A model for gene evolution of the ets-1/ets-2 transcription factors based on structural and functional homologies.

The chicken c-ets-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that differ in their N-termini, encoded respectively by the I54 and alpha beta exons. p68c-ets-1 equivalents are only found in birds and reptiles while p54c-ets-1 is widely conserved in vertebrates, from amphibians to mammals. Thus, the classical view concerning the evolution of the c-ets-1 gene has been to consider that I54 is of ancient origin whereas alpha and beta, which provide an additional activating domain in p68c-ets-1, would have been acquired much more recently. Sequencing the alpha and beta exons in various species pinpointed a highly conserved region of 13 amino acids which is rich in acidic and hydrophobic residues, a feature of some other transactivating domains. Strikingly, this subdomain is also present in the otherwise unrelated N-terminal activating region of p58c-ets-2 and was thus named BEC for Ets-1-beta/Ets-2-Conserved sequence. Moreover, the two N-termini share the BEC sequence at a homologous position in their highly similar genomic organization indicating a common origin. This structural homology underlies a functional similarity since fusion of the heterologous GAL4 DNA-binding domain with either of the two isolated domains demonstrates that BEC is essential in both cases for the transactivating activity. The function of the alpha beta domain in the context of p68c-ets-1 also strictly depends on the presence of the BEC sequence. Finally, the whole N-terminus of p58c-ets-2 can functionally substitute for its counterpart in p68c-ets-1 further demonstrating that p68c-ets-1 and p58c-ets-2 are structurally and functionally more closely related than previously thought. Besides, we also found BEC in the N-terminus of the Drosophila pointed gene which may be considered as closely related to the uncommitted 'ets1/2' common ancestor. These data demonstrate that the alpha and beta exons are not a recent and specific acquisition but stem, like the p58c-ets-2 N-terminus, from the invertebrate unduplicated 'ets 1/2' gene. This work unravels a new model for the ets-1/ets-2 gene's evolution, based for the first time on both structural and functional evidences. Accordingly, p68c-ets-1 and p58c-ets-2 are the direct descendants of the ancestral 'ets1/2' gene whereas I54 may have been acquired as a second promoter in the c-ets-1 gene after the duplication. Indeed, I54 is not found in the Drosophila pointed gene. The high degree of similarity, and hence of functional redundancy, between p68c-ets-1 and p58c-ets-2 may have led to the rapid divergence (and even loss in mammals) of alpha and beta during evolution whereas I54, which provided a novel function unique to c-ets-1, was maintained within the presently widespread p54c-ets-1 version.

Colocalization and heteromerization between the two human oncogene POZ/zinc finger proteins, LAZ3 (BCL6) and PLZF.

Most acute promyelocytic leukemia (APL) cases are associated with recurrent translocations between the gene of retinoic receptor alpha and that of PML (t(15;17)) or PLZF (t(11;17)). PML localizes onto discrete intranuclear domains, the PML-nuclear bodies, and displays anti-oncogenic and pro-apoptotic properties. PLZF encodes a transcription factor belonging to the POZ/domain and Krüppel zinc finger (POK) family which interacts directly with PML. PLZF is related to another POK protein, LAZ3(BCL6), which is structurally altered, and presumably misexpressed, in many non-Hodgkin lymphoma (NHL) cases. PLZF and LAZ3 share many functional properties: both inhibit cell growth, concentrate into punctated nuclear subdomains and are sequence-specific transcriptional repressors recruiting a histone deacetylase-repressing complex. Given these similarities, we tested whether both proteins could be targeted by each other. Here, LAZ3 and PLZF are shown to colocalize onto nuclear dots. Moreover, truncated derivatives of one protein, which display a diffuse nuclear localization, are recruited onto nuclear dots by the full-length other. The colocalization and the reciprocal 'rescue' is the result of a direct interaction between LAZ3 and PLZF, as indicated by yeast two hybrid assays, in vitro immunoprecipitations, and GST pull down experiments. In contrast to LAZ3 homomerization, LAZ3/PLZF heteromerization in yeast does not solely depend on POZ/POZ contacts but rather also relies on interactions between the two zinc finger regions and 'cross' contacts between the zinc finger region and the POZ domain of each partner. Likewise, LAZ3 shows some colocalization with the PLZF partner PML upon stable overexpression of both proteins in CHO cells and interacts with PML in yeast. Finally, endogenous LAZ3 and PLZF are co-induced and partially colocalized in myeloid MDS cells. These data indicate that a physical interaction between LAZ3 and PLZF underlies their simultaneous recruitment onto multiproteic nuclear complexes, presumably involved in transcriptional silencing and whose integrity (for APL) and/or function (for APL and NHL) may be altered in oncogenesis.

