Immunoglobulin Heavy Chain Gene Enhancer Research Articles

Complex sex-biased antibody responses: estrogen receptors bind estrogen response elements centered within immunoglobulin heavy chain gene enhancers.

Nuclear hormone receptors including the estrogen receptor (ERα) and the retinoic acid receptor regulate a plethora of biological functions including reproduction, circulation and immunity. To understand how estrogen and other nuclear hormones influence antibody production, we characterized total serum antibody isotypes in female and male mice of C57BL/6J, BALB/cJ and C3H/HeJ mouse strains. Antibody levels were higher in females compared to males in all strains and there was a female preference for IgG2b production. Sex-biased patterns were influenced by vitamin levels, and by antigen specificity toward influenza virus or pneumococcus antigens. To help explain sex biases, we examined the direct effects of estrogen on immunoglobulin heavy chain sterile transcript production among purified, lipopolysaccharide-stimulated B cells. Supplemental estrogen in B-cell cultures significantly increased immunoglobulin heavy chain sterile transcripts. Chromatin immunoprecipitation analyses of activated B cells identified significant ERα binding to estrogen response elements (EREs) centered within enhancer elements of the immunoglobulin heavy chain locus, including the Eµ enhancer and hypersensitive site 1,2 (HS1,2) in the 3' regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus. Taken together, the results highlight: (i) the important targets of ERα in regulatory regions of the immunoglobulin heavy chain locus that influence antibody production, and (ii) the complexity of mechanisms by which estrogen instructs sex-biased antibody production profiles.

Mutant p53 Enhances the Development and Sustained Growth of MYC-Driven Lymphoma and Exerts a Dominant Negative Effect Preferentially Deregulating Pathways for Metabolism and DNA Repair

Over-expression of the c-MYC oncogene and Trp53 gene mutations are among the most common genetic alterations in human cancer and, when combined, result in highly aggressive malignant disease. Trp53 gene mutations produce over-expressed mutant TRP53 proteins that drive cancer growth through both loss of wild-type Trp53 tumor suppressor function and gain-of-function oncogenic properties. The Eμ-Myc mouse model provides a setting to study the functional interplay between c-Myc over-expression and mutant TRP53 proteins. Eμ-Myc transgenic mice carry a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma, and develop aggressive pre-B/B cell lymphoma with a high (~20%) spontaneous rate of Trp53 mutation.The effect of five mouse mutant TRP53 proteins (V170M, I192S, G280, R246Q, R270H) was initially examined in three settings (Trp53-/-, Trp53+/- and Trp53+/+;Eμ-Myc) using a hematopoietic stem and progenitor cell (HSPC) reconstitution model. Each mutant TRP53 protein studied corresponds to a commonly re-occurring Tp53 mutation in human cancer. Retroviral over-expression enabled the comparison of mutant-specific and genotype-specific features for each mutant TRP53 protein. Mutant TRP53 expression did not accelerate lymphoma development in mice receiving Trp53-/- or Trp53+/- HSPCs. However, mice reconstituted with Trp53+/- HSPCs expressing the TRP53 mutants displayed an altered tumor spectrum compared to mice reconstituted with control Trp53+/- HSPCs. In contrast, mutant TRP53 markedly accelerated lymphoma development in mice receiving Trp53+/+;Eμ-Myc HSPCs, highlighting a synergy between c-Myc over-expression and Trp53 mutations in neoplastic transformation. Furthermore, inducible mutant TRP53 expression demonstrated a dependency on sustained expression of mutant TRP53 in established MYC-driven lymphomas. Notably, none of the c-MYC plus mutant TRP53 driven lymphomas exhibited spontaneous endogenous Trp53 mutations.Despite the enhanced tumorigenesis, most established lymphomas from this model displayed sensitivity to TRP53-activating drugs consistent with a weak dominant negative effect over wild-type Trp53-induced apoptosis. Consistent with this finding, pre-malignant Trp53+/+;Eμ-Myc primary B-cells expressing mutant TRP53 were not protected against Trp53-induced apoptosis. Pre-malignant B-cells displayed a small increase in cell cycling and an expansion of the tumor-initiating pre/pro-B cell population. Most significantly, functional assessment of DNA damage in pre-malignant cells, using single cell gel electrophoresis (comet assay) and γ-H2AX staining, revealed increased DNA damage, suggesting an important role for defects in DNA repair during mutant TRP53-driven lymphoma development.To investigate the nature of the dominant negative effect, mutant TRP53 protein was exogenously expressed in mouse Eµ-Myc Trp53+/+ lymphoma cell lines. The impact of mutant TRP53 on the transcriptional function of the endogenous wild-type TRP53 protein was then studied using the TRP53-activating compound, nutlin-3a. Surprisingly, in established lymphoma cell lines, mutant TRP53 impaired nutlin-3a-induced apoptosis despite substantial induction of the critical pro-apoptotic effector, PUMA. To explore this finding further, we globally characterized the dominant negative effect, and assessed for mutant TRP53-specific transcriptional targets, by performing whole transcriptome sequence (RNAseq) analysis after treatment with nutlin-3a. Analysis of known wild-type Trp53 target genes (n=283) demonstrated that the induction of these genes as a group was repressed in the presence of the mutant TRP53 protein (ROAST test, p=6.7e-04). Remarkably, however, mutant TRP53 significantly repressed only 57% of the nutlin-3a-induced Trp53 target genes. Analysis of these strongly repressed genes highlighted the importance of several pathways, including metabolism, DNA damage repair and negative feedback loops in TRP53 signaling. This suggests a previously unrecognized selectivity of the dominant-negative-effect for certain p53 pathways that may be important in cancer initiation. Additional mutant TRP53-specific transcriptional targets were also identified and are under further investigation. DisclosuresNo relevant conflicts of interest to declare.

