additional C/EBPα mutants to induce bcl-2 and inhibit apoptosis was assessed. The BRM2 mutant, which does not bind E2F, Δ3–8, lacking transactivation domains, GZ, having the GCN4 leucine zipper, and L12V, which cannot dimerize, each retained these activities. In contrast, BR3, with 4 mutant residues in the basic domain, did not induce bcl-2 and only weakly prevented cell death. The BR3 mutations were then studied individually. Only R300G prevented DNA-binding; this mutant did not induce bcl-2 and only weakly inhibited cell death. K298T and K302N each potently induced bcl-2 and prevented apoptosis. Finally, R297G only weakly induced bcl-2 and had an intermediate effect on survival. Perhaps R300G alters the conformation of R297, explaining R300G's inability to induce bcl-2. In summary, C/EBPα stimulates cell survival, in part via direct activation of the bcl-2 P2 promoter. This induction is optimal when C/EBPα retains its ability to bind DNA, but can also occur via protein:protein interaction, perhaps mediated in part by R297 with the Cdx:A-Myb complex or with other proteins which regulate the bcl-2 gene, such as NF-κB. Targeting this interaction with the outer surface of the C/EBPα basic region may be an effective means to identify novel therapeutics for AMLs expressing C/EBPα oncoproteins.

HDAC2 Plays a Role in Protecting t(14;18) Lymphoma Cells from Apoptosis by Up-Regulation of Bcl-2.

Histone deacetylases (HDACs) usually cause repression of gene expression through deacetylation of histones H3 and H4. Inappropriate transcriptional repression by HDACs has been observed in some malignancies, and several HDAC inhibitors are in clinical trials to treat solid tumors and hematologic malignancies. While the transcriptional activation of genes by HDAC inhibitors has been consistently associated with increased histone acetylation, transcriptional repression by HDAC inhibitors is less well understood. Previously, we showed that two structurally unrelated HDAC inhibitors, Trichostatin A (TSA) and Sodium Butyric Acid (NaB), induced apoptosis and down-regulated bcl-2 expression in follicular lymphoma cells, in which the t(14;18) translocation results in increased bcl-2 expression and resistance to apoptosis. In the current study we further explored the mechanisms underlying the HDAC inhibitor-induced bcl-2 transcriptional repression. Transient transfection of expression vectors encoding three Class I HDACs and real-time reverse transcriptase PCR analysis showed that while overexpression of HDAC2 consistently increased bcl-2 mRNA transcription, overexpression of HDAC1 and HDAC3 did not have a significant effect on bcl-2 levels. These results indicate that HDAC2 has a functional role in the regulation of bcl-2 expression. Furthermore, immunoprecipitation and Western blot analysis showed that TSA treatment dramatically increased the acetylation of two bcl-2 promoter sequence-specific transcription factors, Sp1 and C/EBPα, which are required for bcl-2 P1 and P2 promoter transcriptional activity as well as for TSA-induced bcl-2 transcriptional repression. Increased acetylation of Sp1 and C/EBPα was accompanied by decreased association of HDAC2 but not HDAC1 with these two transcription factors. More importantly, quantitative chromatin immunoprecipitation assays revealed decreased binding of HDAC2 but not HDAC1 to the bcl-2 promoter regions in vivo. These results suggest that posttranslational acetylation of Sp1 and C/EBPα by TSA disrupts the association of HDAC2 with them and leads to decreased binding to the bcl-2 promoters. The current study along with our previous findings suggest that HDAC2 may be an important target of HDAC inhibitor-induced bcl-2 transcriptional repression and provide a mechanistic rationale for the use of HDAC inhibitors in the treatment of human follicular lymphoma.

Regulation of Bcl-2 expression by C/EBP in t(14;18) lymphoma cells.

