ETV4 Expression Research Articles

Transcriptional Plasticity Compensates for Ikaros and Aiolos Proteasomal Degradation and Mediates Resistance to IMiDs in Multiple Myeloma (MM)

Background: Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, TXNDC5, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, MAFB, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in MM. However, the identity of the transcription factors (TFs) or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. As such the discovery of the BET family member BRD4 as the master histone acetyl mark reader at enhancers loci regulating MYC lead to promising therapeutic developments in MM and numerous other cancers. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKAROS (IKZF1) and AIOLOs (IKZF3) leading to the transcriptional repression of MYC and the suppression of MM cells survival and proliferation. However, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes.Methods and Results: In order to define how IMiDs repress MYC transcription, we first defined IKZF1, BRD4, the lysine acetyl transferase P300 and the mediator complex subunit MED1 mapping within the MM genome using ChIPseq. In MM cell lines (MM1S, RPMI8226, ARP1 and AMO1), IKZF1 predominantly mapped to intronic and intergenic loci which are typically enriched with enhancer and superenhancer elements. Indeed, IKZF1 mapping to the genome nearly completely (96.5%) overlapped that of P300, MED1 and BRD4 co-occupied enhancer and superenhancer loci. We also confirmed that in the MM1S sensitive cell lines IMiDs (lenalidomide 10 μM, 24h) exposure efficiently depleted IKZF1, BRD4, P300 and MED1 at enhancer loci with ensuing MYC (and MAF) downregulation. In contrast, in resistant cell lines (RPMI8226) and in spite efficient IKZF1 displacement, BRD4, P300 and MED1 were retained at the oncogenic enhancer (IGLL5) driving MYC (and MAF). These findings lead us to postulate that in IMiDs resistant cells retention of BRD4 and MED1 at oncogenic enhancers in the absence of IKZF1 likely results from rewiring of the TFs regulating MYC. To identify TFs that may co-localize with BRD4 and IKZF1, we analyzed the enrichment of DNA motifs at IKZF1and BRD4 co-occupied enhancers using the MEME suite motif-finding algorithms. This computational analysis revealed a strong enrichment at these MM enhancers of the GGAA motif recognized by the ETS family of TFs (P= 3.2 e-743) and other motifs boxes for the RUNX (P= 9.6 e-725), MYC/MYB (P= 8.8 e-52) and interferon regulatory (IRF) (P= 3.1 e-293) TFs. We next confirmed that the ETS family TF ETV4 was indeed expressed in IMiDs resistant, but not sensitive, MM cell lines. ChiPseq occupancy profiles in IMiDs resistant RPMI8226 cell line revealed co-localization of ETV4 with IKZF1, P300 and BRD4. As predicted, lenalidomide treatment induced global depletion of IKZF1 but not ETV4 at BRD4 occupied enhancers in resistant cell lines (RPMI8226 and ARP1). Importantly, Cas9-mediated knock out of ETV4 in RPMI8226 cells sensitized them to lenalidomide with ensuing MYC downregulation and cell death.Confirming its role in MM, ETV4 transcript was indeed detectable in primary patients' samples in the CoMMpass data repository (ETV4 FPKM >1.0 in 112/724) and its expression was associated with significantly reduced survival outcomes (HR 0.64; P=0.0008). Similarly, high expression (top quartiles) of RUNX2 or MYB, TFs with enriched motifs at IKZF1 co-occupied enhancer loci, was also associated with decreased survival. Of note RNAseq analysis of paired patient samples pre- and post-IMiDs treatment (n=14 pairs) revealed significant upregulation of ETV4 at the time of acquired IMiDs resistance (7/14). Lastly transcriptome analysis of 101 patients enrolled in the RD arm (lenalidomide and dexamethasone) of the POLLUX trial (NCT02076009) confirmed the reduced survival of patients with top quartiles expression of ETV4 as well as MYB and RUNX2 (Fig.1)Conclusion: Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. [Display omitted] DisclosuresNeri:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Soong:Jannsen: Employment. Chiu:Janssen: Employment. Bahlis:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads

The precise timing and sequence of changes in expression of key genes and proteins during human sex-differentiation and onset of steroidogenesis was evaluated by whole-genome expression in 67 first trimester human embryonic and fetal ovaries and testis and confirmed by qPCR and immunohistochemistry (IHC). SRY/SOX9 expression initiated in testis around day 40 pc, followed by initiation of AMH and steroidogenic genes required for androgen production at day 53 pc. In ovaries, gene expression of RSPO1, LIN28, FOXL2, WNT2B, and ETV5, were significantly higher than in testis, whereas GLI1 was significantly higher in testis than ovaries. Gene expression was confirmed by IHC for GAGE, SOX9, AMH, CYP17A1, LIN28, WNT2B, ETV5 and GLI1. Gene expression was not associated with the maternal smoking habits. Collectively, a precise temporal determination of changes in expression of key genes involved in human sex-differentiation is defined, with identification of new genes of potential importance.

MiR-17-5p suppresses cell proliferation and invasion by targeting ETV1 in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the malignancy with the worst outcome among all breast cancer subtypes. We reported that ETV1 is a significant oncogene in TNBC tumourigenesis. Consequently, investigating the critical regulatory microRNAs (miRNAs) of ETV1 may be beneficial for TNBC targeted therapy.MethodsWe performed in situ hybridization (ISH) and immunohistochemistry (IHC) to detect the location of miR-17-5p and ETV1 in TNBC patient samples, respectively. miR-17-5p expression in TNBC tissues and cell lines was assessed by quantitative real-time PCR (qRT-PCR). ETV1 expression was evaluated by qRT-PCR, western blotting and IHC. Cell Counting Kit-8 (CCK-8), colony formation, Transwell and wound closure assays were utilized to determine the TNBC cell proliferation and migration capabilities. In vivo tumour metastatic assays were performed in a zebra fish model.ResultsThe abundance of miR-17-5p was significantly decreased in TNBC cell lines and clinical TNBC tissues. The miR-17-5p expression levels were closely correlated with tumour size (P < 0.05) and TNM stage (P < 0.05). By contrast, the expression of ETV1 was significantly up-regulated in TNBC cell lines and tissues. There is an inverse correlation between the expression status of miR-17-5p and ETV1 (r = −0.28, P = 3.88 × 10−3). Luciferase reporter assay confirmed that ETV1 was a direct target of miR-17-5p. Forced expression of miR-17-5p in MDA-MB-231 or BT549 cells significantly decreased ETV1 expression and suppressed cell proliferation, migration in vitro and tumour metastasis in vivo. However, rescuing the expression of ETV1 in the presence of miR-17-5p significantly recovered the cell phenotype. High miR-17-5p expression was associated with a significantly favourable prognosis, in either the ETV1-positive or ETV1-negative groups (log-rank test, P < 0.001; P < 0.001). Both univariate and multivariate analyses showed that miR-17-5p and ETV1 were independent risk factors in the prognosis of TNBC patient.ConclusionsOur data indicate that miR-17-5p acts as a tumour suppressor in TNBC by targeting ETV1, and a low-abundance of miR-17-5p may be involved in the pathogenesis of TNBC. These findings indicate that miR-17-5p may be a therapeutic target for TNBC.

