Ets Family Proteins Research Articles

Genetic Study of Elf5 and Ehf in the Mouse Salivary Gland

Salivary gland (SG) development, maturation, and homeostasis require coordinated roles of transcription factors (TFs) that dictate specific cell identities and fate. The ETS family of proteins are important transcriptional drivers of diverse cell lineages, tissue development, and differentiation programs and hence are also likely to play an important role in the SG. Here we have leveraged genomic and epigenomic data of the SG to examine the expression profile of ETS genes and identified 2 closely related paralogs, Elf5 and Ehf, that are highly expressed in distinct epithelial subpopulations. By using a well-defined mouse knockout model of Elf5, we show that Elf5, despite its enriched expression in the acinar cells, is functionally dispensable for maintaining the homeostatic state of the adult SG epithelium. The lack of a discernible phenotype of the Elf5-null SG might be due to possible functional redundancy with Ehf or other ETS factors. To probe this possibility and to examine the specific consequences of Ehf loss in the SG, we used CRISPR-Cas9 to generate mice in which the DNA-binding ETS domain of Ehf is disrupted due to an insertion mutation. We demonstrate that the Ehf mutant (EhfMut) mice exhibit a distinct cellular phenotype with decreased granular convoluted tubules that are accompanied by an increased accumulation of the intercalated Sox9-positive ductal cell population. Interestingly, the ductal phenotype of the EhfMut animals is highly pronounced in males, reaffirming the established sexual dimorphism of the SG that exists in rodents. Our results show that unlike Elf5, Ehf plays a nonredundant role in directing ductal cell differentiation of the SG and highlights the phenotypic subtlety in mutant mice of closely related TFs and the importance of careful consideration of cell type–specific studies.

Ets family proteins regulate the EMT transcription factors Snail and ZEB in cancer cells.

The epithelial–mesenchymal transition (EMT) is a crucial morphological event that occurs during epithelial tumor progression. Snail and ZEB1/2 (ZEB1 and ZEB2), known as EMT transcription factors, are key regulators of this transition. ZEB1/2 are positively correlated with EMT phenotypes and the aggressiveness of cancers. On the contrary, Snail is also correlated with the aggressiveness of cancers, but is not correlated with the expression of EMT marker proteins. Snail is induced by transforming growth factor‐β (TGF‐β), a well‐known inducer of EMT, in various cancer cells. Interestingly, Snail induction by TGF‐β is markedly enhanced by active Ras signals. Thus, cancer cells harboring an active Ras mutation exhibit a drastic induction of Snail by TGF‐β alone. Here, we found that members of the E26 transformation‐specific (Ets) transcription factor family, Ets1 and Ets2, contribute to the upregulation of both Snail and ZEB1/2. Snail induction by TGF‐β and active Ras is dramatically inhibited using siRNAs against both Ets1 and Ets2 together, but not on their own; in addition, siRNAs against both Ets1 and Ets2 also downregulate the constitutive expression of Snail and ZEB1/2 in cancer cells. Examination of several alternatively spliced variants of Ets1 revealed that p54‐Ets1, which includes exon VII, but not p42‐Ets1, which excludes exon VII, regulates the expression of the EMT transcription factors, suggesting that Ets1 is a crucial molecule for regulating Snail and ZEB1/2, and thus cancer progression is mediated through post‐translational modification of the exon VII domain.

ETS factors in prostate cancer.

The ETS family of proteins consists of 28 transcription factors, many of which play critical roles in both normal tissue development and homeostasis and have been implicated in development and progression of a variety of cancers. In prostate cancer, gene fusion and overexpression of ETS factors ERG, FLI1, ETV1, ETV4 and ETV5 have been found in half of prostate cancer patients in Caucasian men and define the largest genetic subtype of prostate cancer. This review summarizes the data on the discovery, modeling, molecular taxonomy, lineage plasticity and therapeutic targeting of ETS family members in prostate cancer.

