TP63 P2 promoter functional analysis identifies ß-catenin as a key regulator of ΔNp63 expression.

The ΔNp63 protein, a product of the TP63 gene that lacks the N-terminal domain, has a critical role in the maintenance of self renewal and progenitor capacity in several types of epithelial tissues. ΔNp63 is frequently overexpressed in squamous cell carcinoma (SCC) and in some other epithelial tumours. This overexpression may contribute to tumour progression through dominant-negative effects on the transcriptionally active (TA) isoforms of the p53 family (TAp63, TAp73 and p53), as well as through independent mechanisms. However, the molecular basis of ΔNp63 overexpression is not fully understood. Here, we show that the expression of ΔNp63 is regulated by the Wnt/ß-catenin pathway in human hepatocellular carcinoma (HCC) and SCC cell lines. This regulation operates in particular through TCF/LEF sites present in the P2 promoter of TP63. In addition, we show that ΔNp63 and ß-catenin are frequently coexpressed and accumulated in oesophageal SCC, but not in HCC. These results suggest that activation of the ß-catenin pathway may contribute to overexpression of ΔNp63 during tumour progression, in a cell type-specific manner.

TWISTing an embryonic transcription factor into an oncoprotein.

Over the past decade, the reactivation of TWIST embryonic transcription factors has been described as a frequent event and a marker of poor prognosis in an impressive array of human cancers. Growing evidence now supports the premise that these cancers hijack TWIST's embryonic functions, granting oncogenic and metastatic properties. In this review, we report on the history and recent breakthroughs in understanding TWIST protein functions and the emerging role of the associated epithelial-mesenchymal transition (EMT) in tumorigenesis. We then broaden the discussion to address the general contribution of reactivating embryonic programs in cancerogenesis.

[Role of the epithelial-mesenchymal transition during tumor progression]. / Rôle de la transition épithéliomésenchymateuse dans la progression tumorale.

The epithelial-mesenchymal transition (EMT) is a morphogenetic program that converts epithelial into mesenchymal cells during the embryonic development. This mechanism is frequently reactivated during tumor progression and provides cells with motility and invasive capabilities favoring the metastatic dissemination from epithelial tumors. Various EMT-inducing transcription factors, such as the TWIST proteins, were also shown to inhibit oncogene-induced fail-safe programs (senescence and apoptosis), thereby promoting the progression from benign to malignant stages. Altogether, these observations suggest that EMT could play an important role in favoring both tumor development and metastatic dissemination.

A twist for survival and cancer progression.

A major obstacle to the expansion of abnormal cells with significant proliferative potential is the induction of programmed cell death. Consequently, oncogene-driven hyperproliferation must be associated with apoptosis inhibition to allow malignant outgrowth. The oncogenic cooperation of N-Myc and Twist-1 in the development of neuroblastoma, the most common and deadly solid tumour of childhood, perfectly illustrates such a process. N-Myc promotes cell proliferation, whereas Twist-1 counteracts its pro-apoptotic properties by knocking-down the ARF/p53 pathway. On the basis of numerous recent studies reporting its overexpression in a variety of human cancers, we discuss in this review the role of Twist-1 as a potent inhibitor of the cell safety programs engaged in response to an abnormal mitogenic activity.

Activation of the Notch-regulated transcription factor CBF1/RBP-Jkappa through the 13SE1A oncoprotein.

Signaling through the Notch pathway controls cell growth and differentiation in metazoans. Following binding of its ligands, the intracellular part of the cell surface Notch1 receptor (Notch1-IC) is released and translocates to the nucleus, where it alters the function of the DNA-binding transcription factor CBF1/RBP-Jkappa. As a result, CBF1/RBP-Jkappa is converted from a repressor to an activator of gene transcription. Similarly, the Epstein Barr viral oncoprotein EBNA2, which is required for B-cell immortalization, activates genes through CBF1. Moreover, the TAN-1 and int-3 oncogenes represent activated versions of Notch1 and Notch4, respectively. Here, we show that the adenoviral oncoprotein 13S E1A also binds to CBF1/RBP-Jkappa, displaces associated corepressor complexes, and activates CBF1/RBP-Jkappa-dependent gene expression. Our results suggest that the central role of the Notch-CBF1/RBP-Jkappa signaling pathway in cell fate decisions renders it susceptible to pathways of viral replication and oncogenic conversion.

The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators.

