DNA-bound Complexes Research Articles

The ever-increasing complexity of TGF-β signaling

It was a long wait between 1992, when the first transmembrane receptor serine/threonine kinase of the TGFsuperfamily was described, and 1996, when the first cytoplasmic mediators of signaling from these receptor were identified from genetic experiments in Drosophila melanogaster and Caenorhabditis elegans. In the interim period, many laboratories, using protein–protein interaction traps, had identified molecules bound to the TGFsuperfamily receptors, but which, disappointingly, could not be shown to mediate direct effects of TGFon known target genes. These included FKBP12, the subunit of farnesyl-protein transferase, two WD-domain containing proteins, TRIP1 and the B subunit of the phosphatase PP2A, clusterin, and more recently TRAP1 and another WD-domain containing protein, STRAP. More subtle roles for these proteins in TGFsignaling are now being found. Most significantly, identification of the Drosophila Mad protein and the C. elegans Sma proteins heralded in an explosive period in which the mammalian counterparts of these proteins, the Smad proteins, were identified and characterized and in which the rudimentary features of the direct signaling pathway mediated by these proteins were mapped out. In this overtly simple pathway, a receptor-activated Smad (Smad1, 2, 3, 5, 8) is a direct substrate of the type I receptor serine/threonine kinase, becoming phosphorylated on two serine residues in its extreme C-terminus. The conformationally activated, phosphorylated R-Smad then forms an oligomeric complex with the obligatory partner of all R-Smads, Smad4, and this complex then translocates to the nucleus where it regulates transcription in the context of additional factors including transcription factors, co-activators, co-repressors, and a host of other molecules which modulate the activity of the transcriptional complex. Specificity of signaling resides both in the preference of the receptor for a particular R-Smad (Smads 2 and 3 for TGF/ activin and Smads 1, 5, and 8 for the BMPs) and in the formation of the nuclear DNA-bound complexes

SMRTe Inhibits MEF2C Transcriptional Activation by Targeting HDAC4 and 5 to Nuclear Domains

The silencing mediator for retinoic acid and thyroid hormone receptors (SMRT) mediates transcriptional repression by recruiting histone deacetylases (HDACs) to the DNA-bound nuclear receptor complex. The full-length SMRT (SMRTe) contains an N-terminal sequence that is highly conserved to the nuclear receptor corepressor N-CoR. To date, little is known about the activity and function of the full-length SMRTe protein, despite extensive studies on separated receptor interaction and transcriptional repression domains. Here we show that SMRTe inhibits MEF2C transcriptional activation by targeting selective HDACs to unique subnuclear domains. Indirect immunofluorescence studies with anti-SMRTe antibody reveal discrete cytoplasmic and nuclear speckles, which contain RARalpha in an RA-sensitive manner. Formation of the SMRTe nuclear speckles results in recruitment of several class I and class II HDACs to these subnuclear domains in a process depending on HDAC enzymatic activity. Intriguingly, although HDAC4 is located primarily in the cytoplasm, coexpression of SMRTe dramatically translocates HDAC4 from the cytoplasm into the nucleus, where HDAC4 prevents MEF2C from activating muscle differentiation. SMRTe also translocates HDAC5 from diffusive nucleoplasm into discrete nuclear domains. Accordingly, SMRTe synergizes with HDAC4 and 5 to inhibit MEF2C transactivation of target promoter, suggesting that nuclear domain targeting of HDAC4/5 may be important in preventing muscle cell differentiation. These results highlight an unexpected new function of the nuclear receptor corepressor SMRTe for its role in regulating cellular trafficking of nuclear receptor and selective HDACs that may play an important role in regulation of cell growth and differentiation.

Transcriptional repression by suppressor of hairless involves the binding of a hairless-dCtBP complex in Drosophila.

