Solution Structure of the N-Terminal Domain of Mediator Subunit MED26 and Molecular Characterization of Its Interaction with EAF1 and TAF7.

MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1). In order to investigate the mechanism of regulation by MED26, MED26-NTD structure was solved by NMR, revealing a 4-helix bundle. EAF1 (239-268) and TAF7 (205-235) peptide interactions were both mapped to the same groove formed by H3 and H4 helices of MED26-NTD. Both interactions are characterized by dissociation constants in the 10-µM range. Further experiments revealed a folding-upon-binding mechanism that leads to the formation of EAF1 (N247-S260) and TAF7 (L214-S227) helices. Chemical shift perturbations and nuclear Overhauser enhancement contacts support the involvement of residues I222/F223 in anchoring TAF7 helix to a hydrophobic pocket of MED26-NTD, including residues L48, W80 and I84. In addition, Ala mutations of charged residues located in the C-terminal disordered part of TAF7 and EAF1 peptides affected the binding, with a loss of affinity characterized by a 10-time increase of dissociation constants. A structural model of MED26-NTD/TAF7 complex shows bi-partite components, combining ordered and disordered segments, as well as hydrophobic and electrostatic contributions to the binding. This study provides molecular detail that will help to decipher the mechanistic basis for the initiation to elongation switch-function mediated by MED26-NTD.

1H, 15N and 13C assignments of the N-terminal domain of the Mediator complex subunit MED26.

MED26 is a subunit of the Mediator, a very large complex involved in regulation of gene transcription by RNA Polymerase II. MED26 regulates the switch between initiation and elongation phases of the transcription. This function requires interaction of its N-terminal domain (NTD) with several protein partners implicated in transcriptional regulation. Molecular details of the structure and interaction mode of MED26 NTD would improve understanding of this complex regulation. As a first step towards structural characterization, sequence specific (1)H, (13)C and (15)N assignments for MED26 NTD was performed based on Nuclear Magnetic Resonance spectroscopy. TALOS+ analysis of the chemical shifts data revealed a domain solely composed of helices. Assignments will be further used to solve NMR structure and dynamics of MED26 NTD and investigate the molecular details of its interaction with protein partners.

Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25.

The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions.

The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members.

PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro. Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.

The coactivator activator CoAA regulates PEA3 group member transcriptional activity.

The PEA3 (polyoma enhancer activator 3) group members [ERM (ETS-related molecule), ER81 (ETS-related 81) and PEA3] of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the hnRNP (heterogeneous nuclear ribonucleoprotein)-like protein CoAA (Coactivator activator) interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RRMs (RNA recognition motifs) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group member activity during metastasis.

NMR structure of the human Mediator MED25 ACID domain.

MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed ß-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the ß-barrel, defined by strands B4-B7-B6.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

ERM transcription factor contains an inhibitory domain which functions in sumoylation-dependent manner.

ERM, PEA3 and ETV1 belong to the PEA3 group of ETS transcription factors. They are involved in many developmental processes and are transcriptional regulators in metastasis. The PEA3 group members share an N-terminal transactivation domain (TAD) whose activity is inhibited by a flanking domain named the negative regulatory domain (NRD). The mechanism of this inhibition is still unknown. Here we show that the NRD maps to residues 73 to 298 in ERM and contains three of the five SUMO sites previously identified in the protein. We demonstrate that these three SUMO sites are responsible for NRD's inhibitory function in the Gal4 system. Although the presence of the three sites is required to obtain maximal inhibition, only one SUMO site is sufficient to repress transcription whatever its localization within the NRD. We also show that NRD is a SUMO-dependent repression domain that can act in cis and in trans to downregulate the powerful TAD of the VP16 viral protein. In addition, we find that the SUMO sites outside the NRD also play a role in the negative regulation of full-length ERM activity. We thus postulate that each SUMO site in ERM may function as an inhibitory motif.

ETV1 is a novel androgen receptor-regulated gene that mediates prostate cancer cell invasion.