Identifying Targets for Therapy in High Risk t(4;14) Myeloma Using Multi-Level Molecular and Phenotypic Analysis of Isogenic MMSET and MMSET Knock out Cell Lines

IntroductionIn a subset of multiple myeloma (MM) patients the t(4;14) deregulates the histone methyltransferase, MMSET and growth factor receptor, FGFR3 and has been associated with poor prognosis. In some patients this has been ameliorated by the introduction of proteasome inhibitors but 50% of t(4;14) cases have a high-risk gene expression profile (GEP70) at presentation and derive less benefit from their use. The t(4;14), therefore, constitutes a very significant target for therapy. Despite international efforts at drug design it has proven difficult to target MMSET directly. Targeting FGFR3 has been attempted and may be effective when the gene is mutated, but this only occurs in a small proportion. We have used multi-level molecular (proteomic, gene expression, metabolomic) and phenotypic analysis to identify downstream molecules upregulated in t(4;14) MM as an alternative approach to defining novel targets for therapy.Materials/MethodsThe MM cell line KMS11 has t(4;14) which re-locates MMSET downstream of the immunoglobulin heavy chain gene enhancer. From KMS11 the isogenic paired cell lines non-translocated allele knock out (NTKO, retains high MMSET expression) and translocated allele knock out (TKO, low MMSET expression) have been derived using homologous recombination (Lauring et al, Blood 2008).We performed Affymetrix GEP (HG_U133_plus_2) on mRNA extracted from KMS11, NTKO and TKO cells in triplicate and analysed using Partek®. Probes were included in the analysis if they had fold change (FC) >2 or <-2 (FDR <0.05). Mass spectrometry was performed on lysates from cells grown in media supplemented with heavy/light labeled amino acids. Proteins meeting quality metrics and FC >2 or <-2 were included. Pathway analysis was performed in WebGestalt.A high throughput CellTitre-Blue® viability screen using 484 targeted anti-cancer compounds (80, 200, 800nM) was performed in KMS11vTKO cells (n=3), and the relative reduction in viability compared.ResultsGEP results from 1349 patients revealed increased expression of DSG2, BACE2, SULF2 and PTP4A3 along with MMSET and FGFR3 in the t(4;14) subgroup, suggesting novel targets for therapy.GEP experiments in KMS11, NTKO and TKO cell lines showed increased expression of DSG2 and BACE2 genes in MMSET high vs low cells, indicating this occurs downstream of MMSET. In total 283 genes had altered expression common between MMSET high and low cell line comparisons (246 increased in MMSET high cells, 37 increased in MMSET low). Kegg pathways including cell adhesion molecules (7 genes, P =0.001), tight junctions (6, P =0.005) and apoptosis (5, P =0.007) were enriched.SILAC mass spectrometry identified 58 proteins with altered expression common between MMSET high and low cell line comparisons (41 increased in MMSET high cells, 17 increased in MMSET low). Kegg pathways including steroid biosynthesis (2 proteins, P =0.0014) and metabolic pathways (8, P =0.0125) were enriched suggesting metabolomic analysis (in progress) may yield interesting results.Combining mRNA and proteomic analysis, 13 genes/proteins were consistently altered in high vs low MMSET cell lines (12 increased in MMSET high). The most readily targetable of those upregulated is carbonic anhydrase 2, for which there are therapeutic agents available. Another, CD55, accelerates the decay of complement components. This hinders the formation of the membrane attack complex, required for the activity of antibody therapies, suggesting their efficacy may be impaired in this subgroup. 5/12 of those upregulated were associated with the actin cytoskeleton highlighting another novel target for therapy.Compounds that resulted in preferential reduction in cell viability in MMSET high cells included histone deacetylase inhibitors. MMSET has been shown to interact with HDAC1 suggesting a mechanism by which this selectivity might be mediated. Other classes associated with selectivity included PI3K/mTOR and topoisomerase inhibitors.ConclusionsUsing a multi-modality approach we have identified a number of potential targets in t(4;14) MM. We have also identified a potential biomarker of antibody-targeted therapy resistance in this subgroup, suggesting molecular subgroup analysis of clinical trials using these agents will be important.Our targets will be further validated as potential avenues for therapeutic intervention with the aim of improving outcomes in this poor prognostic group. DisclosuresPawlyn:Celgene: Honoraria, Other: Travel support; The Institute of Cancer Research: Employment. Stein:University of Arkansas for Medical Sciences: Employment. Kaiser:Chugai: Consultancy; BristolMyerSquibb: Consultancy; Janssen: Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Morgan:Takeda-Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Arkansas for Medical Sciences: Employment; Weisman Institute: Honoraria; CancerNet: Honoraria; MMRF: Honoraria. Davies:Onyx-Amgen: Honoraria; Celgene: Honoraria; University of Arkansas for Medical Sciences: Employment; Takeda-Milenium: Honoraria.