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OBJECTIVES/SPECIFIC AIMS: The goal of this project is to determine the role of ETV6 in early B-cell development and define how germline ETV6 mutations result in predisposition to leukemia. METHODS/STUDY POPULATION: Gene expression commons were queried for expression levels of Etv6 and Pax5 at different stages of hematopoiesis. Mouse bone marrow was isolated and fractioned into cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19−), Fraction B (CD19+, CD24+, BP1−), and Fraction C (CD19+ CD24+ BP1+). Wild-type or germline mutant P214L ETV6 were cloned in an MiG vector and expressed in Ba/F3 cells. ChIP-PCR was performed by cross-linking proteins to DNA with 1% formaldehyde for 10 minute at room temperature, followed by cell lysis with RIPA buffer. Lysates were sonicated to shear DNA to a length of 200–1000 base pairs, then Protein A agarose beads were used to clean and immunoprecipitate chromatin. RESULTS/ANTICIPATED RESULTS: We observed that Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (p&lt;0.0001). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=0.9993; p=0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19−, and CD20−) compared to the pre-B cell fraction (CD10+, CD34−, CD19+, CD20−). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared with the early B cell fraction. In Ba/F3 cells expressing ETV6 constructs, ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (p≤0.05). ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We detected association of ETV6, SIN3A, and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results provide a mechanism of interaction for ETV6 and PAX5, 2 genes often disrupted in B-cell leukemia. These findings are significant because PAX5 misregulation results in a B cell development halt, lineage infidelity, and leukemogenesis. In continuing our studies, we have generated a transgenic mouse endogenously expressing the ETV6 P214L mutation by CRISPR/Cas9 editing, and these mice appear to have a thrombocytopenic phenotype similar to that observed in patients carrying the ETV6 P214L mutation. These animals will be the focus of our continued investigation of the mechanism by which ETV6 germline mutation results in a predisposition to leukemia. Our ultimate goal is a comprehensive understanding of how this process may be targeted more efficiently in patients with both heritable and sporadic forms of leukemia involving ETV6.

Nasopharyngeal carcinoma super-enhancer–driven ETV6 correlates with prognosis

Nasopharyngeal carcinoma (NPC) most frequently occurs in southern China and southeast Asia. Epidemiology studies link NPC to genetic predisposition, Epstein-Barr virus (EBV) infection, and environmental factors. Genetic studies indicate that mutations in chromatin-modifying enzymes are the most frequent genetic alterations in NPC. Here, we used H3K27ac chromatin immune precipitation followed by deep sequencing (ChIP-seq) to define the NPC epigenome in primary NPC biopsies, NPC xenografts, and an NPC cell line, and compared them to immortalized normal nasopharyngeal or oral epithelial cells. We identified NPC-specific enhancers and found these enhancers were enriched with nuclear factor κB (NF-κB), IFN-responsive factor 1 (IRF1) and IRF2, and ETS family members ETS1 motifs. Normal cell-specific enhancers were enriched with basic leucine zipper family members and TP53 motifs. NPC super-enhancers with extraordinarily broad and high H3K27ac signals were also identified, and they were linked to genes important for oncogenesis including ETV6. ETV6 was also highly expressed in NPC biopsies by immunohistochemistry. High ETV6 expression correlated with a poor prognosis. Furthermore, we defined the EBV episome epigenetic landscapes in primary NPC tissue.

Inactivation of Capicua in adult mice causes T-cell lymphoblastic lymphoma.

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.

Capicua deficiency induces autoimmunity and promotes follicular helper T cell differentiation via derepression of ETV5

High-affinity antibody production through the germinal centre (GC) response is a pivotal process in adaptive immunity. Abnormal development of follicular helper T (TFH) cells can induce the GC response to self-antigens, subsequently leading to autoimmunity. Here we show the transcriptional repressor Capicua/CIC maintains peripheral immune tolerance by suppressing aberrant activation of adaptive immunity. CIC deficiency induces excessive development of TFH cells and GC responses in a T-cell-intrinsic manner. ETV5 expression is derepressed in Cic null TFH cells and knockdown of Etv5 suppresses the enhanced TFH cell differentiation in Cic-deficient CD4+ T cells, suggesting that Etv5 is a critical CIC target gene in TFH cell differentiation. Furthermore, we identify Maf as a downstream target of the CIC–ETV5 axis in this process. These data demonstrate that CIC maintains T-cell homeostasis and negatively regulates TFH cell development and autoimmunity.

Human microvascular endothelial cell promotes the development of dorsal root ganglion neurons via BDNF pathway in a co-culture system.

