Mass production of functional human pancreatic ß-cells: why and how?

Diabetes (either type 1 or type 2) is due to insufficient functional ß-cell mass. Research has, therefore, aimed to discover new ways to maintain or increase either ß-cell mass or function. For this purpose, rodents have mainly been used as model systems and a large number of discoveries have been made. Meanwhile, although we have learned that rodent models represent powerful systems to model ß-cell development, function and destruction, we realize that there are limitations when attempting to transfer the data to what is occurring in humans. Indeed, while human ß-cells share many similarities with rodent ß-cells, they also differ on a number of important parameters. In this context, developing ways to study human ß-cell development, function and death represents an important challenge. This review will describe recent data on the development and use of convenient sources of human ß-cells that should be useful tools to discover new ways to modulate functional ß-cell mass in humans.

Constitutive instability of muscle regulatory factor Myf5 is distinct from its mitosis-specific disappearance, which requires a D-box-like motif overlapping the basic domain.

Transcription factors Myf5 and MyoD play critical roles in controlling myoblast identity and differentiation. In the myogenic cell line C2, we have found that Myf5 expression, unlike that of MyoD, is restricted to cycling cells and regulated by proteolysis at mitosis. In the present study, we have examined Myf5 proteolysis through stable transfection of myogenically convertible U20S cells with Myf5 derivatives under the control of a tetracycline-sensitive promoter. A motif within the basic helix-loop-helix domain of Myf5 (R93 to Q101) resembles the "destruction box" characteristic of substrates of mitotic proteolysis and thought to be recognized by the anaphase-promoting complex or cyclosome (APC). Mutation of this motif in Myf5 stabilizes the protein at mitosis but does not affect its constitutive turnover. Conversely, mutation of a serine residue (S158) stabilizes Myf5 in nonsynchronized cultures but not at mitosis. Thus, at least two proteolytic pathways control Myf5 levels in cycling cells. The mitotic proteolysis of Myf5 is unlike that which has been described for other destruction box-dependent substrates: down-regulation of Myf5 at mitosis appears to precede that of known targets of the APC and is not affected by a dominant-negative version of the ubiquitin carrier protein UbcH10, implicated in the APC-mediated pathway. Finally, we find that induction of Myf5 perturbs the passage of cells through mitosis, suggesting that regulation of Myf5 levels at mitosis may influence cell cycle progression of Myf5-expressing muscle precursor cells.

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor.

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G(1), while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

Phylogeny of the p68c-ets-1 amino-terminal transactivating domain reveals some highly conserved structural features.

The chicken c-ets-1 locus gives rise to two distinct transcription factors differing only in their structurally and functionally unrelated N-termini. One of these transcription factors, p54c-ets-1, contains a specific, short (27 amino acids), hydrophilic N-terminus encoded by a single exon, I54, that is widely conserved among vertebrates. The other one, p68c-ets-1, the cellular counterpart of the viral ets oncogene product, differs in the replacement of the I54 by two exons, termed alpha and beta, encoding a larger (71 amino acids), hydrophobic N-terminus which, in contrast to I54, exhibits properties of a transactivating domain. To date the alpha and beta exons have only been found in chicken. Here, we demonstrate the existence of the alpha and beta exons in other avian species (quail and duck) and the existence of the alpha exon in reptiles (turtle). However, none of them could be detected in mammals. Our results strongly suggest that, in contrast to the phylogenetically well-conserved I54 exon, the alpha exon is restricted to reptilian species (birds and 'true' reptiles), whereas the beta exon is detectable so far only in birds. Comparison of their amino acid sequences reveals that the alpha exon and to a much greater extent the beta exon have diverged faster than the I54 exon. In addition, we show that the N- and C-terminal thirds of the alpha exon and the highly hydrophobic nature of the alpha beta-encoded sequence are heavily conserved features and thus likely to be required for function as a transactivating domain in p68c-ets-1 and possibly in the viral P135gag-myb-ets transforming protein.

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.

Two functionally distinct domains responsible for transactivation by the Ets family member ERM.

The recently cloned human Ets transcription factor ERM is closely related to the ER81 and PEA3 genes. Here, we report the functional analysis of the DNA-binding and transactivation properties of ERM. Specific DNA-binding by ERM requires the ETS domain, conserved in all members of the Ets family and is inhibited by an 84 residue long central region and the carboxy-terminal tail. Two fragments of ERM are transferrable activation domains: alpha, which sits in the 72 first residues and encompasses the acidic domain conserved between ERM, ER81 and PEA3, and the carboxy-terminal tail which also bears a DNA-binding inhibition function. Deletion of alpha strongly reduces transactivation by ERM. Moreover, alpha and the carboxy-terminal tail exhibit functional synergism, suggesting that they activate transcription through different mechanisms. In support of this idea, we demonstrate that VP16 squelches transactivation by alpha but not by the carboxy-terminal tail. This result also indicates that alpha and VP16 may share common limiting cofactors. alpha and the carboxy-terminal tail do not seem to be conserved within the whole Ets family, indicating that the specificity of ERM may rely on interactions with distinct cofactors.