Androgens and the androgen receptor (AR) act in cells by modulating gene expression. Through gene microarray studies, we have identified Ets Variant Gene 1 (ETV1) as a novel androgen-regulated gene. Our data demonstrate that ETV1 mRNA and protein are up-regulated in response to ligand-activated AR in androgen-dependent LNCaP cells, but there is no detectable ETV1 expression in normal prostate cells. The ETV1 promoter is induced by androgens and recruits the AR in the context of chromatin. ETV1-regulated endogenous matrix metalloproteinase genes can be induced by ligand-activated AR. In contrast to the hormone-induced expression in androgen-dependent LNCaP cells, ETV1 expression in androgen-independent LNCaP cells is high and unresponsive to androgen. This androgen-independent ETV1 expression contrasts with the hormone-dependent expression observed for TMPRSS2 in these androgen-independent prostate cancer cells. ETV1 is overexpressed in prostate cancer independent of the TMPRSS2:ETV1 translocation. Disruption of ETV1 expression in both androgen-dependent and androgen-independent prostate cancer cells significantly compromises the invasion capacity of these cells, suggesting an important role for ETV1 in prostate cancer metastasis. Collectively, these results demonstrate that ETV1 expression transitions from androgen-induced to androgen-independent as prostate cancer cells switch from hormone-dependent to hormone-refractory and suggest that this transition may be in part responsible for the elevated levels of ETV1 observed in prostate tumors. Additionally, our data provide an indirect mechanism of AR regulation of gene expression, via the transactivation of the transcription factor ETV1.

[PEA3 family of transcription factors and the regulation of oncogenesis]. / Les facteurs de transcription du groupe PEA3: régulateurs transcriptionnels dans le processus de cancérisation.

Erm, Er81, and Pea3 are the three members of the PEA3 group which belong to the Ets transcription factors family. These proteins regulate transcription of multiple target genes, such as those encoding several matrix metalloproteinases (MMP), which are enzymes degrading the extracellular matrix during cancer metastasis. In fact, PEA3-group genes are often overexpressed in different types of human cancers that also over-express these MMP and display a disseminating phenotype. In experimental models, regulation of PEA3 group member expression has been shown to influence the metastatic process, thus suggesting that these factors play a key role in metastasis.

Expression, purification, and structural prediction of the Ets transcription factor ERM.

The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors. Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an alpha-helix. In contrast, the 250-residue central domain seems to have very little structure.

The Ets transcription factors of the PEA3 group: transcriptional regulators in metastasis.

The PEA3 group is composed of three highly conserved Ets transcription factors: Erm, Er81, and Pea3. These proteins regulate transcription of multiple genes, and their transactivating potential is affected by post-translational modifications. Among their target genes are several matrix metalloproteases (MMPs), which are enzymes degrading the extracellular matrix during normal remodelling events and cancer metastasis. In fact, PEA3-group genes are often over-expressed in different types of cancers that also over-express these MMPs and display a disseminating phenotype. Experimental regulation of the synthesis of PEA3 group members influences the metastatic process. This suggests that these factors play a key role in metastasis.

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Expression of the Ets transcription factor Erm is regulated through a conventional PKC signaling pathway in the Molt4 lymphoblastic cell line.

Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.

Transcriptional regulation of the murine brca2 gene by CREB/ATF transcription factors.

The brca2 gene encodes a nuclear protein which is mainly involved in DNA repair and, when mutated, is responsible for some of the hereditary breast cancers. However, brca2 expression is also deregulated in sporadic breast tumors. In the mouse brca2 gene we had earlier identified a region of 148bp upstream of the transcription start site sufficient to activate its expression. In the present report, we show that the -92 to -40bp region is essential for the transcription of brca2 in murine mammary cells and that this nucleotide sequence contains one putative CREB/ATF consensus site (cAMP responsive element: CRE). We demonstrated that the mutation of this binding site led to a highly significant reduction of the mouse brca2 transcription, and that CREB, CREM, and/or ATF-1 functionally bound to and regulated this promoter. Therefore, the regulation of the promoter of the mouse brca2 gene is driven by this family of transcription factors.

