Ets Family Research Articles

DNMT3b affects colorectal cancer development by regulating FLI1 through DNA hypermethylation.

Friend leukemia integration 1 (FLI1) is an ETS transcription factor family member. Here, we identified cg11017065 as the most hyper-methylated cytosine and guanine (CpG) in colorectal cancer (CRC), which belongs to the FLI1 gene. Moreover, integrated bioinformatics prediction and analysis of our cohort showed that FLI1 expression was downregulated and DNA methylation was elevated in CRC. Bioinformatics prediction also indicated that patients overexpressing FLI1 had higher survival rates than those with low FLI1 expression. CRC cells with ectopic expression of FLI1 had reduced invasion, migration, cloning ability and increased apoptosis. Furthermore, DNA-methyltransferase 3b (DNMT3b) was found to be significantly overexpressed in CRC, and low DNMT3b expression predicted a prolonged survival. DNMT3b bound to the FLI1 promoter. Inhibition of DNMT3b increased FLI1 expression and inhibited the malignant phenotype of CRC cells. Inhibition of FLI1 reversed the phenotypic modulation by DNMT3b depletion in vitro and in vivo. In conclusion, our data indicate that DNMT3b potentiates CRC cell proliferation, migration, and invasion through downregulating FLI1.

The ETS transcription factor ETV6 constrains the transcriptional activity of EWS–FLI to promote Ewing sarcoma

Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS–FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS–FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.

Solithromycin inhibits IL-13-induced goblet cell hyperplasia and MUC5AC, CLCA1, and ANO1 in human bronchial epithelial cells.

Solithromycin is a novel fluoroketolide antibiotic belonging to the class of macrolide antibiotics. Activation of the interleukin (IL)-13 receptor leads to STAT6 activation and subsequent induction of SAM pointed domain containing ETS transcription factor (SPDEF), chloride channel accessory 1 (CLCA1), and anoctamin-1 (ANO1), all of which are associated with the induction of MUC5AC. We examined the effects of solithromycin on mucin production led by IL-13 signaling. Normal human bronchial epithelial cells were grown at the air-liquid interface with IL-13 with/without solithromycin for 14 days. Histochemical analysis was performed using hematoxylin and eosin staining and MUC5AC immunostaining. MUC5AC, SPDEF, CLCA1, and ANO1 mRNA expressions were examined using real-time polymerase chain reaction. Western blot analysis was performed to assess CLCA1 and ANO1 proteins, and phosphorylation of STAT6 and ERK. Solithromycin attenuated IL-13 induction of goblet cell hyperplasia and MUC5AC, CLCA1 and ANO1 mRNA and protein expression induced by IL-13, but had no effect on the phosphorylation of STAT6 and ERK. Our results indicate that solithromycin could attenuate goblet cell hyperplasia and MUC5AC induced by IL-13 through inhibition of CLCA1 and ANO1 mRNA and protein expression. However, much more information is required to clarify the molecular mechanisms underlying the inhibition of CLCA1 and ANO1 by solithromycin.

Abstract B041: Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states

Abstract Solid tumors are complex ecosystems comprised of subpopulations of heterogeneous cancer cells that are altered by interactions with diverse cellular and acellular factors present in tumor microenvironments (TMEs). Accruing evidence suggests that heterogeneity of tumor cells and the TME, and bidirectional crosstalk between these spatially and temporally dynamic components, contributes to the generation of metastatic and therapy-resistant cell subpopulations. Ewing sarcomas are highly metastatic bone and soft tissue tumors driven by pathognomonic FET:ETS transcription factor fusions, predominantly EWS::FLI1. Despite sharing this single oncogenic driver, we and others have shown that Ewing sarcoma tumor cells are highly plastic cells that exist in transcriptionally and phenotypically distinct states. Notably, the extent of EWS::FLI1 transcriptional activity, ranging from low to high, has been shown to in part define the Ewing sarcoma cell state. Cells with high levels of fusion protein activity are generally proliferative and non-motile, while cells with lower levels of EWS::FLI1 expression or activity display enhanced metastatic potential. In this study we have leveraged single cell mRNA-seq and CITE-seq of Ewing sarcoma cell lines, gene expression and immunohistochemical profiling of primary tumor samples, and 3D culture invasion assays to define the complex Ewing sarcoma ecosystem and to investigate the role of tumor cell subpopulations and tumor: TME cross talk on tumor biology. Our results show cells with low levels of EWS::FLI1 activity activate an epithelial-mesenchymal transition (EMT)-like shift in gene expression. This activation of a partial EMT program is notable for an increase in expression and secretion of extracellular matrix (ECM) proteins by subpopulations of tumor cells and by increased matrix invasion. These ECM-related genes serve as transcriptional markers of the EMT-like cell state in both Ewing sarcoma models and in primary patient tumors. Moreover, the local TME surrounding these subpopulations is rich in tumor cell-derived ECM proteins including Tenascin-C, biglycan, and SPARC, proteins implicated in promotion of metastasis. We propose that subpopulations of ECM-secreting Ewing sarcoma tumor cells remodel the TME in a manner reminiscent of cancer associated fibroblasts (CAFs) in carcinomas. Further, we propose that this CAF-like matrix deposition contributes to local tumor progression and metastatic dissemination. Significantly, this ECM-secreting tumor cell state can be induced by canonical Wnt and TGF-beta ligands, suggesting that factors present in the bone TME can serve as inducers of a metastatic cell state. Together these findings demonstrate that Ewing sarcoma metastasis may be promoted by specific cell subpopulations which respond to and remodel the bone tumor microenvironment. Citation Format: Emma Wrenn, April Apfelbaum, Trisha Lipson, Erin Rudzinski, Xuemei Deng, Katherine Braun, Nicolas Garcia, Shruti Bhise, Olivia Waltner, Scott Furlan, Beth Lawlor. Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B041.

ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Regulation of EWSR1-FLI1 Function by Post-Transcriptional and Post-Translational Modifications.

Ewing sarcoma is the second most common bone tumor in childhood and adolescence. Currently, first-line therapy includes multidrug chemotherapy with surgery and/or radiation. Although most patients initially respond to chemotherapy, recurrent tumors become treatment refractory. Pathologically, Ewing sarcoma consists of small round basophilic cells with prominent nuclei marked by expression of surface protein CD99. Genetically, Ewing sarcoma is driven by a fusion oncoprotein that results from one of a small number of chromosomal translocations composed of a FET gene and a gene encoding an ETS family transcription factor, with ~85% of tumors expressing the EWSR1::FLI1 fusion. EWSR1::FLI1 regulates transcription, splicing, genome instability and other cellular functions. Although a tumor-specific target, EWSR1::FLI1-targeted therapy has yet to be developed, largely due to insufficient understanding of EWSR1::FLI1 upstream and downstream signaling, and the challenges in targeting transcription factors with small molecules. In this review, we summarize the contemporary molecular understanding of Ewing sarcoma, and the post-transcriptional and post-translational regulatory mechanisms that control EWSR1::FLI1 function.

Transcriptional Regulation of Siglec-15 by ETS-1 and ETS-2 in Hepatocellular Carcinoma Cells.

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) has been identified as a crucial immune suppressor in human cancers, comparable to programmed cell death 1 ligand (PD-L1). However, the regulatory mechanisms underlying its transcriptional upregulation in human cancers remain largely unknown. Here, we show that the transcription factors ETS-1 and ETS-2 bound to the Siglec-15 promoter to enhance transcription and expression of Siglec-15 in hepatocellular carcinoma (HCC) cells and that transforming growth factor β-1 (TGF-β1) upregulated the expression of ETS-1 and ETS-2 and facilitated the binding of ETS-1 and ETS-2 to the Siglec-15 promoter. We further demonstrate that TGF-β1 activated the Ras/C-Raf/MEK/ERK1/2 signaling pathway, leading to phosphorylation of ETS-1 and ETS-2, which consequently upregulates the transcription and expression of Siglec-15. Our study defines a detailed molecular profile of how Siglec-15 is transcriptionally regulated which may offer significant opportunity for therapeutic intervention on HCC immunotherapy.

Ganglioneuronal Differentiation in Ewings Sarcoma of the Chest Wall (Askin Tumour): An Uncommon Post-Chemotherapy Change

Ewing sarcoma is classified under undifferentiated small round cell sarcomas of bone and soft tissue. Such tumours arising in the chest wall were previously called Askin’s tumour, however this terminology is not preferred at present. These tumours classically have a small round cell morphology and fusion of FET family of genes (usually EWSR1) with a member of the ETS family of transcription factors. Rare cases of Ewing sarcoma are found to have extensive neural differentiation with ganglion cells and neuropil at diagnosis as well as following chemotherapy. We report a 12 year old boy who presented with a large mass in the right hemi-thorax, which was diagnosed as Ewing sarcoma with EWSR1 translocation by FISH. However, following four cycles of chemotherapy (Vincristine, Doxorubicin, Cyclophosphamide alternating with ifosphamide and etoposide), the resection specimen showed predominantly neural differentiation mimicking a ganglioneuroma with only small areas of small round cell component. EWSR1 translocation was also demonstrated in the resection specimen. After a period of nine months, the child presented with recurrence at the same site and was treated with Irinotecan, cyclophosphamide with topotecan and temozolamide giving excellent clinical response. In this paper, we emphasize the rare occurrence of extensive neural differentiation in Ewing sarcoma and the need to revisit the upfront chemotherapy protocols for patients who exhibit this rare phenomenon

Distinct Prognostic and Immunological Roles of ETS1 and ETS2: A Pan-Cancer Analysis.

