Ets regulation of the Fos promoter

A transcription factor ternary complex composed of Serum Response Factor (SRF) (Homologous to Drosophila Serum response factor) and Ternary Complex Factor (TCF) mediates the response of the c-fos (Drosophila homolog: Fos-related antigen) Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1 (Drosophila homolog: Pointed), SAP-1 and ERP/NET, have been reported to have the properties of TCF. Elk-1 and SAP-1a were compared with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation (Price, 1995).

Mitogenic and stress signals result in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which has not been clear. Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions (Strahl, 1996).

TCFs, which are members of the Ets family of transcription factors, are recruited to the Serum Response Element (SRE) in the c-fos promoter by SRF. These Ets proteins, which are substrates for the MAP kinases, are direct targets of the Ras/MAP kinase signal transduction pathway. One of the TCFs, SAP-1a, displays a significant level of autonomous binding to the SRE Ets box. In contrast to previous observations, deletion of the SRF binding domain does not modulate the autonomous binding of SAP-1a. The autonomous binding was not modulated by the phosphorylation of SAP-1a by MAP kinases. The autonomous binding is also detected in live cells: transfected SAP-1a is able to restore the response of a CArG-less SRE in PC12 cells. The response occurs in the absence of SRF recruitment since a mutant of SAP-1a, in which the B-box (a domain required for interaction with SRF) has been deleted, is still able to transactivate the CArG-less SRE. The transactivation is repressed by a Ras transdominant negative mutant, indicating the involvement of the Ras/MAP kinase pathway. Taken together, these data demonstrate that SAP-1a is capable of binding to the c-fos SRE in the absence of SRF (Masutani, 1997).

A study was made of the promoter of the c-fos gene, specifically the serum response element. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2-10 min transiently increases binding of the two complexes; binding returns to control values within 30 min. The two GH-stimulated complexes are supershifted by antibodies against the serum response factor (SRF), indicating that they contain SRF or an antigenically related protein. One of the GH-stimulated complexes is supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH is lost when the SRF binding site in the SRE is mutated, and mutation of either the SRF or TCF binding site alters the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibits the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 (an ETS-domain protein) is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression is mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulates the serine phosphorylation of Elk-1. The increase is evident within 10 min and subsides after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1, contributing to GH-promoted transcriptional activation via the SRE (Liao, 1997).

The transcription factor Elk-1, a nuclear target of extracellular-regulated kinases (ERKs), plays a pivotal role in immediate early gene induction by external stimuli. Notably, the degree of phosphorylation of Elk-1 is tightly correlated with proliferative signals to the level of activation of c-fos transcription. No data yet indicate the role of Elk-1 in the adult brain in vivo. The present work examined the following: (1) Elk-1 mRNA and protein expression in the adult rat brain, and (2) the regulation of Elk-1 (i.e., its phosphorylation state) in an in vivo model of immediate early gene (IEG) induction: an electrical stimulation of the cerebral cortex leading to c-fos and zif268 mRNA induction in the striatum. Elk-1 mRNA is expressed in various brain structures of the adult rat, and this expression is exclusively neuronal. Elk-1 protein is not only nuclear (as shown previously in transiently transfected cell lines) but is also present in soma, dendrites, and axon terminals. On electrical stimulation of the glutamatergic corticostriatal pathway, a strict spatiotemporal correspondence among ERK activation, Elk-1 phosphorylation, and IEG mRNA induction is seen. Both activated proteins are found in cytosolic and nuclear comparments of neuronal cells in the activated area. These data suggest that the ERK signaling pathway plays an important role in regulating genes controlled by serum response element sites via phosphorylation of Elk-1 in vivo (Sgambato, 1998).

The rapid and transient induction of the human proto-oncogene c-fos in response to a variety of stimuli depends on the serum response element (SRE). In vivo footprinting experiments show that this promoter element is bound by a multicomponent complex that includes the serum response factor (SRF) and a ternary complex factor such as Elk-1. SRF is thought to recruit a ternary complex factor monomer into an asymmetric complex. A quaternary complex that binds the SRE is described that, in addition to an SRF dimer, contains two Elk-1 molecules. Its formation at the SRE is strictly dependent on phosphorylation of S-383 in the Elk-1 regulatory domain and appears to involve a weak intermolecular association between the two Elk-1 molecules. The influence of mutations in Elk-1 on quaternary complex formation in vitro correlates with their effect on the induction of c-fos reporter expression in response to mitogenic stimuli in vivo (Gille, 1998).

SAP-1 is a member of the Ets transcription factors and cooperates with SRF protein to activate transcription of the c-fos protooncogene. The crystal structures of the conserved ETS domain of SAP-1 bound to DNA sequences from the E74 and c-fos promoters reveal that a set of conserved residues contact a GGA core DNA sequence. Discrimination for sequences outside this core is mediated by DNA contacts from conserved and nonconserved protein residues and sequence-dependent DNA structural properties characteristic of A-form DNA structure. Comparison with the related PU.1/DNA and GABPalpha/beta/DNA complexes provides general insights into DNA discrimination between Ets proteins. Modeling studies of a SAP-1/SRF/DNA complex suggest that SRF may modulate SAP-1 binding to DNA by interacting with its ETS domain (Mo, 1998).

