E26 Transformation-specific Factors Research Articles

Genome-wide identification and characteristic analysis of ETS gene family in blood clam Tegillarca granosa

BackgroundETS transcription factors, known as the E26 transformation-specific factors, assume a critical role in the regulation of various vital biological processes in animals, including cell differentiation, the cell cycle, and cell apoptosis. However, their characterization in mollusks is currently lacking.ResultsThe current study focused on a comprehensive analysis of the ETS genes in blood clam Tegillarca granosa and other mollusk genomes. Our phylogenetic analysis revealed the absence of the SPI and ETV subfamilies in mollusks compared to humans. Additionally, several ETS genes in mollusks were found to lack the PNT domain, potentially resulting in a diminished ability of ETS proteins to bind target genes. Interestingly, the bivalve ETS1 genes exhibited significantly high expression levels during the multicellular proliferation stage and in gill tissues. Furthermore, qRT-PCR results showed that Tg-ETS-14 (ETS1) is upregulated in the high total hemocyte counts (THC) population of T. granosa, suggesting it plays a significant role in stimulating hemocyte proliferation.ConclusionOur study significantly contributes to the comprehension of the evolutionary aspects concerning the ETS gene family, while also providing valuable insights into its role in fostering hemocyte proliferation across mollusks.

CCRG-05. A UNIFYING AND SELF-REINFORCING MECHANISM OF TUMOR IMMORTALITY IN TERT PROMOTER MUTANT CANCERS

Abstract Telomerase Reverse Transcriptase promoter mutations enable tumor cell immortality in millions of cancer patients annually. While prior therapies targeting telomerase lacked tumor selectivity and were poorly tolerated, TERT promoter mutations and their regulation present a unique opportunity for tumor specific reversal of cellular immortality. The two hotspot mutations, G228A and G250A, generate identical de novo E26 transformation specific (ETS) transcription factor binding sites. However, the 28 ETS factors share a similar binding site preference, raising the question of whether all ETS factors can reactivate TERT. In glioblastoma and a few other cancers, we have shown the de novo ETS site along with a nearby native ETS site (ETS-195/200) recruit one ETS factor, the GA-binding protein (GABP) tetramer, to reactivate TERT. This GABP-mediated mechanism may be restricted to specific cancer types and the two hotspot mutations or could be widely relevant to any cancer with a de novo ETS TERT promoter mutation. We have found that the GABP tetramer activates 6 distinct mutant TERT promoters across 16 different cancer types. However, TERT expression is maintained in most tetramer depleted tumor cells as the tetramer is serially replaced by GABP dimers and weakly by a paralogous tetramer complex that increase following disengagement of GABP tetramer-mediated negative feedback loops. Elimination of both the tetramer and dimer reinstates epigenetic repression of TERT, shortens telomeres, and prevents cancer cell division. However, some knockout cancer cells avoid this fate through weak activation of TERT by the paralogous GABP tetramer complex. The compensatory maintenance of TERT expression can be overcome with a dominant negative GABPB1, leading to TERT silencing, evidenced by gene expression and non-invasive metabolic imaging correlates, and telomere shortening in TERT promoter mutant glioblastoma. We are currently investigating the delivery of this dominant negative to tumor cells via clinically approved retroviral replicating vectors.

ETS transcription factors: Multifaceted players from cancer progression to tumor immunity.

The E26 transformation specific (ETS) family comprises 28 transcription factors, the majority of which are involved in tumor initiation and development. Serving as a group of functionally heterogeneous gene regulators, ETS factors possess a structurally conserved DNA-binding domain. As one of the most prominent families of transcription factors that control diverse cellular functions, ETS activation is modulated by multiple intracellular signaling pathways and post-translational modifications. Disturbances in ETS activity often lead to abnormal changes in oncogenicity, including cancer cell survival, growth, proliferation, metastasis, genetic instability, cell metabolism, and tumor immunity. This review systematically addresses the basics and advances in studying ETS factors, from their tumor relevance to clinical translational utility, with a particular focus on elucidating the role of ETS family in tumor immunity, aiming to decipher the vital role and clinical potential of regulation of ETS factors in the cancer field.

Multiple ETS Factors Participate in the Transcriptional Control of TERT Mutant Promoter in Thyroid Cancers.

Hotspot mutations in the TERT (telomerase reverse transcriptase) gene are key determinants of thyroid cancer progression. TERT promoter mutations (TPM) create de novo consensus binding sites for the ETS ("E26 transformation specific") family of transcription factors. In this study, we systematically knocked down each of the 20 ETS factors expressed in thyroid tumors and screened their effects on TERT expression in seven thyroid cancer cell lines with defined TPM status. We observed that, unlike in other TPM-carrying cancers such as glioblastomas, ETS factor GABPA does not unambiguously regulate transcription from the TERT mutant promoter in thyroid specimens. In fact, multiple members of the ETS family impact TERT expression, and they typically do so in a mutation-independent manner. In addition, we observe that partial inhibition of MAPK, a central pathway in thyroid cancer transformation, is more effective at suppressing TERT transcription in the absence of TPMs. Taken together, our results show a more complex scenario of TERT regulation in thyroid cancers compared with other lineages and suggest that compensatory mechanisms by ETS and other regulators likely exist and advocate for the need for a more comprehensive understanding of the mechanisms of TERT deregulation in thyroid tumors before eventually exploring TPM-specific therapeutic strategies.