The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

Cloning of two novel human importin-alpha subunits and analysis of the expression pattern of the importin-alpha protein family.

The import of many proteins into the nucleus is mediated by the importin-alpha/beta heterodimer. While only one importin-beta gene has been found, several forms of importin-alpha have been described. In addition to the three human importin-alphas already identified, we report here the primary structure of two new human importin-alpha proteins. The five known human importin-alpha subunits can be classified into three subfamilies that appear conserved in higher eukaryotic organisms. We show by immunoblotting that the different importin-alpha subfamilies are expressed in a variety of human tissues and mammalian cell lines.

Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein.

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

B-Myb, a repressed trans-activating protein.

B-Myb belongs to a family of related transcription factors which share a unique DNA binding domain. B-Myb plays an important role in regulation of the cell cycle. Its expression is upregulated by the human papilloma virus HPV16 E7 oncoprotein. Overexpression of B-Myb can bypass p53-mediated cell cycle arrest. The founding member of the myb gene family, c-Myb, and A-Myb are involved in hematopoiesis and neurogenesis, respectively, and are both activators of gene transcription. Whether B-Myb is a transactivator or a repressor, however, has remained a matter of discussion. We reviewed the transactivation potential of B-Myb in yeast, taking advantage of the fact that inducible gene activation is an evolutionarily conserved process. By mutational analysis we localized a conserved activation domain in B-Myb. In vertebrate cells the transactivation potential of B-Myb is concealed by the C-terminal part of the protein. We show that the cell cycle regulators cyclin A and cyclin E activate B-Myb by eradicating the inhibition mediated by its carboxy-terminus. Our data suggest that in vertebrates the trans-activating function of B-Myb is regulated during the cell cycle and link Myb functions to cell cycle progression.

Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation.

CD30 is a member of the tumor necrosis factor receptor superfamily, which can transduce signals for proliferation, death, or nuclear factor kappa B (NF-kappa B) activation. Investigation of CD30 signaling pathways using a yeast two-hybrid interaction system trapped a cDNA encoding the tumor necrosis factor receptor-associated factor (TRAF)-2 TRAF homology domain. TRAF-1 and TRAF-3 also interacted with CD30, and > 90% of in vitro-translated TRAF-1 or -2, or 50% of TRAF-3, bound to the CD30 cytoplasmic domain. TRAF-1, -2, and -3 bound mostly, but not exclusively, to the carboxyl-terminal 36 residues of CD30. The binding was strongly inhibited by a CD30 oligopeptide centered around a PXQXT (where X is any amino acid) motif shared with CD40 and the Epstein-Barr virus transforming protein LMP1, indicating that this motif in CD30 is an important determinant of TRAF-1, -2 or -3 interaction. At least 15% of TRAF-1, -2, or -3 associated with CD30 when coexpressed in 293 cells. The association was not affected by CD30 cross-linking. However, cross-linking of CD30 activated NF-kappa B. NF-kappa B activation was dependent on the carboxyl-terminal 36 amino acids of CD30 that mediate TRAF association. TRAF-2 has been previously shown to have a unique role in TRAF-mediated NF-kappa B activation, and NF-kappa B activation following CD30 cross-linking was blocked by a dominant negative TRAF-2 mutant. These data indicate that CD30 cross-linking-induced NF-kappa B activation is predominantly TRAF-2-mediated.

Regulation of C/EBP beta/NF-M activity by kinase oncogenes.

CAAT Enhancer Binding proteins (C/EBP) belong to a family of transcription factors which are implicated in a number of developmental and growth regulatory processes. One member of this family known as C/EBP beta (called NF-M in the chicken system) is particularly important in myelomonocytic cells because it is targeted by kinases and collaborates with the Myb oncoprotein to induce the expression of myeloid specific genes. Experiments dissecting the structure of NF-M suggest that it is a repressed transcription factor. Using the yeast two-hybrid system we showed that a negative regulatory domain masks the transactivation domain. Examination of NF-M mutants suggests that kinase (proto-) oncogenes uncover the concealed transcriptional activity by phosphorylation of the negative regulatory domain.

The BTB/POZ domain targets the LAZ3/BCL6 oncoprotein to nuclear dots and mediates homomerisation in vivo.