Notch is the receptor for a conserved signaling pathway that regulates numerous cell fate decisions during development [1]. Signal transduction involves the presenilin-dependent intracellular processing of Notch and the nuclear translocation of the intracellular domain of Notch, NICD [2-6]. NICD associates with Suppressor of Hairless [Su(H)], a DNA binding protein, and Mastermind (Mam), a transcriptional coactivator [7-9]. In the absence of Notch signaling, Su(H) acts as a transcriptional repressor [10, 11]. Repression by Su(H) is relieved by the activation of Notch [12-16]. In the Drosophila embryo, this transcriptional switch from repression to activation is important for patterning the expression of the single-minded (sim) gene along the dorsoventral axis [12]. Here, we investigate the mechanisms by which Su(H) inhibits the expression of Notch target genes in Drosophila. We show that Hairless, an antagonist of Notch signaling [17-19], is required to repress the transcription of the sim gene. Hairless forms a DNA-bound complex with Su(H). Furthermore, it directly binds the Drosophila C-terminal Binding Protein (dCtBP), which acts as a transcriptional corepressor. The dCtBP binding motif of Hairless is essential for the function of Hairless in vivo. We propose that Hairless mediates transcriptional repression by Su(H) via the recruitment of dCtBP.

FokI requires two specific DNA sites for cleavage

FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5′ overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity.

Cooperativity in transcriptional control.

The regulation of eukaryotic genes almost always depends on cooperative interactions between factors. This is essential to allow the rich array of transcriptional responses required for both development and adaptation to the environment. We are only beginning to decipher the mechanisms governing this cooperativity, which include processes such as the cooperative assembly of large DNA-bound protein complexes and the cooperative recruitment of positively and negatively acting co-regulatory proteins.One of the hopes of the genomics revolution is that we will eventually be able to predict how a gene will function, for example, in development, by simply analyzing its sequence. Given the conservation in protein coding sequences between diverse organisms, it is highly likely that the evolution of developmental complexity is due more to the evolution of enhancers than to the evolution of coding regions. If we are ever to predict developmental phenotypes from gene sequences, it will be essential to gain a better understanding of the mechanisms by which transcription factors cooperate with one another to define transcriptional states.Suggestions for further reading

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Interleukin-6-induced Tethering of STAT3 to the LAP/C/EBPβ Promoter Suggests a New Mechanism of Transcriptional Regulation by STAT3

LAP/C/EBPbeta is a member of the C/EBP family of transcription factors and contributes to the regulation of the acute phase response in hepatocytes. Here we show that IL-6 controls LAP/C/EBPbeta gene transcription and identify an IL-6 responsive element in the LAP/C/EBPbeta promoter, which contains no STAT3 DNA binding motif. However, luciferase reporter gene assays showed that STAT3 activation through the gp130 signal transducer molecule is involved in mediating IL-6-dependent LAP/C/EBPbeta transcription. Southwestern analysis indicated that IL-6 induces binding of a 68-kDa protein to the recently characterized CRE-like elements in the LAP/C/EBPbeta promoter. Transfection experiments using promoter constructs with mutated CRE-like elements revealed that these sites confer IL-6 responsiveness. Further analysis using STAT1/STAT3 chimeras identified specific domains of the protein that are required for the IL-6-dependent increase in LAP/C/EBPbeta gene transcription. Overexpression of the amino-terminal domain of STAT3 blocked the IL-6-mediated response, suggesting that the STAT3 amino terminus has an important function in IL-6-mediated transcription of the LAP/C/EBPbeta gene. These data lead to a model of how tethering STAT3 to a DNA-bound complex contributes to IL-6-dependent LAP/C/EBPbeta gene transcription. Our analysis describes a new mechanism by which STAT3 controls gene transcription and which has direct implication for the acute phase response in liver cells.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences.

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional 'transcriptosome' has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Id helix-loop-helix proteins antagonize pax transcription factor activity by inhibiting DNA binding.

The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.

The DNA-bound orientation of Cu(II)·Xaa-Gly-His metallopeptides

The DNA-bound orientations of Cu(II)·Xaa-Gly- l-His metallopeptides (where Xaa is Gly, l-Lys or l-Arg) were investigated by DNA fiber EPR spectroscopy and molecular modeling. Observed and calculated EPR spectra indicated that the g // axes of 1:1 Cu(II) complexes of the tripeptides tilted about 50° from the DNA fiber axis. These results suggest that the complexes are stereospecifically oriented in the DNA minor groove. Although the side chain of the N-terminal amino acid residue did not affect the orientation of the DNA-bound complexes, it contributed to their stability in the presence of DNA; the Cu(II) complex of Gly-Gly- l-His was found to dissociate to hydrated Cu(II) ion more extensively than the respective l-Lys-Gly- l-His and l-Arg-Gly- l-His complexes. The ionic interaction between the positively charged lysine or arginine residues and the negatively charged DNA phosphodiester backbone may result in the reduced dissociation of these complexes when bound to the DNA minor groove.