ETS1 and ETS2, the main ETS family of transcription factors, have been found to act as downstream effectors of the RAS/MAPK pathway. This study explores the expression and prognostic values of ETS1 and ETS2 across cancers. We also aimed to explore the significance of ETS1 and ETS2 expression in normal immune cells with relation to tumorigenesis. The expression of ETS1 and ETS2 was examined in the HPA and GEPIA2 databases. The KM plotter was applied to examine prognostic value of ETS1 and ETS2. Correlation between ETS1/ETS2 and infiltrating immune cells and immune checkpoints was assessed using TIMER2.0. The mutation landscape of ETS1/ETS2 was explored using the cBioPortal. STRING and GEPIA2 were used to screen ETS1/ETS2 binding and correlated genes. Enrichr was applied to perform GO and KEGG enrichment analyses. ETS1 showed enhanced expression in lymphoid tissue, while ETS2 showed low tissue specificity. ETS1 was increased in 12 and decreased in 6 cancers, while ETS2 was increased in 4 and decreased in 13 cancers. Both ETS1 and ETS2 were favorable prognostic markers in LIHC and KIRC, while they showed different prognostic roles in more cancers. ETS1 showed stronger correlation with several infiltrating immune cells and immune checkpoints compared with ETS2. Both ETS1 and ETS2 harbored low mutation ratio. ETS1 interacting and correlated genes were enriched in GO terms in response to cadmium ion and response to oxidative stress, while those of ETS2 were enriched in transcription regulation. ETS1 and ETS2 showed different patterns in expression, prognostic values, correlation with immune infiltrating, and immune checkpoints. ETS1 and ETS2 play distinct roles across cancer.

Abstract C049: Investigating cancer racial disparities through TCGA transcriptomic and proteomic database

Abstract In the United States, epidemiological studies have highlighted a disparity in cancer incidence and outcome rates between racial groups. This disparity is attributed to numerous factors, including gene polymorphisms, differences in lifestyle and environmental exposures, and socioeconomic factors. Although an abundance of tumor molecular data exists, the effect of race on gene expression signatures often remains unexplored. In this project, we investigated racial molecular differences in tumors of 10 carcinoma types. We used publicly available data from The Cancer Genome Atlas (TCGA) in conjunction with online analysis tools to identify patterns of differential gene expression in tumors obtained from 4,112 White, Black/African American, and Asian patients. We identified race-dependent expression of numerous genes whose mRNA transcript levels were significantly correlated with patient survival outcomes. A small subset of these genes was differentially expressed in multiple carcinomas, including genes involved in cell cycle progression such as CCNB1, CCNE1, CCNE2, and FOXM1. This suggests that cell cycle dysregulation is a major culprit in the health disparity. In contrast, genes such as transcriptional factor ETS1 and apoptotic gene BAK1 were differentially expressed and clinically significant only in specific cancer types. In numerous cancer types we also observed race-dependent regulation of cancer-relevant biological pathways, especially DNA repair mechanisms. This large-scale pan-cancer study refines our understanding of the molecular basis for the cancer racial health disparity and can help inform the use of novel biomarkers in clinical settings as well as the future development of precision therapies. Citation Format: Brian Lei, Anjana Saxena. Investigating cancer racial disparities through TCGA transcriptomic and proteomic database [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C049.

Isoform specific knockdown of the ETS transcription factor Pointed in Drosophila S2 cells.

Alternate splicing of the pointed ( pnt ) gene locus produces two major isoforms, PntP1 and PntP2. Understanding their individual contributions to key developmental processes and identification of their genome-wide transcriptional targets has been hampered by a number of factors including their essential roles during embryonic development, and co-expression in several tissues. siRNAs were designed to target isoform-specific exons that code for the unique N-terminal region of either PntP1 or PntP2. The efficacy and specificity of the siRNAs were examined by co-transfection of isoform specific siRNAs with plasmids encoding epitope tagged PntP1 or PntP2 in Drosophila S2 cells. All P1-specific siRNAs were demonstrated to knockdown PntP1 protein level to greater than 95%, while having nominal impact on PntP2 level. Similarly, PntP2 siRNAs while ineffective at eliminating PntP1, were shown to reduce PntP2 protein level by 87-99%.