The LAZ3/BCL6 gene was identified by its disruption in 3q27 translocations associated with diffuse large cell lymphomas. It is predicted to be a transcription factor as it contains six Krüppel-like Zinc finger motifs and a N-terminal BTB/POZ domain, a protein/protein interaction interface that is widely conserved in Metazoans. Using two antisera raised against non overlapping regions of the predicted ORF, we demonstrate that the LAZ3/BCL6 protein appears as a close ca. 79 kDa doublet in B lymphoid cell lines with either a rearranged or a non rearranged LAZ3/BCL6 locus. By immunofluorescence experiments on transiently transfected COS-1 or NIH3T3 cells, we show that the LAZ3/BCL6 protein displays a punctuated nuclear localisation. This appears to rely on LAZ3/BCL6 proper folding and/or activities as it is impaired in a hormone reversible-fashion through fusion of LAZ3/BCL6 to the ligand-binding domain of the oestrogen receptor. Moreover, deletion of its BTB/POZ domain leads to the disappearance of the nuclear dots although the protein remains nuclear. In addition, by using the yeast two-hybrid system, we show that the LAZ3/BCL6 BTB/POZ domain homomerises in vivo. Thus, the LAZ3/BCL6 BTB/POZ domain has the capability to self-interact and target the protein to discrete nuclear substructures.

Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression.

Phosphorylation of transcription factors is regarded as a major mechanism to control their activity in regulation of gene expression. C/EBP beta is a transcription factor that becomes activated after phosphorylation to induce genes involved in inflammation, acute-phase response, cytokine expression, cell growth, and differentiation. The chicken homolog NF-M collaborates with Myb and various kinase oncogenes in normal myeloid differentiation as well as in the leukemic transformation of myelomonocytic cells. Here, we examined the structure of NF-M and its mechanism of activation. We show that NF-M is a repressed transcription factor with concealed activation potential. Derepressed NF-M exhibits enhanced transcriptional efficacy in reporter assays. More importantly, NF-M activates resident chromatin-embedded, myelomonocyte-specific target genes, even in heterologous cell types such as fibroblasts or erythroblasts. We identified two regions within NF-M that act to repress trans-activation. Repression is abolished by deletion of these regions, activation of signal transduction kinases including v-erbB, polyoma middle T, ras and mil/raf, or point mutation of a critical phosphorylation site for MAP kinases. We provide evidence that phosphorylation plays a unique role to derepress rather than to enhance the trans-activation domain as a novel mechanism to regulate gene expression by NF-M/C/EBP beta.

Identification and analysis of the chicken c-mil promoter: possible involvement of Sp1- and Ets-related proteins.

We have determined the exon organization in the 5' region of the chicken c-mil gene and identified its promoter. A 0.44-kb fragment containing the 5' terminus of the c-mil gene showed strong promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into chicken embryo fibroblasts (CEF). By primer extension analysis, multiple transcriptional start sites were detected within the promoter region. Nucleotide sequence analysis revealed that the c-mil promoter had a high G + C content (71.8%) and contained multiple GC box-like sequences, but no TATA or CAAT boxes. Deletion analysis of 5' upstream sequences showed that the minimal region required for maximal promoter activity in CEF resides in the 99 bp located immediately upstream of the major initiation site. This region contains two putative Sp1 binding sites and one PU box/PEA3 motif, defined as a recognition element for members of the Ets gene family. These sequences bound proteins present in nuclear extracts of CEF as well as in vitro synthesized Ets-related proteins, suggesting that the binding of Sp1 or related proteins and of Ets-related proteins within the promoter is important for modulation of the mil gene.

An alternatively spliced c-mil/raf mRNA is predominantly expressed in chicken muscular tissues and conserved among vertebrate species.

The chicken c-mil gene produces two mRNA species generated by an alternative splicing mechanism. These two transcripts differ at least by the presence or absence of a 60 nucleotide exon (E7a) localized at the splice junction of c-mil exons 7 and 8. By using RNAase protection assays, we have analysed the pattern of expression of these two mRNA species in several chicken tissues. Here we report that the two c-mil mRNAs are differentially expressed in chicken tissues: the mRNA lacking E7a is detected in all tissues tested, while the mRNA containing E7a is detected only in the skeletal muscle, heart and brain. Sequences homologous to E7a have also been detected in DNAs from quail, mouse and human cells and their sequencing revealed that the alternative E7a is structurally preserved in these species. By PCR analyses performed on RNAs extracted from muscular tissues of these species, we also show that the alternative splicing mechanism described in chicken also occurs in these species.

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression.

A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.