Our previous study found that co-culture with human vascular endothelial cells (HMVECs) is beneficial for dorsal root ganglion cells (DRGCs). The goal of the present study is to investigate whether co-culture with HMVECs could promote the development of DRGCs, and whether this effect is induced by the secretion of BDNF by HMVECs. DRGCs were mono-cultured, co-cultured with HMVECs or co-cultured with HMVECs that pre-transfected with BDNF siRNA, the expression of neurite formation and branching factors were determined. The results showed that transfecting with BDNF siRNA inhibited BDNF expression and reduced BDNF secretion. Co-culture with HMVECs increased the expression of Etv4, Etv5, FN-L, FN-M, and GAP-43 in DRGCs that accompanied by the activation of ERK pathway. However, these changes were all reversed by the inhibition of BDNF in HMVECs. In conclusion, our data demonstrate that HMVECs potentiated DRGCs development at least partly by the secretion of BDNF in the co-culture system.

NANOG gene expression is regulated by the ETS transcription factor ETV4 in human embryonic carcinoma NCCIT cells

We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (−285 to −138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (−196AGGATT−191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.

Etv5 Regulates IL-10 Production in Th Cells.

IL-10 is an immunoregulatory cytokine that has broad effects across the immune system. In Th cell subsets, Th2 cells produce considerable amounts of IL-10. The transcription factors that regulate IL-10 production in Th2 cells are still incompletely described. In this study, we demonstrate that the ETS family transcription factor ETS variant (Etv)5 regulates IL-10 production in Th2 cells. T cell-specific Etv5-deficient and littermate control mice demonstrated that IL-10 production and gene expression were significantly decreased in the absence of Etv5. In an Aspergillus fumigatus extract-induced inflammation model, IL-10-producing CD4+ T cells in bronchoalveolar lavage and lung were significantly decreased in mice that lacked Etv5 in T cells, compared with control mice. We showed that Etv5 directly binds to the Il10 locus conserved noncoding sequence 3 site and that it activates gene expression in a luciferase reporter assay and following retroviral transduction. Etv5 deficiency did not affect the expression of other transcription factors known to be important for expression of IL-10, including Jun family members, GATA3, E4BP4, and IFN regulatory factor 4. However, in the absence of Etv5, binding of these transcription factors to the Il10 locus was dramatically reduced. Ectopic Etv5 expression in Th2 cells that lack Etv5 restored IL-10 production and the binding of IL-10-inducing transcription factors including E4BP4, IFN regulatory factor 4, and GATA3. Taken together, we conclude that Etv5 plays a crucial role in regulating IL-10 production in Th2 cells by facilitating the binding of IL-10-inducing transcription factors at the Il10 locus.

ATXN1L, CIC, and ETS Transcription Factors Modulate Sensitivity to MAPK Pathway Inhibition

Intrinsic resistance and RTK-RAS-MAPK pathway reactivation has limited the effectiveness of MEK and RAF inhibitors (MAPKi) in RAS- and RAF-mutant cancers. To identify genes that modulate sensitivity to MAPKi, we performed genome-scale CRISPR-Cas9 loss-of-function screens in two KRAS mutant pancreatic cancer cell lines treated with the MEK1/2 inhibitor trametinib. Loss of CIC, a transcriptional repressor of ETV1, ETV4, and ETV5, promoted survival in the setting of MAPKi in cancer cells derived from several lineages. ATXN1L deletion, which reduces CIC protein, or ectopic expression of ETV1, ETV4, or ETV5 also modulated sensitivity to trametinib. ATXN1L expression inversely correlates with response to MAPKi inhibition in clinical studies. These observations identify the ATXN1L-CIC-ETS transcription factor axis as a mediator of resistance to MAPKi.

Prorenin receptor controls renal branching morphogenesis via Wnt/β-catenin signaling.