ETS variant transcription factor 5 and c-Myc cooperate in derepressing the human telomerase gene promoter via composite ETS/E-box motifs

The human telomerase gene (hTERT) is repressed in most somatic cells. How transcription factors activate the hTERT promoter in its repressive chromatin environment is unknown. Here, we report that the ETS family protein ETS variant transcription factor 5 (ETV5) mediates epidermal growth factor (EGF)-induced hTERT expression in MCF10A cells. This activation required MYC proto-oncogene bHLH transcription factor (c-Myc) and depended on the chromatin state of the hTERT promoter. Using chromatinized bacterial artificial chromosome (BAC) reporters in human fibroblasts, we found that ETV5 and c-Myc/MYC-associated factor X (MAX) synergistically activate the hTERT promoter via two identical, but inverted, composite Ets/E-box motifs enclosing the core promoter. Mutations of Ets or E-box sites in either DNA motif abolished the activation and reduced or eliminated the synergism. ETV5 and c-Myc facilitated each other's binding to the hTERT promoter. ETV5 bound to the hTERT promoter in both telomerase-negative and -positive cells, but it activated the repressed hTERT promoter and altered histone modifications only in telomerase-negative cells. The synergistic ETV5/c-Myc activation disappeared when hTERT promoter repression became relieved because of the loss of distal regulatory elements in chimeric human/mouse BAC reporters. Our results suggest that the binding of c-Myc and ETS family proteins to the Ets/E-box motifs derepresses the hTERT promoter by inducing an active promoter configuration, providing a mechanistic insight into hTERT activation during tumorigenesis.

Mithramycin analogues with unique mechanism of action in Ewing sarcoma

IntroductionChromosomal translocations involving ETS family proteins are common in Ewing Sarcoma Family of Tumors (ESFT) and form aberrant ETS‐fusion transcription factors. EWS‐FLI1 and EWS‐ERG are the most common transcription factors in ESFT and function by binding DNA at microsatellite regions, characterized by GGAA repeats, which are found at promoters of genes that drive ESFT growth. These fusion products also interact with the DNA damage response protein and transcriptional co‐regulator PARP‐1, which allows for the development and combination treatments with novel targeted therapeutics in ESFT. Mithramycin (MTM) has been identified as a potent inhibitor of EWS‐ETS, but this drug has a very narrow therapeutic window and its clinical use is marked by severe liver and hematological toxicities; likely the result of interference with the ubiquitously expressed Sp1 transcription factor as well as the pharmacokinetic and biodistribution characteristics of MTM. Here, we sought to identify analogues of MTM that selectively perturb the ETS transcription factor complex and afford efficacy in Ewing sarcoma as a monotherapy and potentially in combination with PARP inhibitors.MethodsQualitative interactions between drug‐DNA‐protein were assessed by electrophoretic mobility shift assays (EMSA). Time‐resolved fluorescence energy transfer (TR‐FRET) assays were used to quantitatively determine DNA bound ERG and Sp1 perturbation by MTM and analogues. Cellular thermal shift assays (CETSA) were used to semi‐quantitatively assess the physical interaction of compounds with EWS‐FLI1 and Sp1. DNA damage (c‐PARP, γ‐H2AX) and phosphorylation at the C‐terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines. mRNA expression of genes involved in DNA damage repair (DDR) was determined by RT‐PCR. Cell viability, by aerobic respiration, in single and combination treatments with a PARP inhibitor were assessed with resazurin.ResultsAnalogues with hydrophobic 3‐side chain modifications showed increased interaction with DNA bound ERG by TR‐FRET, CETSA, and luciferase reporter assays. Specifically, CETSA demonstrated that MTM analogues physically interact and stabilize EWS‐FLI1, but not with Sp1, which is a unique mechanism of action. MTM analogues enhanced DNA damage in Ewing Sarcoma cells and inhibited RNAP2 phosphorylation. As compared to MTM, treatment with analogues resulted in higher expression of DNA damage markers and down regulation of DDR genes specifically in cell lines containing EWS‐ETS translocations in a concentration dependent manner. Cell viability assays showed strong synergy when MTM analogues were combined with Olaparib, a PARP inhibitor. This effect was only seen in Ewing sarcoma cells and was abolished with EWS‐FLI1 silencing.ConclusionThese studies demonstrate that novel MTM analogues selectively perturb the EWS‐ETS transcription factor complex function. This interaction affects transcription of downstream genes and the DDR apparatus specifically in EWS‐ETS expressing cell lines. The recruitment of PARP to DNA damage sites induced by MTM analogues provides the opportunity for a rational and synergistic treatment combination with a PARP inhibitor.