FGF19-mediated ELF4 overexpression promotes colorectal cancer metastasis through transactivating FGFR4 and SRC.

Background: Metastasis accounts for the high lethality of colorectal cancer (CRC) patients. Unfortunately, the molecular mechanism manipulating metastasis in CRC is still elusive. Here, we investigated the function of E74-like factor 4 (ELF4), an ETS family member, in facilitating CRC progression. Methods: The expression of ELF4 in human CRC samples and CRC cell lines was determined by quantitative real-time PCR, immunohistochemistry and immunoblotting. The migratory and invasive phenotypes of CRC cells were evaluated by in vitro transwell assays and in vivo metastatic models. The RNA sequencing was used to explore the downstream targets of ELF4. The luciferase reporter assays and chromatin immunoprecipitation assays were used to ascertain the transcriptional regulation related to ELF4. Results: We found elevated ELF4 was positively correlated with distant metastasis, advanced AJCC stages, and dismal outcomes in CRC patients. ELF4 expression was also an independent predictor of poor prognosis. Overexpression of ELF4 boosted CRC metastasis via transactivating its downstream target genes, fibroblast growth factor receptor 4 (FGFR4) and SRC proto-oncogene, non-receptor tyrosine kinase, SRC. Fibroblast growth factor 19 (FGF19) upregulated ELF4 expression through the ERK1/2/SP1 axis. Clinically, ELF4 expression had a positive correlation with FGF19, FGFR4 and SRC, and CRC patients who positively coexpressed FGF19/ELF4, ELF4/FGFR4, or ELF4/SRC exhibited the worst clinical outcomes. Furthermore, the combination of the FGFR4 inhibitor BLU-554 and the SRC inhibitor KX2-391 dramatically suppressed ELF4-mediated CRC metastasis. Conclusions: We demonstrated the essentiality of ELF4 in the metastatic process of CRC, and targeting the ELF4-relevant positive feedback circuit might represent a novel therapeutic strategy.

Chromatin Immunoprecipitation (ChIP) to Study the Transcriptional Regulatory Network that Controls Iron Homeostasis in Arabidopsis thaliana.

In plants, gene expression is orchestrated by thousands of transcription factors (TFs). For instance, a large set of bHLH TFs are involved in the regulation of iron homeostasis in Arabidopsis thaliana. The identification of the direct target genes of TFs through uncovering the interaction between the TFs and cis-regulatory elements has become an essential step toward a comprehensive understanding of the iron homeostasis transcriptional regulatory network in Arabidopsis. Chromatin immunoprecipitation (ChIP) followed by qRT-PCR (ChIP-qPCR), sequencing (ChIP-seq), or chip hybridization (ChIP-chip) is a robust tool to investigate protein-DNA interactions in plants in a physiological context. The procedure generally includes six steps: DNA-protein crosslink, isolation of nuclei, shearing of chromatin, immunoprecipitation, DNA purification, and qRT-PCR analyses. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP experiment in Arabidopsis. This protocol focuses on seedlings grown in control and iron deficiency conditions, but can readily be adapted for use with other Arabidopsis tissues or samples. In addition, the protocol could also be applied to perform ChIP-chip or ChIP-seq experiments.

Ets1 gene (Ets Proto-Oncogene1) expression changes in patients with celiac disease under a gluten-free diet

Background. One of the effective genes in the pathogenesis of the celiac disease is the Ets1 gene, which encodes the transcription factor Ets1 and is highly conserved during evolution. The Ets1 gene inhibits the differentiation of T helper 17 (Th17) cells and the production of interleukin-17A (IL-17A) by these cells and decreased expression of the Ets1 gene can lead to autoimmune disorders. The aim of this study is to evaluate the changes in Ets1 gene expression in the blood samples of patients with celiac disease compared with the control group. Methods. Blood samples were collected from twenty patients with celiac disease under a gluten-free diet and also from twenty healthy people. After RNA extraction and cDNA synthesis, a specific primer pair of the Ets1 gene was designed and its expression changes were examined by real-time PCR. Results. The expression of the Ets1 gene in patients with celiac disease on a gluten-free diet did not show a significant difference compared with healthy individuals (p-value= 0.54).. Conclusion. Failure to observe a significant difference between the patient and the control group can be due to the effect of the duration of the gluten-free diet and also the inadvertent entry of gluten from hidden sources into the diet of patients under treatment. Practical Implications. According to the results observed in this study, it is possible that if the gluten-free diet is followed more strictly and over a longer period of time by patients with celiac disease, the expression of the Ets1 gene will proceed as we expected. This issue needs to be evaluated in future studies with a larger community.