Evidence for Mutant p53 Gain-of-Function Effects in Normal Haemopoietic Cells and Myc-Driven Lymphoma

Deregulated c-MYC expression and mutations in p53 are among the most common changes detected in human cancer. It is now established that mutant p53 proteins confer a poor prognosis in human cancer through both loss of wild-type p53 activity as well as various proposed gain-of-function properties. The specific role of mutant p53 in MYC-driven tumorigenesis is not known. The Eμ-Myc mouse model carries a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma (BL). These mice develop aggressive pre-B or B cell lymphomas and ~20% of those tumours exhibit p53 mutations. We have shown that MYC-driven lymphomas are exquisitely dependent on the pro-survival BCL-2 family member MCL-1 such that loss of a single allele of Mcl-1 leads to dramatic tumour regression and prolonged animal survival. Interestingly, we found that this dependency on MCL-1 is reduced, but not completely ablated, by the presence of a p53 mutation. This suggests an important role for mutant p53 in the sustained survival of MYC-driven lymphomas. We are investigating the effects of five different mutant mouse p53 proteins (V170M, I192S, G280, R246Q, R270H) on tumour initiation, sustained growth and chemoresistance in the Eμ-Myc mouse model.We are further examining the effect of p53 mutations on MCL-1 dependence by using a floxed Mcl-1 gene and a tamoxifen-inducible Cre-recombinase in established Eμ-Myc lymphomas. Preliminary data suggest that both loss of wild-type p53 function as well as retroviral over-expression of mutant p53 can compensate for reduced levels of MCL-1 (loss of one Mcl-1 allele). The underlying mechanisms for this are under investigation.The role of mutant p53 in lymphoma cell survival has been further examined in Eμ-Myc lymphoma-derived cell lines. Enforced over-expression of mutant p53 in cell lines containing wild-type p53 impaired induction of apoptosis by Nutlin3A, an inhibitor of Mdm-2 (the major negative regulator of p53). Remarkably, Nutlin-3a-induced apoptosis was impaired although it caused substantial transcriptional induction of the p53 apoptosis effectors, Puma and Noxa. Importantly, different mutant p53 proteins conferred different levels of protection against cell death. The observed protection against cell death may be partly due to dominant-negative effects of mutant p53, however, it does not appear to be robust enough to account for the extent of cell survival. Furthermore, mutant p53 conferred resistance to docetaxol, which is thought to induce cell death through predominantly p53-independent mechanisms. These data suggest that mutant p53 can protect against both p53-dependent and p53-independent cell death processes. Conversely, transcriptional induction of Noxa and Puma implies that “p53-restoration therapy” may remain a feasible treatment strategy even in tumours that bear mutations in p53 and that the role of a dominant-negative effect for some mutant p53 proteins may be less important than previously considered, at least in lymphoma cells.We are also examining the effect of mutant p53 on lymphoma development utilizing a hematopoietic reconstitution model and retroviral over-expression of mutant p53 proteins. The different mutant p53 proteins investigated exhibited distinct effects during tumorigenesis. The R246Q mutant p53 protein markedly accelerated lymphoma development in the context of MYC over-expression. The R246Q mutant p53 protein demonstrated strong selection in p53-deficient (p53-/-) hematopoietic cells during reconstitution indicative of an advantageous activity in emergency hematopoiesis.Overall, these findings provide evidence for a positive oncogenic role of mutant p53 in hematopoietic cells that provides a particularly potent selective advantage in the context of MYC driven lymphoma development. Importantly, different p53 mutations exhibit different functional properties such that different p53 mutations are likely to be associated with distinct risk in human malignant disease. DisclosuresNo relevant conflicts of interest to declare.