Transcriptional regulation of HSPB1 by Friend leukemia integration-1 factor modulates radiation and temozolomide resistance in glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor and is invariably fatal. Heat shock proteins (HSPs) provide protein signatures/biomarkers for GBM that afford potential as targets for developing anti-GBM drugs. In GBM, elevated expression of hypoxia inducible factors under the influence of Ets family proteins significantly promotes the expression of HSPs. RNAseq analysis identified HSPB1 as a prominent upregulated HSP in GBM and in radiation resistant/temozolomide resistant (radio/TMZR) GBM. Here, we established friend leukemia integration 1 (Fli-1), a member of Ets family to be playing a transcriptional regulatory role on the HSPB1 gene. Fli-1 binds to nucleotide residues GGAA at binding sites 3, 6 and 7 in the 5-kb upstream region of HSPB1. Fli-1 has been linked to oncogenic transformation with upregulation in radio/TMZR GBM. Overexpression of Fli-1 in GBM promotes resistance, whereas Fli-1 knockdown in radio/TMZR GBM cells suppresses resistance. We identify the underlying molecular mechanisms of Fli-1-mediated regulation of HSPB1 that drive extracellular matrix remodeling and epithelial to mesenchymal transition in radio/TMZR GBM cells. This study uncovers Fli-1 as a potential therapeutic target for combating radiation and temozolomide resistance in GBM.

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma

SummaryThe glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR’s transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

Ets-1 enhances tumor migration through regulation of CCR7 expression

Ets-1 is a prototype of the ETS protein family. Members of the ETS protein family contain a unique ETS domain. Ets-1 is associated with cancer progression and metastasis in many types of cancer. Many studies have shown a link between elevated expression of Ets-1 in cancer biopsies and poor survival. CCR7 is a chemokine that binds to specific ligand CCL21/CCL19. CCR7 expression is associated with tumor metastasis and infiltration into lymph nodes. The objective of this study was to test whether Ets-1 could regulate CCR7 expression and enhance tumor metastasis. Our data showed that CCR7 expression was downregulated in Ets-1-deficient T cells upon T-cell stimulation. Overexpression of Ets-1 increased CCR7 expression in breast cancer cell lines. In contrast, knockdown of Ets-1 reduced CCR7 expression. Ets-1 could directly bind to CCR7 promoter and mediate CCR7 expression in luciferase reporter assays and chromatin immunoprecipitation assays. Transactivation activity of Ets-1 was independent of the Pointed domain of Ets-1. Ets-1 could also enhance NF-κB and CBP transactivation of CCR7 promoter. Our results also showed that Ets-1 could modulate cancer cell transmigration by altering CCR7 expression in transwell assay and wound healing assay. Taken together, our data suggest that Ets-1 can enhance CCR7 expression and contribute to tumor cell migration.

Abstract 476: MEK inhibitors for treatment of oligodendroglioma

Abstract Oligodendroglioma (OD) is defined by the 2016 WHO classification as IDH mutant gliomas with 1p19q co-deletion. Although ODs are sensitive to chemotherapy, they eventually recur leading to functional deficits and ultimately death. CIC gene mutations, located on chromosome 19q are seen in 60% of 1p19q co-deleted OD and have been linked to worse survival. Despite our growing knowledge of the molecular characteristics of OD, all recurrent OD patients are treated similarly mainly using radiation and/or poorly tolerated cytotoxic chemotherapy with modest benefit. There is therefore an urgent unmet medical need to understand the molecular consequence of recurrent mutations in OD in order to improve patient outcomes. CIC gene mutations result in truncated, degraded or non-functional CIC protein, disrupting CIC protein repressor functions. This leads to upregulation of ETS-Pea3 family of transcription factors which are known oncogenes in a variety of solid cancers. MAPK signaling pathway has been shown to directly activate ETS family of proteins in drosophila. However, the link between MAPK signaling, ETS proteins and CIC in OD is elusive. Here, we propose a model of aggressiveness in CIC mutant OD in which these cells are dependent on MAPK axis for activation of their augmented ETS proteins (i.e ETV1, ETV4) and therefore are sensitive to MAPK inhibition. To test our model, we evaluated mRNA and protein expression of ETS family of proteins in CIC mutant and CIC wild-type mouse OD cell lines and show that ETV1 and ETV4 have elevated protein and mRNA expression in CIC mutant OD cells, respectively. Consistent with our hypothesis, two different MEK inhibitors, trametinib and pimasertib, reduce cell viability of OD cell lines with greater reduction in CIC mutant than wild-type cells. As proof of concept, we demonstrate that CIC mutant cells have increased pERK expression when compared with CIC wild-type cells indicating that MAPK pathway is more active in CIC mutant cells. Overall, we were able to show that MEK inhibitors have anti-tumor effects in OD cell lines, with greater sensitivity seen in CIC mutant cells. This work lays the foundation for repurposing the widely clinically used MEK inhibitors for treatment oligodendroglioma patients with CIC mutation. Citation Format: Nazanin Majd, Soon Young Park, Marisela Martinez de Kraatz, Veerakumar Balasubramaniyan, Mittal Sandeep, Jian Hu, Jihye Paik, John de Groot. MEK inhibitors for treatment of oligodendroglioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 476.

