ETS Domain Research Articles

GA-Binding Protein Is Dispensable for Neuromuscular Synapse Formation and Synapse-Specific Gene Expression

The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and other "synaptic" genes is required for synapse-specific transcription. The Ets domain transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression. Inactivation of GABPalpha, the DNA-binding subunit of GABP, leads to early embryonic lethality, preventing analysis of synapse formation in gabpalpha mutant mice. To study the role of GABP at neuromuscular synapses, we conditionally inactivated gabpalpha in skeletal muscle and studied synaptic differentiation and muscle gene expression. Although expression of rb, a target of GABP, is elevated in muscle tissue deficient in GABPalpha, clustering of synaptic AChRs at synapses and synapse-specific gene expression are normal in these mice. These data indicate that GABP is dispensable for synapse-specific transcription and maintenance of normal AChR expression at synapses.

Transcriptional Repressor Erf Determines Extraembryonic Ectoderm Differentiation

Extraembryonic ectoderm differentiation and chorioallantoic attachment are fibroblast growth factor (FGF)- and transforming growth factor beta-regulated processes that are the first steps in the development of the placenta labyrinth and the establishment of the fetal-maternal circulation in the developing embryo. Only a small number of genes have been demonstrated to be important in trophoblast stem cell differentiation. Erf is a ubiquitously expressed Erk-regulated, ets domain transcriptional repressor expressed throughout embryonic development and adulthood. However, in the developing placenta, after 7.5 days postcoitum (dpc) its expression is restricted to the extraembryonic ectoderm, and its expression is restricted after 9.5 dpc in a subpopulation of labyrinth cells. Homozygous deletion of Erf in mice leads to a block of chorionic cell differentiation before chorioallantoic attachment, resulting in a persisting chorion layer, a persisting ectoplacental cone cavity, failure of chorioallantoic attachment, and absence of labyrinth. These defects result in embryo death by 10.5 dpc. Trophoblast stem cell lines derived from Erf(dl1/dl1) knockout blastocysts exhibit delayed differentiation and decreased expression of spongiotrophoblast markers, consistent with the persisting chorion layer, the expanded giant cell layer, and the diminished spongiotrophoblast layer observed in vivo. Our data suggest that attenuation of FGF/Erk signaling and consecutive Erf nuclear localization and function is required for extraembryonic ectoderm differentiation, ectoplacental cone cavity closure, and chorioallantoic attachment.

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions.

Analysis of transcription factor expression during oogenesis and preimplantation development in mice

The transition from a differentiated germ cell into a totipotent zygote during oogenesis and preimplantation development is critical to the creation of a new organism. During this period, cell characteristics change dynamically, suggesting that a global alteration of gene expression patterns occurs, which is regulated by global changes in various epigenetic factors. Among these, transcription factors (TFs) are essential in the direct regulation of transcription and also play important roles in determining cell characteristics. However, no comprehensive analysis of TFs from germ cells to embryos had been undertaken. We used mRNA amplification systems and microarrays to conduct a genomewide analysis of TFs at various stages of oogenesis and preimplantation development. The greatest alteration in TFs occurred between the 1- and 2-cell stages, at which time zygotic genome activation (ZGA) occurs. Our analysis of TFs classified by structure and function revealed several specific patterns of change. Basic transcription factors, which are the general components of transcription, increased transiently at the 2-cell stage, while homeodomain (HD) TFs were expressed specifically in the oocyte. TFs containing the Rel homology region (RHR) and Ets domains were expressed at a high level in 2-cell and blastocyst embryos. Thus, the global TF dynamics that occur during oogenesis and preimplantation development seem to regulate the transition from germ-cell-type to embryo-type gene expression.