In follicular lymphomas with the t(14;18) translocation, there is increased expression of the bcl-2 gene, which is dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy-chain gene enhancers. We found that t(14;18) lymphomas expressed C/EBPalpha, which is not normally expressed in B lymphocytes. Expression of C/EBPalpha increased bcl-2 expression, and two regions of the bcl-2 P2 promoter that mediated this effect were identified. C/EBPbeta was also able to increase bcl-2 promoter activity through these sites. The 5' site was GC-rich and did not contain a C/EBP consensus sequence; however, C/EBP was observed to interact with this site both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. The 3' region contained the Cdx site, which mediates the effect of A-Myb on the bcl-2 promoter. In vivo binding studies revealed that C/EBP interacted with this region of the bcl-2 promoter as well. Decreased expression of C/EBP factors due to targeting of their transcripts by siRNA molecules resulted in downregulation of Bcl-2 protein. We conclude that C/EBPalpha and C/EBPbeta contribute to the deregulated expression of Bcl-2 in t(14;18) lymphoma cells.

NF-κB1 (p50) Homodimers Contribute to Transcription of thebcl-2 Oncogene

The bcl-2 proto-oncogene is frequently expressed in human cancer. Although bcl-2 was first cloned as the t(14;18) translocation breakpoint from human follicular B-cell lymphoma, it has become apparent that many cell types express bcl-2 because of transcriptional regulation. As such, several transcription factors have been demonstrated to activate expression of bcl-2, including NF-kappaB. We investigated the role of NF-kappaB1 (p50) homodimers in the expression of Bcl-2 in two murine B-cell lymphoma cell lines: LY-as, an apoptosis-proficient line with low Bcl-2 protein expression and no nuclear NF-kappaB activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. We found that nuclear p50 homodimer activity correlated with Bcl-2 expression in these cell types and identified several sites within the bcl-2 5'-flanking region that p50 was capable of binding. In vitro transcription revealed that recombinant p50 enhanced the production of run-off transcripts from the bcl-2 P1 promoter. Additional in vitro transcription experiments suggested the sites by which p50 afforded this effect. We conclude that the p50 homodimer is capable of transcriptional activation of the bcl-2 gene and suggest that its nuclear activity contributes to the expression of bcl-2 in LY-ar cells.

Transcriptional regulation of bcl-2 by nuclear factor kappa B and its significance in prostate cancer.

This work presents direct evidence that the bcl-2 gene is transcriptionally regulated by nuclear factor-kappa B (NF-kappa B) and directly links the TNF-alpha/NF-kappa B signaling pathway with Bcl-2 expression and its pro-survival response in human prostate carcinoma cells. DNase I footprinting, gel retardation and supershift analysis identified a NF-kappa B site in the bcl-2 p2 promoter. In the context of a minimal promoter, this bcl-2 p2 site 1 increased transcription 10-fold in the presence of the p50/p65 expression vectors, comparable to the increment observed with the consensus NF-kappa B site, while for the full p2 promoter region transcriptional activity was increased sixfold by over-expression of NF-kappa B, an effect eliminated by mutating the bcl-2 p2 site 1. The expression of Bcl-2 has been linked to the hormone-resistant phenotype of advanced prostate cancer. Here we show that an increase in the level of expression of Bcl-2 in the human prostate carcinoma cell line LNCaP observed in response to hormone withdrawal is further augmented by TNF-alpha treatment, and this effect is abated by inhibitors of NF-kappa B. Concomitantly, bcl-2 p2 promoter studies in LNCaP cells show a 40-fold increase in promoter activity after stimulation with TNF-alpha in the absence of hormone.

Nerve growth factor- and epidermal growth factor-regulated gene transcription in PC12 pheochromocytoma and INS-1 insulinoma cells

PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA and p75NTR and the epidermal growth factor receptor (EGF). In PC12 cells, NGF treatment initiates a signaling cascade that ultimately leads to a change of the genetic program of the cell. We have investigated the role of NGF in regulating gene transcription in PC12 and INS-1 cells, in order to define if there are NGF-regulated genes per se. Furthermore, to distinguish between growth factor stimulation via receptor tyrosine kinases in general and NGF-specific changes in gene transcription, we analyzed the effects of EGF on gene transcription. First, we tested the biological activities of fusion proteins consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the phosphorylation-dependent activation domains of the transcription factors Elk1, CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells. We found a striking increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is a major substrate for the extracellular signal-regulated protein kinase (ERK). This effect was observed in NGF- as well as in EGF-treated PC12 cells. In INS-1 cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF, but not by EGF. The effects of NGF and EGF on gene transcription were subsequently studied with plasmids containing reporter genes under control of the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences. NGF stimulated Egr-1 promoter activities in PC12 and INS-1 cells, although the effect was much more pronounced in PC12 cells than in INS-1 cells. EGF also stimulated Egr-1 promoter activity in both PC12 and INS-1 cells. Stimulation of c-jun promoter activity by NGF was observed only in PC12 cells. Deletion mutagenesis demonstrated the importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within the c-jun promoter for basal and NGF-mediated transcriptional induction. Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but not in INS-1 cells and EGF did not show any effects on these promoters. We conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of the ERK subtype of mitogen-activated protein kinases in the nucleus and a subsequent activation of Egr-1 gene transcription. The NGF-induced transcription of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific, indicating that NGF can trigger different gene expression programs dependent on the signaling pathways present in a particular cell type. EGF is clearly able to activate gene transcription, suggesting that the differences in the biological activities of EGF and NGF cannot be explained by the inability of EGF to stimulate gene transcription.

A-Myb Up-regulates Bcl-2 through a Cdx Binding Site in t(14;18) Lymphoma Cells

In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.

Bcl-2 Transcription from the Proximal P2 Promoter Is Activated in Neuronal Cells by the Brn-3a POU Family Transcription Factor

The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.

A negative regulatory element in the bcl-2 5'-untranslated region inhibits expression from an upstream promoter.

bcl-2 mRNA is present at high levels in pre-B-cell lines but is down-regulated in most mature B-cell lines. To investigate the mechanisms responsible for its developmental control, we studied the regulation of bcl-2 expression in human B-lineage cell lines. Using nuclear run-on assays, we found that bcl-2 transcription decreases in parallel with levels of steady-state mRNA during B-cell development. To define cis-acting elements that regulate bcl-2 transcription, we analyzed the expression of transiently transfected promoter-reporter constructs. We identified a novel negative regulatory element (NRE) in the bcl-2 5'-untranslated region that decreased expression from the bcl-2 P1 promoter or heterologous promoters in a position-dependent fashion. The NRE functions in either orientation but contains distinct orientation-dependent subfragments. Additional analyses demonstrated that multiple, functionally redundant sequence elements mediate NRE activity. Though the bcl-2 NRE is active in pre-B- and mature B-cell lines, chromatin structure of the endogenous NRE differs in these cells, suggesting that its activity or effect may vary during B-cell development. Our results indicate that negative control of transcription initiated at the P1 promoter is an important determinant of the differential expression of bcl-2.

A Negative Regulatory Element in the bcl-2 5'-Untranslated Region Inhibits Expression from an Upstream Promoter

bcl-2 mRNA is present at high levels in pre-B-cell lines but is down-regulated in most mature B-cell lines. To investigate the mechanisms responsible for its developmental control, we studied the regulation of bcl-2 expression in human B-lineage cell lines. Using nuclear run-on assays, we found that bcl-2 transcription decreases in parallel with levels of steady-state mRNA during B-cell development. To define cis-acting elements that regulate bcl-2 transcription, we analyzed the expression of transiently transfected promoter-reporter constructs. We identified a novel negative regulatory element (NRE) in the bcl-2 5'-untranslated region that decreased expression from the bcl-2 P1 promoter or heterologous promoters in a position-dependent fashion. The NRE functions in either orientation but contains distinct orientation-dependent subfragments. Additional analyses demonstrated that multiple, functionally redundant sequence elements mediate NRE activity. Though the bcl-2 NRE is active in pre-B- and mature B-cell lines, chromatin structure of the endogenous NRE differs in these cells, suggesting that its activity or effect may vary during B-cell development. Our results indicate that negative control of transcription initiated at the P1 promoter is an important determinant of the differential expression of bcl-2.