ETS transcription factors as emerging drug targets in cancer.

The ETS family of proteins consists of 28 transcription factors, many of which have been implicated in development and progression of a variety of cancers. While one family member, ERG, has been rigorously studied in the context of prostate cancer where it plays a critical role, other ETS factors keep emerging as potential hallmark oncodrivers. In recent years, numerous studies have reported initial discoveries of small molecule inhibitors of ETS proteins and opened novel avenues for ETS-directed cancer therapies. This review summarizes the state of the art data on therapeutic targeting of ETS family members and highlights the corresponding drug discovery strategies.

Translocations involving ETS family proteins in human cancer.

The ETS transcription factors regulate expression of genes involved in normal cell development, proliferation, differentiation, angiogenesis, and apoptosis, consisting of 28 family members in humans. Dysregulation of these transcription factors facilitates cell proliferation in cancers, and several members participate in invasion and metastasis by activating certain gene transcriptions. ETS1 and ETS2 are the founding members of the ETS family and regulate transcription by binding to ETS sequences. Three chimeric genes involving ETS genes have been identified in human cancers, which are EWS-FLI1 in Ewing's sarcoma, TMPRSS2-ERG in prostate cancer, and ETV6-RUNX1 in acute lymphocytic leukemia. Although these fusion transcripts definitely contribute to the pathogenesis of the disease, the impact of these fusion transcripts on patients' prognosis is highly controversial. In the present review, the roles of ETS protein translocations in human carcinogenesis are discussed.

Identification and functional analysis of pointed homologs in Bombyx mori

Using gene-knockdown techniques, we searched for endogenous Ets family proteins involved in the regulation of Escherichia coli-dependent lebocin promoter activation in the E. coli-responsive silkworm cell line NIAS-Bm-aff3. Results showed that the gene knockdown of BmPointeds (BmPNTs), Drosophila Pointed orthologs, enhanced E. coli-dependent lebocin promoter activation, suggesting that endogenous BmPNTs repress the activation of this promoter. Furthermore, we found that i) the BmPNT gene produced at least two alternative splicing isoforms, BmPNT1 and BmPNT2, both of which function as repressors; ii) BmPNTs were not associated with an already-reported repressor element, most proximal GGAA/T motif (EtsRE3), in lebocin promoter, which plays a role in the repression of E. coli- and BmRelish1-dependent lebocin promoter activation; iii) although BmPNTs did not directly affect BmRelish1-dependent lebocin promoter activation, they were able to directly repress its activation on the promoter lacking EtsRE3, probably because of competitive inhibition of binding of BmRelish1 to κB sites by BmPNTs; and iv) BmPNTs were mainly expressed in larval hemocytes, and the gene expression levels of BmPNT2, but not of BmPNT1, were decreased in response to E. coli and Bacillus subtilis. These findings suggest that endogenous BmPNTs are directly and indirectly involved in the repression of E. coli-mediated lebocin promoter activation in NIAS-Bm-aff3 cells.

Abstract 4223: Novel strategy for diagnosis of solid tumors by visualization of fusion transcripts