SCFMet30-mediated control of the transcriptional activator Met4 is required for the G1-S transition

Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs.

Thrombopoietin induces the generation of distinct Stat1, Stat3, Stat5a and Stat5b homo- and heterodimeric complexes with different kinetics in human platelets

Objective Thrombopoietin (TPO) is the pivotal regulator of thrombocytopoiesis and megakaryocytopoiesis, and binding to its receptor c-Mpl leads to activation of at least two different signaling pathways: the Jak-Stat pathway and the Ras-MAPK pathway. Our aim was to elucidate which Stat-complexes are formed in TPO signal transduction in human blood platelets. Materials and Methods We used electrophoretic mobility shift assays (EMSA) in order to analyze the formation of distinct Stat complexes on two distinct oligonucleotide probes. Furthermore, we used immunoprecipitation and Western blotting of protein lysates from TPO-stimulated platelets. Results We found homodimers of Stat1α , Stat3, Stat5a, and Stat5b, as well as heterodimers of Stat1/Stat3 and Stat5a/Stat5b, but no Stat1/Stat5 or Stat3/Stat5 heterodimers are formed in platelets in response to TPO. Stat5 complexes bound to labeled DNA with a fast kinetic followed by Stat3 and Stat1. The adapter protein CrkL is present in DNA-bound Stat5 complexes and predominantly bound to Stat5b. The kinase ERK2 is also tyrosine phosphorylated after TPO-stimulation of platelets but this activation does not modulate the phosphorylation of the serine residues in the PXSP motif present in Stat1 and Stat3. Conclusion Our findings thus emphasize the differential regulation of Stat1, Stat3, Stat5a, and Stat5b in platelets and may be an appropriate model of c-Mpl signaling in mega-karyopoiesis.

Differential Expression of the Transcription Factor NF-κB during Human Mononuclear Phagocyte Differentiation to Macrophages and Dendritic Cells

An important role for the Rel/NF-κB family of transcription factors in the differentiation process of dendritic cells (DC) and macrophages (MAC) was recently suggested by a number of mouse knockout studies but only little information is available for defined populations of human cells. To investigate the role of individual NF-κB proteins [p50, p52, p65 (RelA), RelB] in the differentiation of monocyte-derived cell types we analyzed and compared the expression pattern and DNA binding activity of NF-κB members in human monocytes (MO), MO-derived MAC, and MO-derived DC. Constitutive expression of p65 and RelB mRNA was found in MO and no significant regulation was observed during differentiation of MO into MAC or immature DC. Only during lipopolysaccharide-induced terminal differentiation of DC was a marked increase in RelB mRNA detected. In DNA binding assays performed with nuclear extracts from blood MO, p50/p50 homodimers were mainly detected, whereas complexes containing p50/RelB and p50/p65 heterodimers were less abundant. DNA-bound protein complexes containing p50/RelB and p50/p65 increased and additional p65/p65 complexes appeared during differentiation of MO into either MAC or immature DC. A strong increase in complexes containing p50/RelB was observed during terminal differentiation of DC. Therefore, gradual differences in the DNA binding activities of different NF-κB homo- and heterodimers correlate with differentiation stages of MO, MAC, and DC and are probably important for the biological role of these cells.