Expression and prognostic significance of Oct2 and Bob1 in multiple myeloma: implications for targeted therapeutics

The B/plasma cell transcription factor Oct2 and its co-activator Bob1 activate the immunoglobulin heavy chain (IgH) gene enhancer. IgH translocations occur in the majority of myeloma cases, leading to overexpression of genes juxtaposed to the IgH enhancers. We hypothesized that Oct2 and Bob1 are determinants of disease behavior and potential therapeutic targets in myeloma. Oct2 and Bob1 gene expressions were measured in CD138+ plasma cells and CD138− cells from bone marrow samples from patients with myeloma (n = 37); gene expression of Oct2 and Bob1 was higher in CD138+ than in CD138− cells (p < 0.0001). Oct2 and Bob1 protein expressions were assessed in bone marrow tissue microarrays from patients with myeloma (n = 169) with fluorescent immunohistochemistry, and correlated to patient survival. High Oct2 protein expression correlated with reduced survival (hazard ratio [HR] 1.74, 95% confidence interval [CI] 1.11–2.73, p = 0.0164), whereas high Bob1 protein expression correlated with increased survival (HR 0.46, 95% CI 0.29–0.71, p = 0.0008). Oct2 should be explored as a potential selective therapeutic target in myeloma.

Mi2β Shows Chromatin Enzyme Specificity by Erasing a DNase I-hypersensitive Site Established by ACF

ATP-dependent chromatin-remodeling enzymes are linked to changes in gene expression; however, it is not clear how the multiple remodeling enzymes found in eukaryotes differ in function and work together. In this report, we demonstrate that the ATP-dependent remodeling enzymes ACF and Mi2beta can direct consecutive, opposing chromatin-remodeling events, when recruited to chromatin by different transcription factors. In a cell-free system based on the immunoglobulin heavy chain gene enhancer, we show that TFE3 induces a DNase I-hypersensitive site in an ATP-dependent reaction that requires ACF following transcription factor binding to chromatin. In a second step, PU.1 directs Mi2beta to erase an established DNase I-hypersensitive site, in an ATP-dependent reaction subsequent to PU.1 binding to chromatin, whereas ACF will not support erasure. Erasure occurred without displacing the transcription factor that initiated the site. Other tested enzymes were unable to erase the DNase I-hypersensitive site. Establishing and erasing the DNase I-hypersensitive site required transcriptional activation domains from TFE3 and PU.1, respectively. Together, these results provide important new mechanistic insight into the combinatorial control of chromatin structure.

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.

Activation of Mammalian Target of Rapamycin Signaling Promotes Cell Cycle Progression and Protects Cells from Apoptosis in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

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-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells.

