The immunoglobulin heavy-chain gene 3' enhancers deregulate bcl-2 promoter usage in t(14;18) lymphoma cells.

In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-kappaB (NF-kappaB) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBPbeta bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBPbeta. Reduced expression of C/EBPbeta by transfection of small interfering RNA or interference with NF-kappaB activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.

Activation of the c-myc p1 promoter in Burkitt's lymphoma by the hs3 immunoglobulin heavy-chain gene enhancer.

The expression of c-myc is deregulated in Burkitt's lymphoma by the translocation t(8;14). Most of the increased c-myc expression is from the P1 promoter, which is normally a minor promoter. How the P1 promoter is activated by the immunoglobulin heavy chain gene enhancers is not understood. We identified a YY1 site in the immunoglobulin heavy-chain gene HS3 enhancer, which increased c-myc P1 promoter activity, and a MARE site, which decreased c-myc P1 activity. Small Maf proteins bound to the MARE site both in vitro and in vivo, recruited histone deacetylase 2, and resulted in deacetylation of histones H3 and H4 at the c-myc promoter region. In contrast, YY1 recruited CBP and increased histone acetylation at the c-myc promoter. Rb interacts with YY1 to prevent DNA binding in normal B cells, but no significant interaction with YY1 was detected in Burkitt's cells, and binding of YY1 to the HS3 enhancer was observed by chromatin immunoprecipitaton. Increased expression of MafK and/or decreased expression of YY1 by silencing RNA downregulated endogenous c-myc mRNA levels and increased the sensitivity of the cells to doxorubicin. Mutation of the major active sites (nuclear factor-kappa B and YY1) in the enhancers prevented c-myc activation.

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.

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt's lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.

Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.

Identification of an inducible regulator of c-myb expression during T-cell activation.

Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation.

The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor.

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.

Interaction of nuclear proteins with muscle-specific regulatory sequences of the human cardiac alpha-actin promoter.

The human cardiac alpha-actin promoter is involved in the muscle-specific transcriptional regulation of the gene. In this study, we utilized gel mobility shift, methylation interference, and DNase I protection assays to examine protein factor interaction with the promoter in vitro. All assays demonstrated specific interaction of nuclear factors with a region of the promoter encompassed by nucleotides -93 to -113 base pairs from the transcriptional start site. This region contains a CC(A + T-rich)6GG element, termed a CArG box, which has previously been implicated in the muscle-specific transcriptional regulation of the gene by functional assays. Although the gene is only expressed in muscle cells, identical binding activity was present in nuclear extracts of all cell types examined, including those of muscle (C2, L8, and L6 cells) and nonmuscle (HeLa, NIH 3T3, HuT12, and L cells) origin. Furthermore, methylation interference assays showed that identical nucleotides interacted with factors isolated from C2 and HeLa cells. Competition studies showed that the CArG-binding factor, designated as CBF, also interacts with the c-fos serum responsive element, which contains a CArG element, but not with the simian virus 40 enhancer and early promoter. Thus, a region of the human cardiac alpha-actin promoter known to be functionally involved in muscle-specific regulation of the gene appears to interact in vitro, and in an identical manner, with a factor(s) which is neither muscle nor gene specific, suggesting a more complex mode of regulation than previously envisioned.

Molecular mechanisms of transcriptional control of bcl-2 and c-myc in follicular and transformed lymphoma.

A synergistic interaction of Bcl-2 and c-Myc plays a role in lymphomagenesis in mice and in some patients as well. Progression of follicular lymphoma to a more aggressive lymphoma is seen in the majority of patients, and approximately 10% of the transformed lymphomas have a translocation of c-myc in addition to the translocation of bcl-2 found in the original follicular lymphoma. We investigated whether transcriptional deregulation of bcl-2 and c-myc could be examined in primary lymphoma cells by in vivo footprinting and in vitro protein-DNA binding studies. A matched pair of follicular and transformed lymphoma samples was examined. The transformed lymphoma had acquired a translocation of c-myc into the immunoglobulin heavy chain locus. High levels of bcl-2 expression were observed in both the follicular and transformed lymphomas, whereas the expression of c-myc was low in the follicular lymphoma and increased in the transformed lymphoma. In vivo footprint analysis revealed that a CRE site and a Cdx site in the bcl-2 promoter were occupied on the translocated alleles but not on the normal alleles in both the follicular and transformed lymphomas. Two nuclear factor kappaB sites were occupied on the translocated c-myc allele in the transformed lymphoma. Gel shift analysis revealed that these proteins bound to their respective sites in the bcl-2 or c-myc promoter. There was no evidence that the presence of one of the translocations in the immunoglobulin heavy chain locus influenced the expression of the other translocated gene.