Different Domains of the Transcription Factor ELF3 Are Required in a Promoter-specific Manner and Multiple Domains Control Its Binding to DNA

Elf3 is an epithelially restricted member of the ETS transcription factor family, which is involved in a wide range of normal cellular processes. Elf3 is also aberrantly expressed in several cancers, including breast cancer. To better understand the molecular mechanisms by which Elf3 regulates these processes, we created a large series of Elf3 mutant proteins with specific domains deleted or targeted by point mutations. The modified forms of Elf3 were used to analyze the contribution of each domain to DNA binding and the activation of gene expression. Our work demonstrates that three regions of Elf3, in addition to its DNA binding domain (ETS domain), influence Elf3 binding to DNA, including the transactivation domain that behaves as an autoinhibitory domain. Interestingly, disruption of the transactivation domain relieves the autoinhibition of Elf3 and enhances Elf3 binding to DNA. On the basis of these studies, we suggest a model for autoinhibition of Elf3 involving intramolecular interactions. Importantly, this model is consistent with our finding that the N-terminal region of Elf3, which contains the transactivation domain, interacts with its C terminus, which contains the ETS domain. In parallel studies, we demonstrate that residues flanking the N- and C-terminal sides of the ETS domain of Elf3 are crucial for its binding to DNA. Our studies also show that an AT-hook domain, as well as the serine- and aspartic acid-rich domain but not the pointed domain, is necessary for Elf3 activation of promoter activity. Unexpectedly, we determined that one of the AT-hook domains is required in a promoter-specific manner.

Cooperative interaction between ETS1 and GFI1 transcription factors in the repression of Bax gene expression

The proto-oncoproteins ETS1 and growth factor independent-1 (GFI1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant transformation. Here, we report that ETS1 and GFI1 interact and affect gene expression through their cross-talk. Co-immunoprecipitation analyses and glutathione-S-transferase pull-down assays revealed that ETS1 bound directly to GFI1 via its Ets domain, and GFI1 bound to ETS1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reporters showed that GFI1 repressed ETS1-mediated transcriptional activation and ETS1 repressed GFI1-mediated transcriptional activation, in a dose-dependent manner. However, in the Bax promoter where the Ets- and Gfi-binding sites (EBS and GBS) are adjacent, ETS1 and GFI1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the Bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses confirmed that an ETS1-GFI1 complex formed on the Bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against ETS1 and/or GFI1 enhanced endogenous Bax gene expression. Our results suggest that the interaction between ETS1 and GFI1 facilitates their binding to specific sites on the Bax promoter and represses Bax expression in vivo.

TLS-ERG Fusion Protein Blocks Terminal Differentiation of Myeloid Progenitor Cells through Deregulation of Cyclin-Dependent Kinase 1 (CDK1).