Coactivators of Ets transcription factors

The Ets-1 transcription factor plays a critical role in cell growth and development, but the means by which it activates transcription remain unclear. It has been shown that Ets-1 binds the transcriptional coactivators CREB binding protein (CBP) and the related p300 protein (together referred to as CBP/p300) and that this interaction is required for specific Ets-1 transactivation functions. The Ets-1- and c-Myb-dependent aminopeptidase N (CD13/APN) promoter and an Ets-1-dependent artificial promoter are repressed by adenovirus E1A, a CBP/p300-specific inhibitor. Ets-1 activity is potentiated by CBP and p300 overexpression. The transactivation function of Ets-1 correlates with its ability to bind an N-terminal cysteine- and histidine-rich region spanning CBP residues 313 to 452. Ets-1 also binds a second cysteine- and histidine-rich region of CBP, between residues 1449 and 1892. Both Ets-1 and CBP/p300 form a stable immunoprecipitable nuclear complex, independent of DNA binding. This Ets-1-CBP/p300 immunocomplex possesses histone acetyltransferase activity, consistent with previous findings that CBP/p300 is associated with such enzyme activity. These results indicate that CBP/p300 may mediate antagonistic and synergistic interactions between Ets-1 and other transcription factors that use CBP/p300 as a coactivator, including c-Myb and AP-1 (Yang, 1998).

Ets factors, development and differentiation

The projection of developing axons to their targets is a crucial step in the assembly of neuronal circuits. In the spinal cord, the differentiation of specific motor neuron pools is associated with the expression of ETS class transcription factors, notably PEA3 and ER81. Their initial expression coincides with the arrival of motor axons in the vicinity of muscle targets and depends on limb-derived signals. In Pea3 mutant mice, the axons of specific motor neuron pools fail to branch normally within their target muscles, and the cell bodies of these motor neurons are mispositioned within the spinal cord. Thus, the induction of an intrinsic program of ETS gene expression by peripheral signals is required to coordinate the central position and terminal arborization of specific sets of spinal motor neurons (Livet, 2002).

Motor neurons in the spinal cord are grouped into motor pools, each of which innervates a single muscle. The ETS transcription factor PEA3 is a marker of a few such motor pools. pea3 is first induced by GDNF in a caudal subset of the motor neurons that will constitute the pea3+ population. Expansion of the pea3 domain subsequently occurs by recruitment of neurons from more anterior segments. Signaling by Met, the HGF receptor, is required for the rostral expansion of the pea3 domain, while the onset of pea3 expression is independent of met function. met expression is observed in pioneer neurons but does not precede that of pea3 in recruited neurons. Genetic evidence is provided for a non-cell-autonomous function of met during the recruitment process. The presence of a relay mechanism is proposed, allowing cells induced by peripheral signals to recruit more anterior neurons to adopt the same motor pool-related phenotype (Helmbacher, 2003).

During the development of the mouse lung, the expression of a number of genes, including those encoding growth factors and components of their downstream signaling pathways, is enriched in the epithelium and/or mesenchyme of the distal buds. In this location, they regulate processes such as cell proliferation, branching morphogenesis, and the differentiation of specialized cell types. The expression of Pea3 and Erm (or Etv5, Ets variant gene 5), that encodes Pea3 subfamily ETS domain transcription factors, is initially restricted to the distal buds of the developing mouse lung. Erm is transcribed exclusively in the epithelium, while Pea3 is expressed in both epithelium and mesenchyme. Erm/Pea3 are downstream of FGF signaling from the mesenchyme, but their responses toward different FGFs are not the same. The functions of the two proteins were investigated by transgenic expression of a repressor form of Erm specifically in the embryonic lung epithelium. When examined at E18.5, the distal epithelium of transgenic lungs is composed predominantly of immature type II cells, while no mature type I cells are observed. In contrast, the differentiation of proximal epithelial cells, including ciliated cells and Clara cells, appears to be unaffected. A model is proposed for the role of Pea3/Erm during the dynamic process of lung bud outgrowth and proximal-distal differentiation, in response to FGF signaling. These results provide the first functional evidence that Pea3 subfamily members play a role in epithelial-mesenchymal interactions during lung organogenesis (Liu, 2003).