The t(14;18) translocation, which is characteristic of follicular lymphoma, results in the overexpression of the bcl-2 gene dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy chain gene enhancers. Conflicting evidence exists on the effects of NF-kappaB expression on Bcl-2 levels in different cell types. Lymphoma cells with the t(14;18) translocation show high levels of nuclear NF-kappaB proteins. We observed decreased levels of endogenous Bcl-2 when the IkappaBalpha-super-repressor was expressed in a t(14;18) cell line. Deletion analysis of the bcl-2 promoter indicated that the repressive effect of the IkappaBalpha-super-repressor occurred through a region that contained no NF-kappaB consensus sequences. This highly active region contained a c-AMP response element (CRE) and several Sp1 binding sites. Chromatin immunoprecipitation assays with antibodies specific for the NF-kappaB and CREB/ATF family members, as well as Sp1, resulted in the isolation of this IkappaBalpha-super-repressor responsive region of the bcl-2 promoter. Mutation of the CRE and the two Sp1 sites in different combinations in bcl-2 reporter constructs resulted in the loss of bcl-2 promoter repression by the IkappaBalpha-super-repressor. We therefore conclude that the activation of bcl-2 by NF-kappaB in t(14;18) lymphoma cells is mediated through the CRE and Sp1 binding sites.

A Myc-associated Zinc Finger Protein-related Factor Binding Site Is Required for the Deregulation of c-mycExpression by the Immunoglobulin Heavy Chain Gene Enhancers in Burkitt's Lymphoma

The deregulation of expression of the c-myc gene in Burkitt's lymphoma results from the translocation that links one c-myc allele to one of the immunoglobulin genes. This physical linkage promotes interactions between c-myc and immunoglobulin gene regulatory elements that affect c-myc transcription initiation and elongation. We have located a region in the c-myc promoter that is required for the complete activation by the immunoglobulin heavy chain gene enhancer. This regulatory element contains a core sequence, GGGAGG, similar to the GA box recognized by the transcription factor Myc-associated zinc finger protein (MAZ). UV cross-link analysis indicated that the mass of this protein did not correspond to that of MAZ, suggesting that a protein related to but distinct from MAZ bound to this site. Mutation of this regulatory element resulted in a loss of promoter activity induced by the immunoglobulin heavy chain gene enhancer. This site was also required for the c-myc promoter shift from P2 to P1. In vivo footprinting revealed that this site was occupied on the translocated c-myc allele but not on the untranslocated allele. Taken together, these findings suggest that this regulatory element is required for the full activation of c-myc promoter activity by the immunoglobulin heavy chain gene enhancer.

Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms

Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms

Stimulation of Tat-associated kinase-independent transcriptional elongation from the human immunodeficiency virus type-1 long terminal repeat by a cellular enhancer.

The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK.

High affinity MAR-DNA binding is a common property of murine and human mutant p53.

We recently reported that murine MethA mutant but not wild-type p53 specifically binds to MAR-DNA elements (MARs) with high affinity. Here we show that this DNA binding activity is exerted not only by MethA mutant p53 but also by other murine mutant p53 proteins isolated from the transformed murine BALB/c cell lines 3T3tx and T3T3 and differing in their conformational status. High affinity MAR-DNA binding was not restricted to the Xbal-IgE-MAR-DNA fragment from the murine immunoglobulin heavy chain gene enhancer locus [Cockerill et al. (1987): J Biol Chem 262:5394-5397] used in previous studies, as MethA p53 also specifically interacted with other A/T-rich bona fide MARs. Not only murine but also human mutant p53 proteins carrying the mutational hot spot amino acid exchanges 175Arg-->His, 273Arg-->Pro, or 273Arg-->His bound to the Xbal-IgE-MAR-DNA fragment. We therefore conclude that high affinity MAR-DNA binding is a property common to a variety of mutant p53 proteins.

An Atypical Homeodomain in SATB1 Promotes Specific Recognition of the Key Structural Element in a Matrix Attachment Region

SATB1 is a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3' MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5'-(C/A)TAATA-3' to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

Expression of a Pim-1 transgene accelerates lymphoproliferation and inhibits apoptosis in lpr/lpr mice.