In a subset of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in blast crisis, the TLS (translocation liposarcoma) gene is fused to the ERG (ets-related gene) gene through a recurrent t(16;21) translocation. This chromosomal translocation results in the generation of TLS-ERG fusion protein. More recently, the same TLS-ERG fusion protein has also been identified in Ewing's sarcoma in children. To investigate the oncogenic mechanism of TLS-ERG, we have stably expressed TLS-ERG in mouse L-G myeloid progenitor cells through retroviral transduction. L-G cells are dependent on IL-3 for proliferation and undergo terminal differentiation into mature neutrophils when treated with G-CSF. Retroviral expression of TLS-ERG fusion protein in L-G cells blocks G-CSF induced terminal differentiation and enables these cells to proliferate in the absence of IL-3. The ability to transform L-G myeloid progenitor cells appears to be an acquired function of the TLS-ERG fusion protein since neither wild-type TLS nor wild-type ERG is able to transform cells. The evolutionarily conserved arginine at position 367 within the ets domain of TLS-ERG is known to be critical to DNA binding, we therefore have replaced arginine at this position with a leucine (R367L). When stably expressed in L-G cells, this TLS-ERG mutant no longer possesses the ability to transform cells. As cellular transformation often reflects a robust cell cycle machinery, we have examined the effects of TLS-ERG on Cdk1, a major protein kinase involved in all four phases of the cell cycle. While the level of Cdk1 protein decreases to an undetectable level during G-CSF-induced terminal differentiation or after IL-3 withdrawal in control L-G cells harboring the empty retroviral vector, the level of Cdk1 protein remains unchanged in TLS-ERG cells following the same treatments. Through RT-PCR analysis, we have found that the steady state level of Cdk1 mRNA is not directly affected by TLS-ERG. Instead, our results show that TLS-ERG increases the stability of Cdk1 protein through repressing genes involved in protein degradation. Using a luciferase reporter construct, we have found that transcriptional repression by TLS-ERG can be reversed by 5-aza-2′-deoxycytidine (also called Decitabine, a DNA methyltransferase inhibitor) or by trichostatin A (a histone acetyltransferase inhibitor). Interestingly, treatment of TLS-ERG-expressing L-G cells with these two epigenetic drugs can also destabilize Cdk1 protein and facilitate terminal differentiation in these TLS-ERG cells. To investigate whether deregulation of Cdk1 protein is indeed correlated with oncogenic transformation by the TLS-ERG fusion protein, we have constructed a lentiviral vector for delivery of a dominant-negative form of Cdk1. In L-G cells harboring TLS-ERG, lentiviral expression of the dominant-negative Cdk1 is found to restore the ability of cells to undergo G-CSF-induced terminal differentiation. In addition, stable siRNA knockdown of Cdk1 is also able to release cells from TLS-ERG blockage of terminal differentiation. Together, these findings suggest that deregulation of Cdk1 activity by TLS-ERG fusion protein plays a critical role in cellular transformation in vitro, and the role of Cdk1 deregulation in leukemogenesis is currently being examined in vivo in a TLS-ERG mouse model.

Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter

It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5' promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

Molecular Analysis of the Interaction between the Hematopoietic Master Transcription Factors GATA-1 and PU.1

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Participation of nuclear localization signal 2 in the 3'-ETS domain of FLI1 in nuclear translocation of various chimeric EWS-FLI1 oncoproteins in Ewing tumor

A t(11;22)(q24;q12) translocation is present in 90% of Ewing's sarcoma, and results in the formation of the EWS-FLI1 fusion gene encoding an oncogenic transcription factor. To clarify the function of chimeric EWS-FLI1 proteins, an identification of a nuclear localization signal (NLS) in the EWS, FLI1 and EWS-FLI1 proteins is important because the chimeric oncoprotein may lose or gain NLS function different from native proteins resulting in different subcellular localization, and in deregulated gene expression. Furthermore, some studies reported that patients with one type of fusion gene ('type 1') had better overall survival than those with other types, suggesting that functional differences may be present among various fusion proteins. There has been only one study reporting a NLS in EWS, but none reporting those in FLI1 and EWS-FLI1. To clarify the molecular mechanisms of Ewing tumor development, we first identified the NLSs of EWS and FLI1. We allocated the NLS to amino acid residues 632-656 near the C-terminal region of EWS that is different from the previous study, and identified two NLSs of FLI1, NLS1 (63-90) in the N-terminal domain and NLS2 (319-360) in the 3'-ETS domain. In addition, the present study showed that all of the EWS-FLI1 fusion proteins completely reside in the nucleus without affecting the frequency of nuclear localization among variants, suggesting that NLS2 of FLI1 was used for nuclear translocation of various EWS-FLI1 fusion proteins.

The Transcriptional ETS2 Repressor Factor Associates with Active and Inactive Erks through Distinct FXF Motifs

The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the alphaF and alphaG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.

