D1 Promoter Research Articles

Promoter Hypermethylation as an Independent Prognostic Factor for Relapse in Patients with Prostate Cancer Following Radical Prostatectomy

To analyze the prognostic significance of six epigenetic biomarkers (APC, Cyclin D2, GSTP1, TIG1, Rassf1A, and RARbeta2 promoter hypermethylation) in a homogeneous group of prostate cancer patients, following radical prostatectomy alone. Biomarker analyses were done retrospectively on tumors from 74 prostate cancer patients all with a Gleason score of 3 + 4 = 7 and minimum follow-up period of 7 years. Using quantitative methylation-specific PCR, we analyzed six gene promoters in primary prostate tumor tissues. Time to any progression was the primary end point, and development of metastatic disease and/or death from prostate cancer was a secondary point. The association of clinicopathologic and biomolecular risk factors to recurrence was done using the log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used. At a median follow-up time of 9 years, 37 patients (50%) had evidence of recurrence: biochemical/prostate-specific antigen relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression (TTP), the significant factors were age > 60 [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.2-0.8; P = 0.01], hypermethylation of GSTP1 (HR, 0.23; 95% CI; 0.09-0.64; P = 0.004), and hypermethylation of APC (HR, 3.0; 95% CI, 1.42-6.32; P = 0.004). In another multivariate analysis, a profile of hypermethylation of APC and cyclin D2 hypermethylation was significant as well: if either any one was hypermethylated (HR, 1.84; 95% CI, 0.92-3.72; P = 0.09) or if both were hypermethylated (HR, 4.3; 95% CI, 1.52-12.33; P = 0.01). Methylation status of selected genes in the prostate cancer specimen may predict for time to recurrence in Gleason 3 + 4 = 7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.

Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes

Lymphocyte proliferation is key to the regulation of the immune system. Cyclin D2 is the first cell cycle protein induced following stimulation through the T-cell receptor, the B-cell receptor or cytokines. The promoter of this cyclin integrates a diverse range of signals. Through investigating the regulation of this promoter by interleukin-2 and phosphatidylinositol 3-kinase, we have identified a role for the transcription factor CREB, cAMP response element-binding protein. Mutation of the CREB-binding site reduced cyclin D2 promoter activity 5-10-fold. CREB-1 is phosphorylated at serine 133, a critical site for activity, in both T cells and Epstein-Barr virus immortalized B cells. The introduction of an S133A mutant of CREB-1 reduces IL-2 induction of cyclin D2 promoter activity, demonstrating a role for this phosphorylation site in promoter activity. Two inhibitors of protein kinase A reduce lymphocyte proliferation and CREB-1 phosphorylation. This study demonstrates that the cyclin D2 promoter is capable of being regulated by PI3K and CREB and identifies CREB-1 and protein kinase A as potential targets for altering lymphocyte proliferation.

Phosphatidylinositol 3‐kinase is required for the transcriptional activation of cyclin D2 in BCR activated primary mouse B lymphocytes

Induction of cyclin D2 is essential for mediating cell cycle entry in B cells activated by BCR cross-linking. In the present study we show that, like B lymphocytes lacking cyclin D2, the p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) or other components of the B cell signalosome, p110delta-null B cells fail to induce cyclin D2 and enter early G1 but not S phase of the cell cycle. The inhibitors of PI3K activity, LY294002 and Wortmannin, also abrogate cyclin D2 induction by BCR cross-linking, confirming that the class IA PI3K is necessary for cyclin D2 induction in response to BCR stimulation. Furthermore, using both p85alpha-null and p110delta-null B cells and inhibitors of PI3K, this study demonstrates for the first time, that BCR cross-linking induces cyclin D2 mRNA expression via transcriptional activation of the cyclin D2 promoter and that this transcriptional activation of cyclin D2 requires PI3K activity. Moreover, we identify a region between nucleotides -1624 and -1303 of the cyclin D2 promoter containing elements responsive to anti-IgM, which are PI3K dependent. Further characterisation of signalling intermediates downstream of the BCR revealed a perturbation of MAPK signalling pathways in p85alpha-null and p110delta-null B cells, and our data suggests that cross-talk exists between the PI3K and JNK pathways.