Abstract The discovery of chromosomal translocations as one major cause of cancers has led to the introduction of numerous diagnostic techniques to detect these chromosomal abnormalities. Chromosomal banding, DNA fluorescence in situ hybridization (DNA FISH), and RT PCR are all techniques used for the diagnosis of cancers. Despite the advancement these techniques have brought to the field, they are primarily optimized for the detection of blood cancers and are not very efficient for the detection of solid tumors which are very heterogeneous. One such cancer, Ewing's sarcoma (ES), is a prevalent pediatric, bone and soft tissue tumor that is caused by a chromosomal translocation between Ewing's sarcoma breakpoint region 1 (EWSR1) on chromosome 22 and various members of the ETS protein family. The most common fusion partner, in 85% of cases, is Friend leukemia integration 1 (FLI1) on chromosome 11 which leads to the formation of the fused EWS/FLI1 gene that codes for an aberrant transcription factor. However, even within this single translocation there are multiple breakpoint-fusion scenarios that can occur which results in heterogeneity and increases the chances of a faulty diagnosis with standard techniques. Considering the diversity that exists within the molecular abnormalities that result in Ewing's sarcoma and other solid tumors, there is a need for an alternative and more robust method of detection. The technique of single molecule fluorescence in situ hybridization (smFISH) has been modified by our group to target the EWS/FLI1 transcript that results from the chromosomal fusion; this new technique is referred to as “Fusion FISH”. In Fusion FISH, two differently labeled probe sets are designed to target each half of the EWS/FLI1 fusion transcript. A co-localization of the two probe sets indicates the presence of the EWS/FLI1 translocation and a positive test for Ewing's sarcoma. Preliminary data has established the success of Fusion FISH in detecting both type 1 and type 2 translocations in several Ewing's sarcoma cell lines. The fusion transcript is expressed at similar levels in all cell lines and clearly distinct from the signal of wild type EWS and FLI1 mRNA. Additional studies will examine this new technique in other solid cancers, patient tumor samples, and for validation of additional relevant mRNA biomarkers. Citation Format: Fatu Badiane Markey, Mona Batish. Novel strategy for diagnosis of solid tumors by visualization of fusion transcripts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4223.

Elk-3 is a KLF4-regulated gene that modulates the phagocytosis of bacteria by macrophages.

ETS family proteins play a role in immune responses. A unique member of this family, Elk-3, is a transcriptional repressor that regulates the expression of HO-1. Elk-3 is very sensitive to the effects of inflammatory mediators and is down-regulated by bacterial endotoxin (LPS). In the present study, exposure of mouse macrophages to Escherichia coli LPS resulted in decreased, full-length, and splice-variant isoforms of Elk-3. We isolated the Elk-3 promoter and demonstrated that LPS also decreased promoter activity. The Elk-3 promoter contains GC-rich regions that are putative binding sites for zinc-finger transcription factors, such as Sp1 and KLFs. Mutation of the GC-rich region from bp -613 to -603 blunted LPS-induced down-regulation of the Elk-3 promoter. Similar to the LPS response, coexpression of KLF4 led to repression of Elk-3 promoter activity, whereas coexpression of Sp1 increased activity. ChIP assays revealed that KLF4 binding to the Elk-3 promoter was increased by LPS exposure, and Sp1 binding was decreased. Thus, down-regulation of Elk-3 by bacterial LPS is regulated, in part, by the transcriptional repressor KLF4. Overexpression of Elk-3, in the presence of E. coli bacteria, resulted in decreased macrophage phagocytosis. To determine whether limited expression of HO-1 may contribute to this response, we exposed HO-1-deficient bone marrow-derived macrophages to E. coli and found a comparable reduction in bacterial phagocytosis. These data suggest that down-regulation of Elk-3 and the subsequent induction of HO-1 are important for macrophage function during the inflammatory response to infection.

Regulation of endothelial cell-specific Robo4 gene expression by DNA methylation and non-lineage specific transcription factors

Studies of tissue-specific gene expression have suggested that tissue-specific transcription factors or tissue-specific combinations of non-cell-type-specific transcription factors regulate tissue-specific gene expression. Although the studies of endothelial cell (EC)-specific gene expression has identified several transcriptional activators such as SP1, ETS family proteins, and GATA proteins, their expression and combinations are not likely to be EC-specific. To investigate the mechanism of EC-specific gene expression, we analyzed the regulation mechanism of an EC-specific gene, Roundabout4 (Robo4). We identified the 3-kb Robo4 promoter and several transcription factors including SP1 and GABP that bind to the Robo4 promoter and promote Robo4 gene expression. However, we could not explain the mechanism for EC-specific Robo4 gene expression with only those factors because their expression and combination are not EC-specific. Therefore, we hypothesized the contribution of other mechanisms, especially epigenetic control, to Robo4 gene regulation and demonstrated the importance of DNA methylation for EC-specific Robo4 expression. In this review, we summarize our recent studies and discuss the novel regulation model of Robo4 gene expression by transcription factors and DNA methylation.