Translocations involving c-myc and c-myc function.

c-MYC is the prototype for oncogene activation by chromosomal translocation. In contrast to the tightly regulated expression of c-myc in normal cells, c-myc is frequently deregulated in human cancers. Herein, aspects of c-myc gene activation and the function of the c-Myc protein are reviewed. The c-myc gene produces an oncogenic transcription factor that affects diverse cellular processes involved in cell growth, cell proliferation, apoptosis and cellular metabolism. Complete removal of c-myc results in slowed cell growth and proliferation, suggesting that while c-myc is not required for cell proliferation, it acts as an integrator and accelerator of cellular metabolism and proliferation.

Differential protein binding to the c-myc promoter during differentiation of hematopoietic cell lines.

In vivo footprinting has been used to examine protein binding sites in the c-myc promoter during differentiation of HL60 and K562 cell lines. The c-myc gene is expressed in proliferating cells, but c-myc levels decrease dramatically during differentiation. A number of potential protein binding sites have been identified from in vitro studies of the c-myc promoter, but very little is known about occupancy of these sites in vivo. We have identified in vivo footprints at DNase hypersensitive sites II2 and III1 which disappear during differentiation, while a footprint at site IV is present only in differentiated cells. Footprints at DNase hypersensitive sites I and II1 do not change with differentiation. A protected region near DNase hypersensitive site III2 is present in both undifferentiated and differentiated cells, but it extends further 5' in undifferentiated cells. From the protected sequences we have been able to identify candidate transcription factors likely to be involved in the control of c-myc expression. By electrophoretic mobility shift assay we have demonstrated that a protein binds to the sequence at site IV. We have also examined the 3' end of the first exon and the 5' end of intron I and do not find any evidence for protein binding sites in this region that was thought to be important for the block to transcription elongation during differentiation.

The critical role of the PE21 element in oncostatin M-mediated transcriptional repression of the p53 tumor suppressor gene in breast cancer cells.

Cytokine oncostatin M (OM) exerts growth-inhibitory and differentiative effects on breast cancer cells. Previously we showed that the transcription from the p53 gene in breast cancer cells was down regulated by OM. To elucidate the molecular mechanisms underlying the OM effect on p53 transcription, in this study, we dissected the p53 promoter region and analysed the p53 promoter activity in breast tumor cells. We showed that treatment of MCF-7 cells with OM induced a dose- and time-dependent suppression of p53 promoter activity. The p53 promoter activity was decreased to 35% of control at 24 h and further decreased to 20% at 48 h by OM at concentrations of 5 ng/ml and higher. Deletion of the 5'-flanking region of the p53 promoter from -426 to -97 did not affect the OM effect. However, further deletion to -40 completely abolished the repressive effect of OM. The p53 promoter region -96 to -41 contains NF-kappaB and c-myc binding sites, and a newly identified UV-inducible element PE21. Mutations to disrupt NF-kappaB binding or c-myc binding to the p53 promoter decreased the basal promoter activity without affecting the OM-mediated suppression, whereas mutation at the PE21 motif totally abolished the OM effect. We further demonstrated that insertion of PE21 element upstream of the thymidine kinase minimal promoter generated an OM response analogous to that of the p53 promoter. Finally, we detected the specific binding of a nuclear protein with a molecular mass of 87 kDa to the PE21 motif. Taken together, we demonstrate that OM inhibits the transcription of the p53 gene through the PE21 element. Thus, the PE21 element is functionally involved in p53 transcription regulated by UV-induction and OM suppression.

Negative regulation of bcl-2 expression by p53 in hematopoietic cells.

The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas.

Suppression of apoptosis and granulocyte colony-stimulating factor-induced differentiation by an oncogenic form of Cbl.

OBJECTIVE: The retroviral oncogene v-Cbl causes pre-B cell lymphomas and myeloid leukemias in mice, and its Drosophila homologue is oncogenic, causing enhanced receptor tyrosine kinase signaling. The human Cbl gene resides at 11q23. The aim of this study is to determine the effect of oncogenic Cbl on growth-regulating responses. MATERIALS AND METHODS: The oncogenic mutant of Cbl (CblDelta1-357) was transfected into factor-dependent 32Dcl3 myeloid cells. Consequently, cell survival and differentiation were measured. Lyn, Syk, MAP kinase, and phosphatidylinositol 3'(PI3')-kinase activities, protein phosphorylation, Bcl-2 promoter activity, ubiquitination, and levels of Bcl-2, Bax, Bad, and Bcl-x(L) were determined. In addition, the effect of v-Cbl on TF-1 cell survival upon granulocyte-macrophage colony-stimulating factor withdrawal was studied. RESULTS: 32Dcl3 and TF-1 cells expressing v-Cbl showed resistance to apoptosis upon growth factor withdrawal, and 32Dcl3 cells completely failed to respond to granulocyte colony-stimulating factor's induction of differentiation. Basal activities of Lyn, Syk, and PI3'-kinase were elevated in the v-Cbl line. There was neither enhanced tyrosine phosphorylation of cellular protein content, Cbl, or Jak2, nor serine phosphorylation of MAP kinase or Akt. After factor withdrawal, the level of Bcl-2 was greater in v-Cbl cells than in control cells. CONCLUSIONS: Neither increased Bcl-2 promoter activity nor decreased ubiquitination of Bcl-2 could account for increased Bcl-2 levels. v-Cbl-expressing 32Dcl3 cells were resistant to differentiation. v-Cbl suppresses apoptosis and differentiation, possibly through enhancement of Lyn, Syk, and PI3'-kinase activities and Bcl-2.