A Human T-Cell Lymphotropic Virus Type 1 Enhancer of Myc Transforming Potential Stabilizes Myc-TIP60 Transcriptional Interactions

The human T-cell lymphotropic virus type 1 (HTLV-1) infects and transforms CD4+ lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. Here, we demonstrate that the HTLV-1 pX splice-variant p30II markedly enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter, dependent upon its conserved Myc-responsive E-box enhancer elements, which are associated with increased S-phase entry and multinucleation. Enhancement of c-Myc transforming activity by HTLV-1 p30II is dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and it requires TIP60 histone acetyltransferase (HAT) activity and correlates with the stabilization of HTLV-1 p30II/Myc-TIP60 chromatin-remodeling complexes. The p30II oncoprotein colocalizes and coimmunoprecipitates with Myc-TIP60 complexes in cultured HTLV-1-infected ATLL patient lymphocytes. Amino acid residues 99 to 154 within HTLV-1 p30II interact with the TIP60 HAT, and p30II transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner, as determined by microarray gene expression analyses. Importantly, these results suggest that p30II functions as a novel retroviral modulator of Myc-TIP60-transforming interactions that may contribute to adult T-cell leukemogenesis.

Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad

Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.

Reading and function of a histone code involved in targeting corepressor complexes for repression.

A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

Promoter Hypermethylation in Benign Breast Epithelium in Relation to Predicted Breast Cancer Risk

The tumor suppressor genes RASSF1A, APC, H-cadherin, RARbeta2, and cyclin D2 are methylated more frequently in breast cancer than in adjacent benign tissue. However, it is unclear whether promoter methylation of tumor suppressor genes in benign breast tissue is associated with an increased risk for breast cancer. Promoter hypermethylation was measured in benign and malignant breast samples obtained by fine needle aspiration biopsy from 27 breast cancer patients and 55 unaffected women whose risk of breast cancer had been defined using the Gail, Claus, and BRCAPRO models. Cyclin D2 methylation occurred in 57% of tumor samples but not in corresponding benign breast samples and in only one sample from an unaffected patient (P < 0.0001). RARbeta2 methylation occurred in 32% of benign breast samples from cancer patients but only 9% of similar samples from unaffected women (P = 0.002). Promoter methylation of RASSF1A and APC occurred more frequently (70% and 56%, respectively) in unaffected women at high-risk for breast cancer as defined by the Gail model than in low/intermediate risk women (29% and 20%, P = 0.04 and P = 0.03). Of the Gail model risk factors, only number of prior breast biopsies was highly correlated with APC and RASSF1A methylation (P = 0.0001 and 0.02, respectively). Since cyclin D2 promoter methylation occurs almost exclusively in tumors, it may be possible to exploit it for the early detection of breast cancer. Promoter methylation of APC, RARbeta2, and RASSF1A in benign breast epithelium is associated with epidemiologic markers of increased breast cancer risk.

FoxO3a and BCR-ABL Regulatecyclin D2Transcription through a STAT5/BCL6-Dependent Mechanism

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

17beta-estradiol stimulates the growth of human keratinocytes by inducing cyclin D2 expression.