Activation of the cyclin D1 Gene by the E1A-associated Protein p300 through AP-1 Inhibits Cellular Apoptosis

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at -954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1(-/-)) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1(+/+) fibroblasts but increased apoptosis in cyclin D1(-/-) cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G(1) analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

Flipping the Molecular Light Switch Off: Formation of DNA-Bound Heterobimetallic Complexes Using Ru(bpy)2tpphz2+ and Transition Metal Ions

Formation of a DNA-bound heterobimetallic complex involving Ru(bpy)2tpphz2+ (tpphz = tetrapyrido[3,2-a:2‘,3‘-c:3‘ ‘,2‘ ‘-h:2‘ ‘,3‘ ‘-j]phenazine) and Cu2+ is proposed. The monomeric Ru(bpy)2tpphz2+ is highly luminescent in B-DNA at [DNA-P]/[Ru] = 50, 10 μM Ru. Upon addition of Cu2+, luminescence loss and spectral shifts consistent with Cu2+ complexation and strong associational quenching are observed. Formation of a Ru(bpy)2tpphz2+/Cu2+ DNA complex which places one metal center in the major groove and the other in the minor groove on the opposing side of the helical axis is proposed.

Direct association and nuclear import of the hepatitis B virus X protein with the NF-kappaB inhibitor IkappaBalpha.

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-kappaB. A recent report indicates that X activates NF-kappaB by acting on two cytoplasmic inhibitors of this family of transcription factors: IkappaBalpha and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IkappaBalpha, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IkappaBalpha (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-kappaB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IkappaBalpha (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IkappaBalpha mediating the interaction with the p50 and p65 subunits of NF-kappaB and is also close to the nuclear export sequence of IkappaBalpha, therefore providing a potential explanation for the nuclear accumulation of IkappaBalpha with X. This association can also be observed upon the induction of endogenous IkappaBalpha by tumor necrosis factor alpha (TNF-alpha) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-kappaB activation following TNF-alpha treatment, probably by preventing the reassociation of newly synthesized nuclear IkappaBalpha with DNA-bound NF-kappaB complexes.

Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and Nuclear Translocation in Human Lymphocytes

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ ions, that.OH generated from H2O2 through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

Specific Androgen Receptor Activation by an Artificial Coactivator

Transcription activation of steroid receptors, such as the androgen receptor (AR), is mediated by coactivators, which bridge the receptor to the preinitiation complex. To develop a tool for studying the role of the AR in normal development and disease, we constructed artificial coactivators consisting of the transcription activation domains of VP16 or p65/RelA and the AR hinge and ligand-binding domain (ARLBD), which has been shown to interact with the AR N-terminal domain. The artificial VP16-ARLBD and ARLBD-p65 coactivators interacted with the AR N terminus and wild-type AR in an androgen-dependent and androgen-specific manner. VP16-ARLBD and ARLBD-p65 enhanced the AR transactivity up to 4- and 13-fold, respectively, without affecting the expression of the AR protein. The coactivators did not enhance the transcription activity of the progesterone receptor (PR) or the glucocorticoid receptor (GR), showing their specificity for the AR. In addition, to construct PR- and GR-specific coactivators, the VP16 activation domain was fused to the PR and GR hinge/ligand-binding domain. Although VP16-PRLBD and VP16-GRLBD interacted with the C-terminal portion of steroid receptor coactivator-1, they did not enhance the transcription activity of their receptor. The presented strategy of directing activation domains or other protein activities into the DNA-bound AR complex provides a novel means of manipulating AR function in vitro and in vivo.

CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes.

Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes.

Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors.

The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. Id proteins are thought to inhibit differentiation mainly through interaction with other HLH proteins and by blocking their DNA-binding activity. Members of the ternary complex factor (TCF) subfamily of ETS-domain proteins have key functions in regulating immediate-early gene expression in response to mitogenic stimulation. TCFs form DNA-bound complexes with the serum response factor (SRF) and are direct targets of MAP kinase (MAPK) signal transduction cascades. In this study we demonstrate functional interactions between Id proteins and TCFs. Ids bind to the ETS DNA-binding domain and disrupt the formation of DNA-bound complexes between TCFs and SRF on the c-fos serum response element (SRE). Inhibition occurs by disrupting protein-DNA interactions with the TCF component of this complex. In vivo, the Id proteins cause down-regulation of the transcriptional activity mediated by the TCFs and thereby block MAPK signalling to SREs. Therefore, our results demonstrate a novel facet of Id function in the coordination of mitogenic signalling and cell cycle entry.