FEV acts as a transcriptional repressor through its DNA-binding ETS domain and alanine-rich domain.

Although most Ets transcription factors have been characterized as transcriptional activators, some of them display repressor activity. Here we characterize an Ets-family member, the very specifically expressed human Fifth Ewing Variant (FEV), as a transcriptional repressor. We show that among a broad range of human cell lines, only Dami megakaryocytic cells express FEV. This nuclear protein binds to Ets-binding sites, such as that of the human ICAM-1 promoter. We used this promoter to demonstrate that FEV can repress both basal transcription and, even more strongly, ectopically Ets-activated transcription. We identified two domains responsible for FEV-mediated repression: the ETS domain, responsible for passive repression, and the carboxy-terminal alanine-rich domain, involved in active repression. In the Ets-independent LEXA system also, FEV acts as a transcriptional repressor via its alanine-rich carboxy-terminal domain. The mechanism by which FEV actively represses transcription is currently unknown, since FEV-triggered repression is not reversed by the histone deacetylase inhibitor trichostatin A. We also showed that long-term overexpression of FEV proteins containing the alanine-rich domain prevents cell clones from growing, whereas clones expressing a truncated FEV protein lacking this domain develop like control cells. This confirms the importance of this domain in FEV-triggered repression.

Genomic organization and expression of the mouse Brca2 gene.

A mutation of the Brca2 gene product is responsible for a large proportion of the inherited breast cancers. Here, we have demonstrated that the mouse Brca2 gene is composed of 27 exons and 26 introns, spanning approximately 48 kbp, almost all intron-exon junctions being classical. The overall mouse Brca2 gene structure is highly similar in the coding sequences to that of the human gene. The predicted 11-kb transcript is predominantly present in testis, spleen, thymus, epididymis, and seminal vesicles. A smaller-size, strong positive hybridization signal, which was obtained by using 5' end exons as probes, is ubiquitously observed in mouse tissues. The exact origin and function of this small transcript is currently unknown. The transcription start site of this gene has been identified at approximately 300 bp upstream from the translation initiation codon, and the first 148 bp proximal TATA-less promoter region is sufficient to activate maximal transcription of this gene in mammary cells.

Ectopic expression of the ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription.

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

ERM transactivation is up-regulated by the repression of DNA binding after the PKA phosphorylation of a consensus site at the edge of the ETS domain.

The final step of the transduction pathway is the activation of gene transcription, which is driven by kinase cascades leading to changes in the activity of many transcription factors. Among these latter, PEA3/E1AF, ER81/ETV1, and ERM, members of the well conserved PEA3 group from the Ets family are involved in these processes. We show here that protein kinase A (PKA) increases the transcriptional activity of human ERM and human ETV1, through a Ser residue situated at the edge of the ETS DNA-binding domain. PKA phosphorylation does not directly affect the ERM transactivation domains but does affect DNA binding activity. Unphosphorylated wild-type ERM bound DNA avidly, whereas after PKA phosphorylation it did so very weakly. Interestingly, S367A mutation significantly reduced the ERM-mediated transcription in the presence of the kinase, and the DNA binding of this mutant, although similar to that of unphosphorylated wild-type protein, was insensitive to PKA treatment. Mutations, which may mimic a phosphorylated serine, converted ERM from an efficient DNA-binding protein to a poor DNA binding one, with inefficiency of PKA phosphorylation. The present data clearly demonstrate a close correlation between the capacity of PKA to increase the transactivation of ERM and the drastic down-regulation of the binding of the ETS domain to the targeted DNA. What we thus demonstrate here is a relatively rare transcription activation mechanism through a decrease in DNA binding, probably by the shift of a non-active form of an Ets protein to a PKA-phosphorylated active one, which should be in a conformation permitting a transactivation domain to be active.