Estrogen is reported to prevent age-associated epidermal thinning in the skin. We examined if 17beta-estradiol (E2) may enhance the growth of human keratinocytes, focusing on its effects on the expression of cell cycle-regulatory proteins. E2 enhanced proliferation, bromodeoxyuridine incorporation of keratinocytes, and increased the proportion of cells in the S phase. The E2-induced stimulation of proliferation and bromodeoxyuridine incorporation was suppressed by antisense oligonucleotide against cyclin D2, which induces G1 to S phase progression. E2 increased protein and mRNA levels of cyclin D2, and resultantly enhanced assembly and kinase activities of cyclin D2-cyclin-dependent kinases 4 or 6 complexes. E2 enhanced cyclin D2 promoter activity, and the element homologous to cAMP response element (CRE) on the promoter was responsible for the effect. Cyclin D2 expression was enhanced by antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, and membrane-impermeable bovine serum albumin-conjugated E2, indicating the effects via membrane E2-binding sites. E2 increased the enhancer activity of CRE-like element and the amount of phosphorylated cAMP response element binding protein (CREB) binding this element, and the increases were suppressed by H-89, an inhibitor of cAMP-dependent protein kinase A. H-89 also suppressed E2-induced cyclin D2 expression, proliferation, and bromodeoxyuridine incorporation in keratinocytes. Antisense oligonucleotide against G-protein-coupled receptor GPR30 suppressed the E2-induced increases of phosphorylated CREB, cyclin D2 level, proliferation, and bromodeoxyuridine incorporation in keratinocytes. These results suggest that E2 may stimulate the growth of keratinocytes by inducing cyclin D2 expression via CREB phosphorylation by protein kinase A, dependent on cAMP. These effects of E2 may be mediated via cell surface GPR30.

Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins.

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. We now show that Myc recruits TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the pre-initiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. We suggest that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes.

A Permissive Role for Phosphatidylinositol 3-Kinase in the Stat5- mediated Expression of Cyclin D2 by the Interleukin-2 Receptor

The interleukin-2 (IL-2) receptor promotes T cell proliferation in part by inducing the expression of D-type cyclins, which enable cells to progress from the G1 to S phase of the cell cycle. We previously showed that the IL-2 receptor induces expression of cyclin D2 by activating the transcription factor Stat5, which binds directly and immediately to a site upstream of the cyclin D2 promoter. We show here that subsequent transcription of the cyclin D2 gene occurs by a delayed, cycloheximide-sensitive mechanism, which implies the involvement of additional regulatory mechanisms. The transcription factor c-Myc is induced by Stat5 and is reported to bind to two E box motifs in the cyclin D2 promoter. However, in IL-2-stimulated T cells, c-Myc does not appear to be involved in cyclin D2 induction, since we found that these two E boxes are preferentially bound by USF-1 and USF-2 and, moreover, are dispensable for cyclin D2 promoter activity. Instead, we found that Stat5 activates the phosphatidylinositol 3-kinase (PI3 kinase) pathway by a delayed, cycloheximide-sensitive mechanism and that PI3 kinase activity is essential for the induction of cyclin D2 by Stat5. Chromatin immunoprecipitation experiments revealed that PI3 kinase is required for the optimal binding of RNA polymerase II to the promoters of cyclin D2 as well as other genes. Our results reveal a novel link between PI3 kinase and RNA polymerase II promoter binding activity and demonstrate discrete, coordinated roles for the PI3 kinase and Stat5 pathways in cyclin D2 transcription.

Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma

The oncogene c-maf is translocated in ∼5%–10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2, integrin β7, and CCR1. c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin β7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.

Promoter methylation of cyclin D2 gene in gastric carcinoma

Methylation of CpG island in the promoter has been recognized as an important mechanism for regulation of gene expression. Although considerable work has been done on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes. The cyclin D2 gene is overexpressed in a subset of gastric carcinoma. To determine whether hypomethylation of cyclin D2 is involved in stomach carcinogenesis, we studied methylation of CpG islands in the cyclin D2 gene by methylation-specific PCR in 34 gastric carcinoma specimens, 21 corresponding non-neoplastic mucosae, and 8 gastric carcinoma cell lines. We also measured levels of cyclin D2 mRNA in 23 of the gastric carcinoma cases and in the gastric carcinoma cell lines. Hypomethylation of the cyclin D2 promoter was found in 24 (71%) of the 34 tumor tissues and in 6 (29%) of the 21 corresponding non-neoplastic mucosa, the incidence being significantly different (p=0.002; Fisher's exact test). Moreover, hypomethylation of cyclin D2 was more common in stage III and IV tumors than in stage I and II tumors (p=00.014; Fisher's exact test). All of three cell lines with promoter hypomethylation expressed detectable levels of cyclin D2 mRNA. Treatment of cyclin D2-negative cells lines harboring promoter hypermethylation with demethylating agent, 5-Aza-2'-deoxycytidine, led to a reactivation of cyclin D2 expression. These results suggest that DNA hypomethylation is a mechanism underlying the increased expression of cyclin D2 in cancer cells and that demethylation of cyclin D2 may be involved in development and progression of gastric carcinoma.