LKB1/STK11 Suppresses Cyclooxygenase-2 Induction and Cellular Invasion through PEA3 in Lung Cancer

We showed that the PEA3 transcriptional factor interacted with LKB1, a serine/threonine kinase, which is somatically mutated in lung cancer. This interaction occurred through the ETS domain of PEA3 and the kinase domain of LKB1. Mutation of LKB1 in lung cancer cells stabilized PEA3. Reintroduction of wild-type (WT) LKB1 into cells induced down-regulation of PEA3 and subsequently resulted in reduced cyclooxygenase-2 RNA and protein expression, whereas germ-line and somatic LKB1 mutants were defective in this activity. LKB1 phosphorylated PEA3 and promoted its degradation through a proteasome-mediated mechanism. Cells expressing mutant LKB1 possessed greater invasive potential compared with cells expressing WT LKB1. Increased invasion of cells with mutant LKB1 was partly due to PEA3 expression, as RNA interference inhibition of PEA3 resulted in dramatic decrease of Matrigel invasion. However, forced expression of PEA3 resulted in down-regulation of epithelial markers and induction of mesenchymal markers. These results suggest that PEA3 stabilization due to LKB1 inactivation could lead to epithelial/mesenchymal transition and greater lung cancer invasion potential.

The LIM domain protein LPP is a coactivator for the ETS domain transcription factor PEA3.

PEA3 is a member of a subfamily of ETS domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression.

Erm/Thyroid Transcription Factor 1 Interactions Modulate Surfactant Protein C Transcription

Expression of surfactant protein C (SP-C), which is restricted to alveolar type II epithelial cells of the adult lung, is critically dependent on thyroid transcription factor 1 (TTF-1). In the present study we have demonstrated that Erm, a member of the Ets family of transcription factors, is expressed in the distal lung epithelium during development and is also restricted to alveolar type II cells in the adult. Erm was up-regulated by fibroblast growth factors (FGFs) in culture, and blocking FGF signaling inhibited Erm expression both in vivo and in vitro. The SP-C minimal promoter was found to contain two potential Ets binding sites, and electrophoretic mobility shift assays showed that two 20-bp wild-type oligonucleotides containing the 5'-GGA(A/T)-3' Ets consensus binding motif were shifted by nuclear extracts from MLE15 cells. Co-transfection assays showed that Erm by itself had little effect on SP-C promoter activity but that Erm significantly enhanced TTF-1-mediated SP-C transcription. Mutation of one of the Ets binding sites reduced SP-C transcription to background levels, whereas mutation of the other site resulted in increased SP-C transcription. Protein-protein interactions between Erm and TTF-1 were demonstrated by mammalian two-hybrid assays and by co-immunoprecipitation assays. Mapping studies showed that the Ets domain of Erm and the combined N terminus and homeodomain of TTF-1 were critical for this interaction. Treatment of primary cultures of adult alveolar type II cells with siRNA targeting Erm diminished expression of both Erm and SP-C but had no effect on beta-actin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Taken together, these results demonstrate that Erm is involved in SP-C regulation, which results from an interaction with TTF-1.

Critical role for the Ets transcription factor ELF-1 in the development of tumor angiogenesis

AbstractThe Ets transcription factors regulate a wide variety of biologic processes. Several members have been shown to play a role in regulating angiogenesis and vascular development. For example, the Ets factor ELF-1 is enriched in the developing vasculature of the embryo, where it regulates the expression of the Tie2 gene. We have determined that ELF-1 and Tie2 expression is also enriched in tumor blood vessels, and have identified a short peptide, 34 amino acids in length, corresponding to the terminal portion of the highly conserved ETS domain that potently blocks the function of ELF-1. A tailored ELF-1 blocking peptide, containing a 12–amino acid HIV-1 TAT protein, readily crosses the cell membrane and enters into the nucleus of endothelial cells, leading to a marked reduction in the expression of ELF-1 gene targets including Tie2 and endothelial nitric oxide synthase. Furthermore, the ELF-1 blocking peptide potently inhibits angiopoietin-1–mediated endothelial cell migration. Systemic administration of this peptide markedly attenuates B16 melanoma tumor growth and tumor-associated angiogenesis in nude mice. These results support the function of ELF-1 in the regulation of Tie2 gene expression during the development of tumor angiogenesis.