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 BTB/POZ domain zinc-finger protein, LRF, is a potential target of the LAZ-3/BCL-6 oncogene.

BTB/POZ-domain C2H2 zinc(Zn)-finger proteins are encoded by a subfamily of genes related to the Drosophila gap gene krüppel. To date, two such proteins, PLZF and LAZ-3/BCL-6, have been implicated in oncogenesis. We have now identified a new member of this gene subfamily which encodes a 62 kDa Zn-finger protein, termed LRF, with a BTB/POZ domain highly similar to that of PLZF. Both human and mouse LRF genes, which localized to syntenic chromosomal regions (19p13.3 and 10B5.3, respectively), were widely expressed in adult tissues and cell lines. At approximately 9.5-10.0 days of embryonic development, the mouse LRF gene was expressed in the limb buds, pharyngeal arches, tail bud, placenta and neural tube. The LRF protein associated in vivo with LAZ-3/BCL-6, but not with PLZF to which it was more related. Although the LRF, or LAZ-3/BCL-6, BTB/POZ domain could readily homodimerize, no heterodimerization was detected in vivo between the LRF and LAZ-3/BCL-6 BTB/POZ domains and interaction between full length LRF and LAZ-3/BCL-6 required the presence of both the BTB/POZ domain and Zn-fingers in each partner protein. As expected from the above results, LRF and LAZ-3/BCL-6 also colocalized with each other in the nucleus. Taken together, our findings suggest that BTB/ POZ-domain Zn-finger proteins may function as homo and heterodimeric complexes whose formation, and hence the resultant effect on transcription of their downstream target genes, is determined by the levels and expression domains of a given partner protein.

Mesodermal expression of the chicken erg gene associated with precartilaginous condensation and cartilage differentiation.

The ets gene superfamily encodes a class of transcription factors that bind to a purine rich sequence through a 85 amino-acid ETS domain. Among them, the human erg gene has been found to be involved in Ewing's sarcoma, primitive neurectodermal tumour of childhood and acute myeloid leukaemia. Nevertheless, little is known about human erg expression. Northern blot analyses have shown a human erg expression restricted to few cell lines and thymus, but the status concerning expression during development remains unknown probably because no homologue of this gene has yet been isolated and studied in other vertebrates. We thus choose to clone the chicken erg gene (ck-erg) and to study its expression during chicken development. We obtained a bona fide clone of ck-erg and defined the transcriptional modulating properties of its product. The ck-Erg protein acts as a transcriptional activator through a conventional consensus ETS binding site. Northern blot studies on various chicken tissues, in situ analyses and comparison with the well-characterised c-ets-1 expression show that ck-erg is expressed in mesoderm- and, to a lesser extent, in ectoderm-derived tissues. During chicken development, two salient features could be observed. From stage E1 to E3.5, ck-erg expression was widely distributed in mesodermal derivatives and neural crest, resembling c-ets-1 expression. However, by E6, the expression of ck-erg exhibited, unlike c-ets-1, a drastically new and strong signal in precartilaginous condensation zones and cartilaginous skeletal primordia. These stages are the first steps of bone formation during skeletal elaboration. Our results show for the first time a possible specific involvement of ck-erg in cartilage morphogenesis.

A residue of the ETS domain mutated in the v-ets oncogene is essential for the DNA-binding and transactivating properties of the ETS-1 and ETS-2 proteins.