Aberrant methylation of the cyclin D2 promoter in primary small cell, nonsmall cell lung and breast cancers.

DNA methylation alteration of several genes contributes to human tumorigenesis. Cyclin D2, a member of the D-type cyclins, is implicated in cell cycle regulation and malignant transformation. In our study, we examined the methylation status of the cyclin D2 promoter in small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), breast tumors and tumor cell lines. We observed that aberrant methylation of cyclin D2 was present in 32 of 56 (57%) SCLC cell lines, 7 of 32 (22%) SCLC tumor tissues; 25 of 61 (47%) NSCLC cell lines, 19 of 48 (40%) NSCLC tumor tissues; 18 of 30 (60%) breast tumor cell lines and 19 of 63 (30%) breast tumor tissues. Methylation was more frequent in the tumor cell lines compared to the primary breast and SCLC tumors (p = 0.007 and p = 0.001, respectively). Methylation was rare in the control tissue samples; 0 of 12 peripheral blood lymphocytes; 0 of 12 buccal epithelial cells; 0 of 18 nonmalignant lung tissues and 3 of 28 (11%) nonmalignant breast tissues. Promoter methylation correlated with loss of transcript by reverse transcription PCR (RT-PCR) in 9 of 11 (6 lung, 5 breast) tumor cell lines tested. Two cell lines that were not methylated also lacked expression, suggesting that other mechanisms of inactivation may be involved. Expression was restored by treatment with the demethylating agent, 5 aza 2' deoxycytidine, in all 9 methylated cell lines. Our results confirm earlier reports in breast cancer and indicate that aberrant methylation of cyclin D2 may contribute to the pathogenesis of the 2 major types of lung cancers.

Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells.

Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the beta-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.

Cyclin D2 expression in familial and sporadic breast cancer

In a differential display experiment comparing normal breast epithelial cells with breast tumors and breast cancer cell lines we isolated a gene, cyclin D2, showing a low expression in tumor cells compared to normal cells. This result was confirmed using Northern blotting and RT-PCR. In total, low expression was seen in 48/109 (44%) tumors. Low cyclin D2 expression was seen in 10/39 (25%) sporadic breast cancer, and in 38/70 (54%) of familial breast cancer. In order to find the explanation for the down-regulation of cyclin D2 we screened for inactivating mutations in tumors with low expression, and studied methylation of the promoter region in breast cancer cell lines. We also used growth factors in an attempt to stimulate cell lines to express cyclin D2, and transfected the cell lines with a vector expressing cyclin D2. Treatment with 5-azacytidin for four days resulted in transcription of cyclin D2. Our result suggests that methylation of the cyclin D2 promoter or another regulating gene is associated with loss of cyclin D2 expression in breast cancer.

Activation of cyclin D1 and D2 promoters by human T-cell leukemia virus type I tax protein is associated with IL-2-independent growth of T cells.