Crystallization and preliminary X-ray analysis of a complex of the FOXO1 and Ets1 DNA-binding domains and DNA

The Ets1 transcription factor is a member of the Ets protein family, a group of evolutionarily related DNA-binding transcriptional factors. Ets proteins activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. FOXO1 is a member of the forkhead-box proteins (FOX proteins), which comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation and differentiation. The FOXO subgroup of FOX proteins regulates the transcription of genes that control metabolism, cell survival, cellular proliferation, DNA damage responses, stress resistance and longevity. The DNA-binding domains (DBDs) of Ets1 and FOXO1 were crystallized in complex with DNA containing a composite sequence for a noncanonical forkhead binding site (AATAACA) and an ETS site (GGAA), FOX:ETS, by the sitting-drop vapor-diffusion method. The FOX:ETS motif has been shown to be a conserved cis-acting element in several endothelial cell-specific genes, including Vegfr2, Tie2, Mef2c and ve-cadherin. Crystals were grown at 291 K using 30% polyethylene glycol 400, 50 mM Tris pH 8.5, 100 mM KCl, 10 mM MgCl2 as the reservoir solution. The crystals belonged to space group C222(1), with unit-cell parameters a = 68.7, b = 104.9, c = 136.3 Å. Diffraction data were collected to a resolution of 2.2 Å.

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1. RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

Elucidation of the mechanism of cell type specific IRF-4-mediated transcription in CD4+ T cells (P1081)

Abstract IL-21 plays a key role in Th17 differentiation. Interferon (IFN) regulatory factor 4 (IRF4) is a critical regulator of differentiation in B and T cells, and Th17 differentiation is defective in Irf4 KO CD4+ T cells. In B cells, IRF4 is known to cooperatively bind DNA in partnership with the Ets family protein PU.1. However, PU.1 is not expressed in most T cells even though IRF4 is active within these cells, and the functional transcription factor complex that regulates Th17-specific genes was not known. Genome-wide Chip-Seq analysis was used to demonstrate that in T cells, IRF4 binds to a novel composite DNA motif that includes AP1 motifs and which we named AP1-IRF4 composite elements (AICEs). Moreover, IL-21-regulated genes in Th17 cells have co-localized binding of IRF4, BATF, and JUN family proteins, and the genes encoding these transcription factors are themselves regulated by IL-21 in CD4+ T cells. RNA-seq analysis revealed subsets of genes whose regulation by IL-21, either positive or negative, depends on IRF4, and reporter studies demonstrated that IL-21-induced transcription of these genes was dependent on both IRF4 and AP1 transcription factor binding. Our results demonstrate that by utilizing distinct binding partners and DNA sequences, IRF4 mediates cell-type and stimulus-specific functions in multiple lymphocyte lineages.

Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.

The SAM Domain of Human TEL2 Can Abrogate Transcriptional Output from TEL1 (ETV-6) and ETS1/ETS2

Regulation of gene expression downstream of the Receptor Tyrosine Kinase signaling pathway in Drosophila relies on a transcriptional effector network featuring two conserved Ets family proteins, Yan and Pointed, known as TEL1 (ETV6) and ETS1/ETS2, respectively, in mammals. As in Drosophila, both TEL1 and ETS1/ETS2 operate as Ras pathway transcriptional effectors and misregulated activity of either factor has been implicated in many human leukemias and solid tumors. Providing essential regulation to the Drosophila network, direct interactions with the SAM domain protein Mae attenuate both Yan-mediated repression and PointedP2-mediated transcriptional activation. Given the critical contributions of Mae to the Drosophila circuitry, we investigated whether the human Ets factors TEL1 and ETS1/ETS2 could be subject to analogous regulation. Here we demonstrate that the SAM domain of human TEL2 can inhibit the transcriptional activities of ETS1/2 and TEL1. Drosophila Mae can also attenuate human ETS1/ETS2 function, suggesting there could be cross-species conservation of underlying mechanism. In contrast, Mae is not an effective inhibitor of TEL1, suggesting the mode of TEL2SAM-mediated inhibition of TEL1 may be distinct from how Drosophila Mae antagonizes Yan. Together our results reveal both further similarities and new differences between the mammalian and Drosophila networks and more broadly suggest that SAM domain-mediated interactions could provide an effective mechanism for modulating output from the TEL1 and ETS1/2 oncogenes.