The c-ets-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that recognize purine-rich motifs. The v-ets oncogene of the avian retrovirus E26 differs from its cellular progenitor p68c-ets-1 by two amino acid substitutions (alanine 285 and isoleucine 445 in c-ets-1 both substituted by valine in v-ets, mutations A and B respectively) and its carboxy-terminal end (mutation C). The B mutation affects a well conserved residue in the carboxy-terminal 85 amino acids, ETS DNA-binding domain. To address the biological relevance of the B mutation found between v-ets and c-ets-1, we have randomly mutagenized isoleucine 445 of p68c-ets-1 by polymerase chain reaction. Using in vitro gel mobility shift assays, we show that this residue is crucial for the binding properties of c-ets-1 since the 12 mutations we have generated at this position, all diminish or even abolish the binding, to the 'optimized' Ets-1 binding site (EBS), of 35 kDa proteins corresponding to the 311 carboxy-terminal residues of c-ets-1. Among them, substitutions of isoleucine to glutamic acid, glycine or proline have the highest inhibitory effects. Similar results were obtained when the same mutations were introduced either in full-length p68c-ets-1 protein or into a carboxy-terminal polypeptide of 109 amino acids encompassing the ETS-domain which has previously been shown to display a very high binding activity as compared with the full-length protein. Consistent with the in vitro results, point mutations in p68c-ets-1 that decrease binding activity to EBS abrogate its ability to transactivate reporter plasmids carrying either the TPA Oncogene Response Unit of the Polyoma virus enhancer (TORU) or a sequence derived from the HTLV-1 LTR. Furthermore, as this isoleucine residue is rather well-conserved within the ETS gene family, we show that mutation of the corresponding isoleucine of c-ets-2 into glycine also abrogates its DNA-binding and hence, transactivating properties. Thus, the v-ets B mutation highlights the isoleucine 445 as an essential amino acid of the c-ets-1 and c-ets-2 DNA-binding domains.

Multiple domains participate in distance-independent LAZ3/BCL6-mediated transcriptional repression.

The LAZ3/BCL6 gene implicated in diffuse large cell lymphomas encodes a transcriptional repressor containing Krüppel-fike zinc fingers. It harbours at its N-terminus a conserved protein/protein interaction motif, the BTB/POZ domain, which is also an autonomous transcriptional repression domain. We demonstrate here using several GAL4-LAZ3/BCL6 chimeras that the BTB/POZ domain plays an important but not exclusive role as its deletion gives rise to a GAL4 chimera that mediates significant, albeit reduced, transcriptional repression. Moreover, the repressive effect mediated either by LAZ3/BCL6 or by the isolated domains occurs with unaltered efficiency even at long distance (1.6 Kbp), ruling out steric hindrance mechanisms. Finally, though the absence of a TATA box appears to weaken this activity, it is largely promoter independent. Taken together, our results demonstrate that multiple domains participate in the promoter and distance-independent LAZ3/BCL6-mediated transcriptional repression.

The BTB/POZ domain: a new protein-protein interaction motif common to DNA- and actin-binding proteins.

The BTB/POZ domain defines a newly characterized protein-protein interaction interface. It is highly conserved throughout metazoan evolution and generally found at the NH2 terminus of either actin-binding or, more commonly, nuclear DNA-binding proteins. By mediating protein binding in large aggregates, the BTB/POZ domain serves to organize higher order macromolecular complexes involved in ring canal formation or chromatin folding.

The LAZ3/BCL6 oncogene encodes a sequence-specific transcriptional inhibitor: a novel function for the BTB/POZ domain as an autonomous repressing domain.