FLI1 mediates the selective expression of hypoxia-inducible factor 1 target genes in endothelial cells under hypoxic conditions.

The selective expression of hypoxia‐inducible factor (HIF) target genes in different physiological and pathological environments forms the basis for cellular adaptation to hypoxia in development and disease. Several E26 transformation‐specific (ETS) transcription factors have been shown to specifically regulate the expression of a subset of HIF‐2 target genes. However, it is unknown whether there are ETS factors that specifically regulate hypoxia‐induced HIF‐1 target genes. The present study was undertaken to explore whether friend leukemia integration 1 (FLI1), an ETS transcription factor, regulates the expression of HIF‐1 target genes. To investigate this possibility, EA.hy926 cells were exposed to 20% O2 (normoxia) or 1% O2 (hypoxia). Western blotting, immunofluorescence staining, and RT‐qPCR revealed that FLI1 mRNA and protein levels increased slightly and that the FLI1 protein co‐localized with HIF‐1α in the nucleus under hypoxic conditions. Further analysis showed that, in the absence of FLI1, the hypoxia‐mediated induction of HIF‐1 target genes was selectively inhibited. The results from immunoprecipitation and luciferase reporter assays indicated that FLI1 cooperates with HIF‐1α and is required for the transcriptional activation of a subset of HIF‐1 target genes with a core promoter region containing FBS in proximity to a functional hypoxia response element (HRE). Furthermore, ChIP analysis further confirmed the direct interaction between FLI1 and the promoter region of FLI1‐dependent HIF‐1 target genes under hypoxia. Together, this study demonstrates that FLI1 is involved in the transactivation of certain HIF‐1 target genes in endothelial cells under hypoxic conditions.

An inhibitor of endothelial ETS transcription factors promotes physiologic and therapeutic vessel regression

During the progression of ocular diseases such as retinopathy of prematurity and diabetic retinopathy, overgrowth of retinal blood vessels results in the formation of pathological neovascular tufts that impair vision. Current therapeutic options for treating these diseases include antiangiogenic strategies that can lead to the undesirable inhibition of normal vascular development. Therefore, strategies that eliminate pathological neovascular tufts while sparing normal blood vessels are needed. In this study we exploited the hyaloid vascular network in murine eyes, which naturally undergoes regression after birth, to gain mechanistic insights that could be therapeutically adapted for driving neovessel regression in ocular diseases. We found that endothelial cells of regressing hyaloid vessels underwent down-regulation of two structurally related E-26 transformation-specific (ETS) transcription factors, ETS-related gene (ERG) and Friend leukemia integration 1 (FLI1), prior to apoptosis. Moreover, the small molecule YK-4-279, which inhibits the transcriptional and biological activity of ETS factors, enhanced hyaloid regression in vivo and drove Human Umbilical Vein Endothelial Cells (HUVEC) tube regression and apoptosis in vitro. Importantly, exposure of HUVECs to sheer stress inhibited YK-4-279-induced apoptosis, indicating that low-flow vessels may be uniquely susceptible to YK-4-279-mediated regression. We tested this hypothesis by administering YK-4-279 to mice in an oxygen-induced retinopathy model that generates disorganized and poorly perfused neovascular tufts that mimic human ocular diseases. YK-4-279 treatment significantly reduced neovascular tufts while sparing healthy retinal vessels, thereby demonstrating the therapeutic potential of this inhibitor.

MRTFA augments megakaryocyte maturation by enhancing the SRF regulatory axis

AbstractSerum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]6GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFAOE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])–induced HEL cells, with and without MRTFAOE. We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFAOE, resulting in an upregulation of 25% of genes. MRTFAOE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFAOE lack ETS binding motifs, and MRTFAOE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP–polymerase chain reaction using HEL cells and primary human CD34+ cell–derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.

ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis.

Half of all prostate cancers are caused by the TMPRSS2-ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2-ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.

Differential Expression of ETS Family Transcription Factors in NCCIT Human Embryonic Carcinoma Cells upon Retinoic Acid-Induced Differentiation

E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

296. Characterization of E74 like factor 3 in the murine pre-implantation embryo

The E26 transformation specific (ETS) family of transcription factors is defined by a highly conserved DNA binding domain containing a hydrophobic core motif 5′-GGA[A/T]-3′. A subfamily of ETS factors, epithelium-specific ETS factor (ESE), includes E74-like factor 3 (Elf3). Elf3 is expressed in many organs including the eye, skin and gastrointestinal tract. By upregulating specific gene transcription, Elf3 is instrumental in causing these organs to become fully differentiated. The presence of Elf3 in the pre-implantation embryo was first reported in 2002.1 Data mining has suggested that the expression of Elf3 increases at the 16-cell embryo and blastocyst stages of pre-implantation development compared to the preceding stages. Reverse transcriptase–polymerase chain reaction (RT-PCR) was used to amplify the 3′ untranslated region (3′UTR) of Elf3. These results suggest that there are two isoforms of Elf3 present in the pre-implantation embryo, which differ by an 88 bp deletion/insertion. The expression and location of these isoforms was investigated using RT-PCR and in-situ hybridisation. (1)Stanton and Green (2002) Mol. Hum. Rep. 8, 149–166.