Transgenic mice expressing the Pim-1 kinase are predisposed to develop T-cell lymphomas with a long latency period of about 7-9 months. However, the exact functional basis of the oncogenic activity of Pim-1 remains obscure. C57BL/6 mice homozygous for the lpr mutation develop a well-described lymphoproliferative syndrome at about 26-30 weeks of age. This syndrome is characterized mainly by the accumulation of abnormal T cells in lymph nodes because of the lack of Fas receptor-induced apoptosis. We find that backcross of E mu-Pim-1 transgenics (mice with a transgene that carries the mouse Pim-1 gene under the transcriptional control of the immunoglobulin heavy chain gene enhancer E mu) into lpr/lpr mice results in strong acceleration of lymphoproliferation and dramatic enlargement of lymph nodes. In addition, we show here that cultured lymph node cells from E mu-Pim-1 lpr/lpr mice are rescued from rapid apoptosis that normally occurs in nontransgenic lpr cells in vitro. We also present evidence that CD4+/CD8+ double-positive thymocytes from lpr/lpr mice are sensitive to dexamethasone-induced apoptosis, although lpr/lpr mice lack the Fas receptor. In contrast, E mu-Pim-1 lpr/lpr animals show considerable protection from dexamethasone-induced apoptosis. These results show that Pim-1 can strongly accelerate lymphoproliferation through inhibition of apoptosis and thereby provide first insight into the functional basis for the oncogenic activity of Pim-1.

TFEC, a Basic Helix-Loop-Helix Protein, Forms Heterodimers with TFE3 and Inhibits TFE3-Dependent Transcription Activation

We have identified a new basic helix-loop-helix (BHLH) DNA-binding protein, designated TFEC, which is closely related to TFE3 and TFEB. The basic domain of TFEC is identical to the basic DNA-binding domain of TFE3 and TFEB, whereas the helix-loop-helix motif of TFEC shows 88 and 85% identity with the same domains in TFE3 and TFEB, respectively. Like the other two proteins, TFEC contains a leucine zipper motif, which has a lower degree of sequence identity with homologous domains in TFE3 and TFEB than does the BHLH segment. Little sequence identity exists outside these motifs. Unlike the two other proteins, TFEC does not contain an acidic domain, which for TFE3 mediates the ability to activate transcription. Like the in vitro translation product of TFE3, the in vitro-translated TFEC binds to the mu E3 DNA sequence of the immunoglobulin heavy-chain gene enhancer. In addition, the product of cotranslation of TFEC RNA and TFE3 RNA forms a heteromeric protein-DNA complex with mu E3 DNA. In contrast to TFE3, TFEC is unable to transactivate a reporter gene linked to a promoter containing tandem copies of the immunoglobulin mu E3 enhancer motif. Cotransfection of TFEC DNA and TFE3 DNA strongly inhibits the transactivation caused by TFE3. TFEC RNA is found in many tissues of adult rats, but the relative concentrations of TFEC and TFE3 RNAs vary considerably in these different tissues. No TFEC RNA was detectable in several cell lines, including fibroblasts, myoblasts, chondrosarcoma cells, and myeloma cells, indicating that TFEC is not ubiquitously expressed.

TFEC, a basic helix-loop-helix protein, forms heterodimers with TFE3 and inhibits TFE3-dependent transcription activation.

We have identified a new basic helix-loop-helix (BHLH) DNA-binding protein, designated TFEC, which is closely related to TFE3 and TFEB. The basic domain of TFEC is identical to the basic DNA-binding domain of TFE3 and TFEB, whereas the helix-loop-helix motif of TFEC shows 88 and 85% identity with the same domains in TFE3 and TFEB, respectively. Like the other two proteins, TFEC contains a leucine zipper motif, which has a lower degree of sequence identity with homologous domains in TFE3 and TFEB than does the BHLH segment. Little sequence identity exists outside these motifs. Unlike the two other proteins, TFEC does not contain an acidic domain, which for TFE3 mediates the ability to activate transcription. Like the in vitro translation product of TFE3, the in vitro-translated TFEC binds to the mu E3 DNA sequence of the immunoglobulin heavy-chain gene enhancer. In addition, the product of cotranslation of TFEC RNA and TFE3 RNA forms a heteromeric protein-DNA complex with mu E3 DNA. In contrast to TFE3, TFEC is unable to transactivate a reporter gene linked to a promoter containing tandem copies of the immunoglobulin mu E3 enhancer motif. Cotransfection of TFEC DNA and TFE3 DNA strongly inhibits the transactivation caused by TFE3. TFEC RNA is found in many tissues of adult rats, but the relative concentrations of TFEC and TFE3 RNAs vary considerably in these different tissues. No TFEC RNA was detectable in several cell lines, including fibroblasts, myoblasts, chondrosarcoma cells, and myeloma cells, indicating that TFEC is not ubiquitously expressed.