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 zinc-finger transcription factor induced by TGF-beta promotes apoptotic cell death in epithelial Mv1Lu cells.

Transforming growth factor-beta (TGF-beta) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc-finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF-beta in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF-beta effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl-2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co-expression of Bcl-2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro-apoptotic properties of TGF-beta.

The cyclic AMP response element in the Bcl-2 promoter confers inducibility by hypoxia in neuronal cells.

In neuronal cells, expression of the anti-apoptotic Bcl-2 gene is induced by hypoxia and produces a protective effect. We show here that this effect is dependent upon the cyclic AMP response element (CRE) in the Bcl-2 promoter since mutation of this element abolishes the response and the isolated CRE can confer the response on a heterologous promoter. Interestingly however, the CRE in the Bcl-2 promoter does not render the promoter responsive to cyclic AMP and is not essential for its response to nerve growth factor. Despite the lack of cyclic AMP responsiveness, activation of the Bcl-2 promoter via the CRE in response to hypoxia requires the CREB transcription factor and is associated with the enhanced phosphorylation of CREB on serine 133 and enhanced transcriptional activation by the CREB-binding protein, CBP, in response to hypoxia. This finding establishes the importance of the CRE in the induction of Bcl-2 gene expression by hypoxia, allowing the Bcl-2 protein to protect neuronal cells against this damaging stimulus.

The immunoglobulin heavy chain gene 3' enhancers induce Bcl2 deregulation and lymphomagenesis in murine B cells.

Human follicular B-cell lymphoma is associated with the t(14;18) chromosomal translocation that juxtaposes the Bcl2 proto-oncogene with the immunoglobulin heavy chain (Igh) locus, resulting in the deregulated expression of Bcl2. Our previous studies have shown that the Igh 3' enhancers deregulate the Bcl2 expression in vitro. However, the effects of the Igh 3' enhancer elements on Bcl2 expression in vivo are not known. To investigate the role of the Igh 3' enhancers in Bcl2 deregulation, we used gene targeting to generate knock-in mice in which four DNase I-hypersensitive regions from the murine Igh 3' region were integrated 3' of the Bcl2 locus. Increased levels of Bcl2 mRNA and protein were observed in the B cells of Igh-3'E-bcl2 mice. B cells from Igh-3'E-bcl2 mice showed an extended survival in vitro compared with B cells from wild-type (Wt) mice. The Bcl2 promoter shift from P1 (the 5' promoter) to P2 (the 3' promoter) was observed in B cells from Igh-3'E-bcl2 mice, similar to human t(14;18) lymphomas. The IgH-3'E-bcl2 mice developed monoclonal B-cell follicular lymphomas, which were slowly progressive. These studies show that the Igh 3' enhancers have an important role in the deregulation of Bcl2 and B-cell lymphomagenesis in vivo.

Functional long-range interactions of the IgH 3' enhancers with the bcl-2 promoter region in t(14;18) lymphoma cells.

To better understand the mechanisms underlying the role of the immunoglobulin heavy-chain gene (IgH) 3' enhancers on bcl-2 transcriptional deregulation in t(14;18) lymphoma, we characterized the physical interactions of the IgH 3' enhancer region with the bcl-2 promoters. Using the chromosome conformation capture technique, we found that the IgH 3' enhancers physically interact with the bcl-2 promoter region over a 350 kb genomic region in t(14;18) lymphoma cells. No interactions of the bcl-2 promoter region with sequences distant to the IgH enhancers were observed. The physical interactions of the IgH enhancers with the bcl-2 5' region are functionally involved in the transcriptional control of bcl-2. The histone deacetylase inhibitor, trichostatin A, repressed bcl-2 transcription and decreased the IgH enhancer-bcl-2 promoter region interactions. We showed by chromatin immunoprecipitation assay and small interference RNA transfection studies that the POU2 family transcription factor Oct-2 and its cofactor Bob-1 have an important function in mediating the IgH enhancer-bcl-2 promoter region interactions. This study reveals a new aspect of the regulatory role of the IgH 3' enhancers on bcl-2 transcription in t(14;18) lymphomas.