Rearrangements and mutations of the LAZ3/BCL6 gene are the most frequent events associated with diffuse large-cell lymphoma, a particular class of non-Hodgkin's lymphomas. This gene encodes a putative regulatory protein with six COOH-terminal Krüppel-like zinc fingers and a NH2-terminal hydrophobic region, the so-called BTB/POZ domain, which mediates homo- as well as heterotypic interactions in other related proteins. Recently, a consensus binding sequence has been defined using the isolated LAZ3/BCL6 zinc finger region produced in bacteria. To understand the normal and oncogenic functions of LAZ3/BCL6, we examined its properties as a transcription factor. We thus demonstrated that its full-length product binds to the same consensus sequence, although the BTB/POZ domain decreases this activity, at least in vitro. In transient transfection experiments, the LAZ3/BCL6 protein exerts a repressive effect, both as a wild-type protein on its own target sequence and as a GAL-4 fusion protein. Furthermore, our results indicate that the BTB/POZ domain plays a prominent role in the mediation of this activity. Indeed, on the LAZ3/BCL6 cognate sequence, deletion of the BTB/POZ domain diminishes the repressive function. Conversely, as a GAL-4 chimera, the isolated LAZ3/BCL6 BTB/POZ domain appears nearly as efficient as the entire protein at inducing transcriptional repression. Taken together, these findings demonstrate that the LAZ3/BCL6 is a sequence-specific transcriptional repressor and point to a novel function for the BTB/POZ region, at least in LAZ3/BCL6, as an autonomous transcriptional inhibitory domain.

The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression.

Recent works demonstrated that some transcriptional repressors recruit histone deacetylases (HDACs) either through direct interaction, or as a member of a multisubunit repressing complex containing other components referred to as corepressors. For instance, the bHLH-Zip transcriptional repressors MAD/MXI recruit HDACs together with the mSIN3 corepressors, whereas unliganded nuclear receptors contact another corepressor, SMRT (or its relative N-CoR), which, in turn, associates with both mSIN3 and HDACs to form the repressor complex. Recently, we reported that SMRT also directly associates with LAZ3(BCL-6), a POZ/Zn finger transcriptional repressor involvedin the pathogenesis of non-Hodgkin lymphomas. However, whether LAZ3 recruits the HDACs-containing repression complex is currently unknown. We report here that LAZ3 associates with corepressor mSIN3A both in vivo and in vitro , and found that a central region, which harbours autonomous repression activity, is mainly responsible for this interaction. Conversely, the N-terminal half of mSIN3A is both necessary and sufficient to bind LAZ3. Moreover, we show that LAZ3 also interacts with an HDAC (HDAC-1) through its POZ domain in vitro while the immunoprecipitation of LAZ3 results in the coretention of an endogenous HDAC activity in vivo . Finally, inhibitors of HDACs significantly reduce the LAZ3-mediated repression. Taken together, we conclude that LAZ3 recruits a repressing complex containing SMRT, mSIN3A and a HDAC, and that its full repressing potential on transcription requires HDACs activity. Our results identify HDACs as molecular targets of LAZ3 oncogene and further strengthen the connection between aberrant chromatin acetylation and human cancers.

Increased expression of the LAZ3 (BCL6) proto-oncogene accompanies murine skeletal myogenesis.

The structural alterations of the LAZ3 (BCL6) gene are one of the most frequent events found in non-Hodgkin lymphoma. LAZ3 encodes a transcriptional repressor with a POZ/zinc finger structure similar to several Drosophila development regulators and to the human promyelocytic leukemia-associated PLZF gene. Consistent with the origin of LAZ3-associated malignancies, LAZ3 is expressed in mature B-cells and required for germinal center formation. However, its ubiquitous expression, with predominant levels in skeletal muscle, suggests that it may act outside the lymphoid system. To study how LAZ3 could be involved in skeletal muscle differentiation, we examined its expression in the C2 muscle cells. We report here that LAZ3 is upregulated at both mRNA and protein levels during the differentiation of proliferating C2 myoblasts into post-mitotic myotubes. This rise in LAZ3 expression is both precocious and sustained, and is not reversed when myotubes are re-exposed to mitogen-rich medium, suggesting that irreversible evens occurring upon myogenic terminal differentiation contribute to lock LAZ3 upregulation. In addition, using two different models, we found that a "simple" growth-arrest upon serum starvation is not sufficient to induce LAZ3 upregulation which rather appears as a feature of myogenic commitment and/or differentiation. Finally, BrdU incorporation assays in C2 cells entering the differentiation pathway indicate that "high" LAZ3 expression strongly correlates with their exit from the cell cycle. Taken as a whole, these findings suggest that LAZ3 could play a role in muscle differentiation. Together with some results reported in other cell types, we propose that LAZ3 may contribute to events common to various differentiation processes, possibly the induction and stabilization of the withdrawal from the cell cycle.

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.