Cooperative DNA Binding with AP-1 Proteins Is Required for Transformation by EWS-Ets Fusion Proteins

A key molecular event in the genesis of Ewing's sarcoma is the consistent presence of chromosomal translocations that result in the formation of proteins in which the amino terminus of EWS is fused to the carboxyl terminus, including the DNA binding domain, of one of five different Ets family proteins. These fusion proteins function as deregulated transcription factors, resulting in aberrant control of gene expression. Recent data indicate that some EWS-Ets target promoters, including the uridine phosphorylase (UPP) promoter, harbor tandem binding sites for Ets and AP-1 proteins. Here we show that those Ets family proteins that participate in Ewing's sarcoma, including Fli1, ERG, and ETV1, cooperatively bind these tandem elements with Fos-Jun while other Ets family members do not. Analysis of this cooperativity in vitro shows that (i) many different spatial arrangements of the Ets and AP-1 sites support cooperative binding, (ii) the bZIP motifs of Fos and Jun are sufficient to support this cooperativity, and (iii) both the Ets domain and carboxy-terminal sequences of Fli1 are important for cooperative DNA binding. EWS-Fli1 activates the expression of UPP mRNA, is directly bound to the UPP promoter, and transforms 3T3 fibroblasts; in contrast, a C-terminally truncated mutant form of EWS-Fli1 that cannot cooperatively bind DNA with Fos-Jun is defective in all of these properties. The results show that the ability of EWS-Ets proteins to cooperatively bind DNA with Fos-Jun is critical to the biologic activities of these proteins. The results have implications for understanding the pathogenesis of Ewing's sarcoma. In addition, they may be relevant to the mechanisms of Ras-dependent activation of genes that harbor tandem Ets and AP-1 binding sites.

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues ( 244KRKR 247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236–252 and 249–267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues ( 318KKK 320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Ets1-Related Protein Is a Key Regulator of Vasculogenesis in Zebrafish

During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp–morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis.

Mae inhibits Pointed-P2 transcriptional activity by blocking its MAPK docking site

During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of a mitogen activated protein tyrosine kinase, called Rolled. Key nuclear targets of Rolled are two antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2), an activator. A critical regulator of this process, Mae, can interact with both Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the mechanism of Pnt-P2 regulation by Mae remained undefined. We find that efficient phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2 activity. Docking site blocking provides a new mechanism whereby the cell can precisely modulate kinase signaling at specific targets, providing another layer of regulation beyond the more global changes effected by alterations in the activity of the kinase itself.

Site-specific DNA methylation by a complex of PU.1 and Dnmt3a/b

The Ets transcription factor PU.1 is a hematopoietic master regulator essential for the development of myeloid and B-cell lineages. As we previously reported, PU.1 sometimes represses transcription on forming a complex with mSin3A-histone deacetyl transferase-MeCP2. Here, we show an interaction between PU.1 and DNA methyltransferases, DNA methyltransferase (Dnmt)3a and Dnmt3b (Dnmt3s). Glutathione-S-transferase pulldown assay revealed that PU.1 directly interacted with the ATRX domain of Dnmt3s through the ETS domain. Dnmt3s repressed the transcriptional activity of PU.1 on a reporter construct with trimerized PU.1-binding sites. The repression was recovered by addition of 5-aza-deoxycitidine, a DNA methyltransferase inhibitor, but not trichostatin A, a histone deacetylase inhibitor. Bisulfite sequence analysis revealed that several CpG sites in the promoter region neighboring the PU.1-binding sites were methylated when Dnmt3s were coexpressed with PU.1. We also showed that the CpG sites in the p16(INK4A) promoter were methylated by overexpression of PU.1 in NIH3T3 cells, accompanied by a downregulation of p16(INK4A) gene expression. These results suggest that PU.1 may downregulate its target genes through an epigenetic modification such as DNA methylation.