Our aim was to examine the involvement of G(1) cell-cycle regulators in cell growth dysregulation induced by HTLV-I. Compared to uninfected cells, higher expression levels of cyclin D1 and D2 mRNA were detected in HTLV-I-infected T-cell lines, which were at least in part mediated by the viral transforming protein Tax since Tax activated both cyclin D1 and D2 promoters in the human T-cell line Jurkat. A Tax mutant that did not activate NF-kappaB failed to activate cyclin D1 and D2 promoters. Inhibitors of NF-kappaB (dominant negative IkappaBs mutants) suppressed Tax-dependent activation of cyclin D1 and D2 promoters, indicating that Tax-induced activation was mediated by NF-kappaB. Wild-type and mutant Tax capable of activating NF-kappaB, but not Tax mutant incapable of activating NF-kappaB, converted cell growth of a T-cell line from being IL-2-dependent to being IL-2-independent; and this conversion was associated with IL-2-independent induction of cyclins D1 and D2. Our data suggest that induction of cyclins D1 and D2 by Tax is involved in IL-2-independent cell-cycle progression as well as IL-2-independent transformation of primary human T cells by HTLV-I. High expression levels of cyclin D1 and D2 mRNAs were also detected in some patients with ATL. Our findings link HTLV-I infection to changes in cellular D-type cyclin gene expression, transformation of T cells and subsequent development of T-cell leukemia.

C-Myc Mediates Activation of the cad Promoter via a Post-RNA Polymerase II Recruitment Mechanism

The c-Myc protein is a site-specific DNA-binding transcription factor that is up-regulated in a number of different cancers. We have previously shown that binding of Myc correlates with increased transcription of the cad promoter. We have now further investigated the mechanism by which Myc mediates transcriptional activation of the cad gene. Using a chromatin immunoprecipitation assay, we found high levels of RNA polymerase II bound to the cad promoter in quiescent NIH 3T3 cells and in differentiated U937 cells, even though the promoter is inactive. However, chromatin immunoprecipitation with an antibody that recognizes the hyperphosphorylated form of the RNA polymerase II carboxyl-terminal domain (CTD) revealed that phosphorylation of the CTD does correlate with c-Myc binding and cad transcription. We have also found that the c-Myc transactivation domain interacts with cdk9 and cyclin T1, components of the CTD kinase P-TEFb. Furthermore, activator bypass experiments have shown that direct recruitment of cyclin T1 to the cad promoter can substitute for c-Myc to activate the promoter. In summary, our results suggest that c-Myc activates transcription of cad by stimulating promoter clearance and elongation, perhaps via recruitment of P-TEFb.

Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter.

Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. We now show that Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc.

Direct and indirect regulation of cytokine and cell cycle proteins by EBNA-2 during Epstein-Barr virus infection.

We have studied the pathways of regulation of cytokine and cell cycle control proteins during infection of human B lymphocytes by Epstein-Barr virus (EBV). Among 30 cytokine RNAs analyzed by the RNase protection assay, tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor, lymphotoxin (LT), and LTbeta were found to be regulated within 20 h of EBV infection of primary B cells. Similar results were obtained using the estrogen-regulated EBNA-2 cell line EREB2.5, in which RNAs for LT and TNF-alpha were induced within 6 h of activation of EBNA-2. Expression of Notch also caused an induction of TNF-alpha RNA. The induction of TNF-alpha RNA by EBNA-2 was indirect, and constitutive expression of either LMP-1 or c-myc proteins did not substitute for EBNA-2 in induction of TNF-alpha RNA. Cyclin D2 is also an indirect target of EBNA-2-mediated transactivation. EBNA-2 was found to activate the cyclin D2 promoter in a transient-transfection assay. A mutant of EBNA-2 that does not bind RBP-Jkappa retained some activity in this assay, and activation did not depend on the presence of B-cell-specific factors. Deletion analysis of the cyclin D2 promoter revealed that removal of sequences containing E-box c-myc consensus DNA binding sequences did not reduce EBNA-2-mediated activation of the cyclin D2 promoter in the transient-transfection assay. The results indicate that cytokines are an early target of EBNA-2 and that EBNA-2 can regulate cyclin D2 transcription in EBV-infected cells by mechanisms additional to the c-myc pathway.