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.

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.

Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci.

One of the most frequent genetic abnormalities associated with non Hodgkin lymphoma is the structural alteration of the 5' non coding/regulatory region of the BCL6 (LAZ3) protooncogene. BCL6 encodes a POZ/Zn finger protein, a structure similar to that of many Drosophila developmental regulators and to another protein involved in a human hematopoietic malignancy, PLZF. BCL6 is a sequence specific transcriptional repressor controlling germinal center formation and T cell dependent immune response. Although the expression of BCL6 negatively correlates with cellular proliferation in different cell types, the influence of BCL6 on cell growth and survival is currently unknown so that the way its deregulation may contribute to cancer remains elusive. To directly address this issue, we used a tetracycline-regulated system in human U2OS osteosarcoma cells and thus found that BCL6 mediates growth suppression associated with impaired S phase progression and apoptosis. Interestingly, overexpressed BCL6 can colocalize with sites of ongoing DNA synthesis, suggesting that it may directly interfere with S phase initiation and/or progression. In contrast, the isolated Zn finger region of BCL6, which binds BCL6 target sequence but lacks transcriptional repression activity, slows, but does not suppress, U2OS cell growth, is less efficient at delaying S phase progression, and does not trigger apoptosis. Thus, for a large part, the effects of BCL6 overexpression on cell growth and survival depend on its ability to engage protein/protein interactions with itself and/or its transcriptional corepressors. That BCL6 restricts cell growth suggests that its deregulation upon structural alterations may alleviate negative controls on the cell cycle and cell survival.

Function of ets genes is conserved between vertebrates and Drosophila.

The Drosophila pointed gene encodes two ETS transcriptional activators, pointedP1 and pointedP2, sharing a common C-terminal ETS domain. In the embryonic central nervous system pointedP2 is required for midline glial cell differentiation, whereas, in the eye, pointedP2 is essential for photoreceptor cell differentiation. Both vertebrate c-ets-1 and c-ets-2 gene ETS domains are highly homologous to the one of pointed. In addition, the N-terminal region of pointedP2 and vertebrate ets products share another homologous domain, the so-called RII/pointed box which appears to mediate the ras-dependent phosphorylation/stimulation. Here, we show that the vertebrate ets genes are functionally homologous to the Drosophila pointed gene. pointedP2 efficiently binds to an optimized c-Ets-1/c-Ets-2 probe in vitro, and stimulates two distinct c-Ets-1/c-Ets-2-responsive sequences when transiently expressed in vertebrate cells. Conversely, when vertebrate ets transgenes are expressed during fly development, they are capable of rescuing the pointed mutant phenotype in both midline glia and photoreceptor development. As ectopically expressed pointedP1 can also rescue pointedP2 deficiency in photoreceptor development, it appears that the ability of ets products to phenocopy each other in vivo does not require the conserved RII/pointed box, but rather, primarily relies on the presence of the highly conserved ETS domain.

The two functionally distinct amino termini of chicken c-ets-1 products arise from alternative promoter usage.

The chicken c-ets-1 locus gives rise to two distinct transcription factors differing by structurally and functionally unrelated N-termini. p54c-ets-1 shows a striking phylogenetic conservation from Xenopus to humans, while p68c-ets-1, the cellular counterpart of the E26-derived v-ets oncogene, is apparently restricted to avian and reptilian species. In the chick embryo, both mRNAs are expressed in a wide array of tissues of mesodermal origin; however, in the embryo and after hatching, p68c-ets-1 is excluded from lymphoid cells where p54c-ets-1 accumulates. In this report, we define the basis of the differential expression of the chicken c-ets-1 products to assess their different potentials as transcription factors. We demonstrate that the two distinct N-termini arise from alternative promoter usage within the chicken c-ets-1 locus. Examination of both promoters reveals that transcription initiates from multiple sites, consistent with the absence of TATA and CAAT elements. Of these two regulatory regions, only the one that initiates the p54c-ets-1 mRNA synthesis is of the G + C-rich type, and its organization is conserved in humans. The avian-specific p68c-ets-1 promoter activity was enhanced by its own product. In addition, we identify numerous potential binding sites for lymphoid-specific transcription factors that might contribute to a tight repressor effect in lymphoid tissues.