In the sea urchin embryo, the skeleton of the larva is built from a population of mesenchymal cells known as the primary mesenchyme cells (PMCs). These derive from the large micromeres that originate from the vegetal pole at fourth cleavage. At the blastula stage, the 32 cells of this lineage detach from the epithelium and ingress into the blastocoel by a process of epithelial-mesenchymal transition. Shortly before ingression, there is a transient and highly localized activation of the MAP-kinase ERK in the micromere lineage. Ingression of the PMCs requires the activity of ERK, MEK and Raf, and depends on the maternal Wnt/ß-catenin pathway. Dissociation experiments and injection of mRNA encoding a dominant-negative form of Ras indicates that this activation is probably cell autonomous. The transcription factors Ets1 and Alx1 were identified as putative targets of the phosphorylation by ERK. Both proteins contain a single consensus site for phosphorylation by the MAP kinase ERK. In addition, the Ets1 protein sequence contains a putative ERK docking site. Overexpression of ets1 by injection of synthetic mRNA in the egg causes a dramatic increase in the number of cells becoming mesenchymal at the blastula stage. This effect could be largely inhibited by treating embryos with the MEK inhibitor U0126. Moreover, mutations in the consensus phosphorylation motif substituting threonine 107 by an aspartic or an alanine residue resulted, respectively, in a constitutively active form of Ets1 that could not be inhibited by U0126 or in an inactive form of Ets1. These results show that the MAP kinase pathway, working through phosphorylation of Ets1, is required for full specification of the PMCs and their subsequent transition from epithelial to mesenchymal state (Röttinger, 2004).

Ets factors and neural crest

The transcription factor Erm (59% identical to Drosophila Pointed over the region of homology) is a member of the Pea3 subfamily of Ets domain proteins that is expressed in multipotent neural crest cells, peripheral neurons, and satellite glia. A specific role of Erm during development has not yet been established. The function of Erm in neural crest development was addressed by forced expression of a dominant-negative form of Erm. Functional inhibition of Erm in neural crest cells interfers with neuronal fate decision, while progenitor survival and proliferation are not affected. In contrast, blocking Erm function in neural crest stem cells does not influence their ability to adopt a glial fate, independent of the glia-inducing signal. Furthermore, glial survival and differentiation were normal. However, the proliferation rate is drastically diminished in glial cells, suggesting a glia-specific role of Erm in controlling cell cycle progression. Thus, in contrast to other members of the Pea3 subfamily that are involved in late steps of neurogenesis, Erm appears to be required in early neural crest development. Moreover, the data point to multiple, lineage-specific roles of Erm in neural crest stem cells and their derivatives, suggesting that Erm function is dependent on the cell intrinsic and extrinsic context (Paratore, 2002).

Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. This report demonstrates a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1(-/-) mice have a nearly complete perinatal lethality. Histologic examination of Ets1(-/-) embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1(-/-) mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, embryonic hearts were cultured in the presence of the MEK inhibitor U0126, and it was found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease (Gao, 2010).

Neural crest cells form diverse derivatives that vary according to their level of origin along the body axis, with only cranial neural crest cells contributing to facial skeleton. Interestingly, the transcription factor Ets-1 is uniquely expressed in cranial but not trunk neural crest, where it functions as a direct input into neural crest specifier genes, Sox10 and FoxD3. This study isolated and interrogated a cis-regulatory element, conserved between birds and mammals, that drives reporter expression in a manner that recapitulates that of endogenous Ets-1 expression in the neural crest. Within a minimal Ets-1 enhancer region, mutation of putative binding sites for SoxE, homeobox, Ets, TFAP2 or Fox proteins results in loss or reduction of neural crest enhancer activity. Morpholino-mediated loss-of-function experiments show that Sox9, Pax7, Msx1/2, Ets-1, TFAP2A and FoxD3, all are required for enhancer activity. In contrast, mutation of a putative cMyc/E-box sequence augments reporter expression, consistent with this being a repressor binding site. Taken together, these results uncover new inputs into Ets-1, revealing critical links in the cranial neural crest gene regulatory network (Barembaum, 2013).

Ets2 and epithelial tumors

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. This study shows that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours (Trimboli, 2009).

Analysis of gene networks in white adipose tissue development reveals a role for ETS2 in adipogenesis

Obesity is characterized by an expansion of white adipose tissue mass that results from an increase in the size and the number of adipocytes. However, the mechanisms responsible for the formation of adipocytes during development and the molecular mechanisms regulating their increase and maintenance in adulthood are poorly understood. This study reports the use of leptin-luciferase BAC transgenic mice to track white adipose tissue (WAT) development and guide the isolation and molecular characterization of adipocytes during development using DNA microarrays. These data reveal distinct transcriptional programs that are regulated during murine WAT development in vivo. By using a de novo cis-regulatory motif discovery tool (FIRE), two early gene clusters were identified whose promoters show significant enrichment for NRF2/ETS transcription factor binding sites. It was further demonstrated that Ets transcription factors, but not Nrf2, are regulated during early adipogenesis and that Ets2 is essential for the normal progression of the adipocyte differentiation program in vitro. These data identify ETS2 as a functionally important transcription factor in adipogenesis and its possible role in regulating adipose tissue mass in adults can now be tested. The approach also provides the basis for elucidating the function of other gene networks during WAT development in vivo. Finally these data confirm that although gene expression during adipogenesis in vitro recapitulates many of the patterns of gene expression in vivo, there are additional developmental transitions in pre and post-natal adipose tissue that are not evident in cell culture systems (Birsoy, 2011).

Other Ets family members

Continued Pointed Evolutionary homologs part 4/4 | back to part 1/4 | part 2/4

pointed : Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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