The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

The transcription factor ELF5 is essential for early preimplantation development.

During early embryonic development, the cell adhesion molecule E-cadherin encoded by the Cdh1 gene plays a vital role in providing proper cell-cell adhesion, ensuring an undifferentiated state critical for maintaining the pluripotency for the development of the preimplantation embryo. The transcriptional regulation of Cdh1 gained attention recently but is not yet fully understood. In a previous study, our team established a correlation between Elf3 and Cdh1 expression and showed its importance in the regulation of MET. Here, the regulation of Cdh1 by Ets transcription factors in early embryogenesis was investigated. A loss-of-function approach was used to study the effect of Elf5 loss on Cdh1 gene expression by small interfering RNAs in fertilized oocytes. Changes in gene expression were measured by qPCR analysis, and developing embryos were visualized by microscopy. Loss of Elf5 arrested the embryos at the 2-cell stage, accompanied by a significant downregulation of Cdh1 expression. The findings presented here illustrate the role of ELF5 in preimplantation development and in regulating the expression of Cdh1. The maintenance of the ELF5 and Cdh1 regulatory node proved essential for the proper development of the early mouse embryos, which is in agreement with the critical role of Elf5 and Cdh1 genes in regulating the early events during embryogenesis.

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

Ewing and Ewing-like sarcomas: A morphological guide through genetically-defined entities.

The fifth edition of the World Health Organization classification of soft tissue and bone tumors redefined Ewing sarcoma by fusions between EWSR1/FUS and ETS family of transcription factors, and recognized three tumor groups among Ewing-like sarcoma: CIC-rearranged sarcoma, sarcoma with BCOR genetic alterations, and round cell sarcoma with EWSR1::non-ETS fusions. Although this classification underscores the critical role of molecular genetics in the diagnosis of small round cell sarcoma, each entry is recognized as a specific entity not only because they have different genetics but because their phenotypes are distinct and reasonably robust to support the diagnosis. This review focuses on the morphological aspects of Ewing sarcoma and a subset of Ewing-like sarcomas (CIC-rearranged sarcoma, BCOR-associated sarcoma, and EWSR1::NFATC2 sarcoma) for which phenotypic characteristics have been well established. Classic histological findings, uncommon variations, and recurrent diagnostic pitfalls are addressed, along with the utility of recently developed immunohistochemical markers (NKX2.2, PAX7, ETV4, BCOR, CCNB3, and NKX3.1). Phenotypic expertise would significantly expedite the diagnostic process and complement (or sometimes outperform) genetic testing, even in well-resourced settings. Morphological knowledge plays an even more substantial role in facilities that do not have easy access to molecular testing.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Cadmium (Cd) pollution in soil has become a major environmental issue worldwide. However, the underlying molecular mechanism of low grain-Cd accumulation (GCA) in maize is still largely unknown. Herein, we report the mechanistic basis for low GCA in maize by a multiomics approach. The low GCA genotype L63 showed normal vacuolar formation and a lower capacity of xylem loading of Cd than the high-accumulator L42 under Cd stress. Transcriptomic sequencing identified 84 low-GCA-associated genes which are mainly involved in the S-adenosylmethionine (SAM) cycle, metal transport, and vacuolar sequestration. A metabolome analysis revealed that L63 plants had a more active SAM cycle and a greater capacity for terpenoid synthesis and phenylalanine metabolism than L42. Combining the analysis of transcriptome and metabolome characterized several genes as key genes involved in the determination of Cd accumulation. Our study identifies a mechanistic basis for low Cd accumulation in maize grains and provides candidate genes for genetic improvement of crops.

Generation of a doxycycline-inducible ETV2 expression cell line using PiggyBac transposase system

E26 transformation-specific variant 2 (ETV2), as a member of Ets family of transcription factors, plays an important role in embryonic vasculogenesis, angiogenesis and hematopoiesis. Here, based on H9 cell line and PiggyBac technology, we generated a doxycycline-inducible ETV2 (Dox-ETV2-H9) embryonic stem cell (ESC) line. The Dox-ETV2-H9 cells maintain normal morphology, karyotype as well as high expression of pluripotent markers. In comparison with previous studies that employing costly ETV2 modRNA to improve differentiation from ESCs into endothelial cells (ECs), the tet-on system in Dox-ETV2-H9 cells could precisely manipulate ETV2 expression, which enables ECs differentiation process more efficient and controllable upon addition of doxycycline.