Cell density-dependent induction of endogenous myogenin (myf4) gene expression by Myf5.

Transcription factors Myf5 and MyoD are critical for myoblast determination. Myogenin is a direct transcriptional target of these factors and its expression is associated with commitment to terminal differentiation. Here, we have used myogenic derivatives of human U20S cells expressing Myf5 or MyoD under control of a tetracycline-sensitive promoter to study expression of endogenous myogenin (myf4). We find that Myf5-mediated induction of myogenin shows striking dependence on cell density. At high cell density, Myf5 is a potent inducer of myogenin expression. At low cell density, Myf5 (unlike MyoD) is a poor inducer of myogenin expression, whilst retaining the capacity to direct expression of other muscle-specific genes. The permissive influence of high cell density on myogenin induction by Myf5 is not a consequence of serum depletion or cell cycle arrest, but is mimicked by a disruption adjacent to the basic region of Myf5 (Myf5/mt) which reduces its DNA binding affinity for E-boxes without compromising its ability to transactivate a reporter gene driven by the myogenin promoter. Coculture of cells expressing wild-type Myf5 and Myf5/mt leads to reduced myogenin induction in Myf5/mt cells. We propose that at low cell density Myf5 inhibits induction of myogenin.

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.

A conditional version of the Ets transcription factor Erm by fusion to the ligand binding domain of the oestrogen receptor.

The fusion of a wide range of proteins to the ligand-binding domain of nuclear receptors has been shown to impart ligand-dependent inducible activity of the resulting chimera. Transcriptional regulators of the ETS family are involved in both normal and oncogenic processes. In order to address the role of Erm, a "PEA3 subgroup" member of this family, we generated a chimera between Erm and the widely used ligand-binding domain of the oestrogen receptor (ER). The chimera, ErmER, consists of Erm protein fused at its C-terminal end to the ER domain. We show that ErmER displays a ligand-dependent transcriptional activity on ets responsive elements. The efficiency of ErmER mediated transactivation is modulated by the hormone concentration while its weak leakiness is reduced by using the steroidal anti-oestrogen EM-139. Our results define ErmER as the first conditional version of an Ets transcription factor, providing a useful tool to decipher Erm biological role and to identify potential Erm target genes.

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 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.

Expression of the PEA3 group of ETS-related transcription factors in human breast-cancer cells.

The PEA3 group of transcription factors belongs to the ets family and is composed of 3 known members, PEA3, ERM and ER81, which are more than 95% identical within the DNA-binding ETS domain and exhibit 50% aa identity overall. Recently, transgenic mice bearing the c-erbB-2/neu oncogene have been shown to over-express PEA3 mRNA in mammary adenocarcinomas, suggesting a role for this gene family in mammary tumorigenesis. In the present work we characterized the mRNA expression levels of PEA3-group genes in a series of human epithelial breast cell lines. Each of the 3 genes was highly expressed in normal human HMEC 1001-7 and HMEC 219-4 cells. In breast-cancer cell lines, the 3 genes were highly expressed in the ER- MDA-MB-436, MDA-MB-330, MDA-MB-231 and BT-20 cell lines, but not in the ER+ MDA-MB-134-VI and ZR-75-1 cells. In an attempt to characterize the PEA3-group proteins in breast-cancer cells, we first produced and characterized specific antibodies against each of these 3 proteins. The anti-ERM and anti-ER81 antibodies recognized specific strong bands at approximately 72 kDa and 62 kDa, corresponding to ERM and ER81, respectively, in MDA-MB-231 and Hs-578T cells expressing significant levels of the 3 mRNAs. No protein was detected in MCF-7 cells expressing low levels of mRNA for PEA3-group-family genes, or in ZR-75-1 cells, where mRNA was undetectable by Northern blot. Although in vitro-translated PEA3 is specifically immunoprecipitated by anti-PEA3 anti-serum, we were unable to immunoprecipitate PEA3 protein from MDA-MB-231 and Hs-578T cells. In order to study the transcription factor activity of ERM, PEA3 and ER81 proteins in mammary-cancer cells, we tested their ability to transactivate a reporter plasmid containing 3 Ets-binding sites, and were able to show that, in all the breast-cancer cells tested, transfected ERM, PEA3 and ER81 are able to transactivate. Although the target genes of the PEA3 group of transcription factors in breast-cancer cells have yet to be determined, these genes have a potential role in the regulation of growth and the progression of human breast cancer.

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.