The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H+-ATPase. Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is essential for mammalian kidney development. Previously, we demonstrated that conditional ablation of the PRR in the UB in PRRUB-/- mice causes severe defects in UB branching, resulting in marked kidney hypoplasia at birth. Here, we investigated the UB transcriptome using whole genome-based analysis of gene expression in UB cells, FACS-isolated from PRRUB-/-, and control kidneys at birth (P0) to determine the primary role of the PRR in terminal differentiation and growth of UB-derived collecting ducts. Three genes with expression in UB cells that previously shown to regulate UB branching morphogenesis, including Wnt9b, β-catenin, and Fgfr2, were upregulated, whereas the expression of Wnt11, Bmp7, Etv4, and Gfrα1 was downregulated. We next demonstrated that infection of immortalized UB cells with shPRR in vitro or deletion of the UB PRR in double-transgenic PRRUB-/-/BatGal+ mice, a reporter strain for β-catenin transcriptional activity, in vivo increases β-catenin activity in the UB epithelia. In addition to UB morphogenetic genes, the functional groups of differentially expressed genes within the downregulated gene set included genes involved in molecular transport, metabolic disease, amino acid metabolism, and energy production. Together, these data demonstrate that UB PRR performs essential functions during UB branching and collecting duct morphogenesis via control of a hierarchy of genes that control UB branching and terminal differentiation of the collecting duct cells.

Screening differentially expressed genes of gastric stromal tumors originated form the muscularis propria by gene chip technology

Objective To screen the related gene expression in gastric muscularis propria tumor by gene chip assay, and to guide the accurate diagnosis and targeted therapygastric of gastric stromal tumors originated form the muscularis propria. Methods 6 cases of samples with gastric muscularis propria mesenchymal tumor and peritumoral tissue were treated by endoscopic treatment and laparoscopic surgery. cDNA was synthesized by reverse transcription with total RNA, then by in vitro transcription synthesised of biotin labeling cRNA that was hybridizationed with the Affymetrix mRNA expression profile chip, screened differentially expressed genes with bioinformatics analysis using PANTHER/KEGG database and Gene Ontology software. Results 3 293 differentially expressed genes were detected in Gastric stromal tumor tissue, including 2 588 up-regulated, and 705 down-regulated genes, especially in DPP10, ETV1, DKK4, CXCL14, MT1M gene expression significantly. These genes were involved in multiple signaling pathways related to tumor, such as Wnt pathway, Rap1 pathway, ECM receptor interaction, Ras signaling pathway, P13K-AKT signaling pathway, and cell adhesion. Conclusion There are significant differences of gene expression between gastric stromal tumor and peritumoral tissue, the aitered expression of DPP10, ETV1, DKK4, CXCL14 and MT1M might be involved in the regulation of gastric stromal tumor, rarely reported before in the literature, and might be new molecular markers or treatment targets. To conduct in-depth study of these genes and related regulation pathways, will contribute to the diagnosis, treatment and prognosis of gastric stromal tumor. Key words: Gastric muscularis propria mesenchymal tumor; Gene chip; Differentially expressed genes

The utility of ETV1, ETV4 and ETV5 RNA in-situ hybridization in the diagnosis of CIC-DUX sarcomas.

A recently characterized group of undifferentiated small round cell sarcomas harbours fusions of the genes CIC and DUX4. Studies report a distinctive gene expression profile for these sarcomas, including expression of E26 transformation-specific (ETS) family proto-oncogenic transcription factors ETV1, ETV4 and ETV5. To test the utility of an ancillary diagnostic technique for these tumours, we evaluated chromogenic RNA in-situ hybridization assays for ETV1, ETV4 and ETV5 as diagnostic adjuncts for this emerging group of highly malignant sarcomas. We tested six confirmed CIC-DUX4 sarcomas and 105 lesions in the differential, including 48 Ewing sarcomas for expression of ETV1, ETV4 and ETV5, scoring expression utilizing a previously validated scale. ETV1 and ETV4 were positive in five of six cases, while ETV5 was positive in six of six. No Ewing sarcoma or other sarcoma tested showed coexpression of these transcripts, while one ETV1/ETV4/ETV5 triple positive previously unclassified round cell sarcoma was identified as harbouring a CIC rearrangement by break-apart fluorescence in-situ hybridization (FISH). We identified overexpression of ETV1, ETV4 and ETV5 transcripts in situ in CIC-DUX4 sarcomas using a robust assay in routine archival sections. One previously unclassified round cell sarcoma showed ETV1/4/5 positivity, and was proved to harbour a CIC rearrangement by break-apart FISH. The sensitivity and specificity observed with our in-situ hybridization assay implies potential utility as an ancillary diagnostic technique, particularly when faced with limited biopsy samples.

Lin28a promotes self-renewal and proliferation of dairy goat spermatogonial stem cells (SSCs) through regulation of mTOR and PI3K/AKT.

Lin28a is a conserved RNA-binding protein that plays an important role in development, pluripotency, stemness maintenance, proliferation and self-renewal. Early studies showed that Lin28a serves as a marker of spermatogonial stem cells (SSCs) and promotes the proliferation capacity of mouse SSCs. However, there is little information about Lin28a in livestock SSCs. In this study, we cloned Capra hircus Lin28a CDS and found that it is evolutionarily conserved. Lin28a is widely expressed in different tissues of Capra hircus, but is expressed at a high level in the testis. Lin28a is specifically located in the cytoplasm of Capra hircus spermatogonial stem cells and may also be a marker of dairy goat spermatogonial stem cells. Lin28a promoted proliferation and maintained the self-renewal of GmGSCs-I-SB in vivo and in vitro. Lin28a-overexpressing GmGSCs-I-SB showed an enhanced proliferation rate, which might be due to increased PCNA expression. Moreover, Lin28a maintained the self-renewal of GmGSCs-I-SB by up-regulating the expression of OCT4, SOX2, GFRA1, PLZF and ETV5. Furthermore, we found that Lin28a may activate the AKT, ERK, and mTOR signaling pathways to promote the proliferation and maintain the self-renewal of GmGSCs-I-SB.

ETV6 Regulates Pax5 Expression in Early B Cell Development

Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia.To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P<0.0001). To confirm relative patterns of Etv6 and Pax5 expression in developing B cells, we isolated bone marrow (BM) from wild type (WT) mice and fractionated cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19-), Fraction B (CD19+, CD24+, BP1-) and Fraction C (CD19+ CD24+ BP1+). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=.9993; P= 0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19-, and CD20-) compared to the preB cell fraction (CD10+, CD34-, CD19+, CD20-). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared to the early B cell fraction.To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression.To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression.In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

ETV4 is a useful marker for the diagnosis of CIC-rearranged undifferentiated round-cell sarcomas: a study of 127 cases including mimicking lesions

Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CIC gene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4 gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3 subfamily genes, notably ETV4 (belonging to the family of ETS transcription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3 sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.

Evaluation of ETV4 and WT1 expression in CIC-rearranged sarcomas and histologic mimics

AbstractA distinct subset of round cell sarcomas harbors capicua transcriptional repressor (CIC) rearrangement. Diagnosing these sarcomas can be difficult owing to their resemblance to Ewing sarcoma and other 'small round blue cell tumors'; molecular techniques are generally required. Recent gene expression studies of CIC-rearranged sarcomas identified the upregulation of ETV4. We assessed the sensitivity and specificity of ETV4 and WT1 immunohistochemistry for CIC-rearranged sarcoma. We evaluated whole-tissue sections from 40 CIC-rearranged sarcomas, 40 Ewing sarcomas, 4 BCOR-CCNB3 sarcomas, 6 unclassified round cell sarcomas, and 150 histologic mimics. Moderate-to-strong nuclear immunoreactivity for ETV4 in at least 50% of cells was observed in 36 (90%) CIC-rearranged sarcomas and 10 (5%) other tumors, including 5 unclassified round cell sarcomas, 2 Wilms tumors, and 1 each desmoplastic small round cell tumor, melanoma, and small cell carcinoma. Thirty-eight (95%) CIC-rearranged sarcomas showed nuclear staining for WT1, and 34 (85%) were positive for both ETV4 and WT1. Of 182 other tumors evaluated, 34 (19%) showed nuclear WT1 positivity, including all Wilms tumors and desmoplastic small round cell tumors, 5 unclassified round cell sarcomas, and a subset of lymphoblastic lymphomas, rhabdomyosarcomas, mesenchymal chondrosarcomas, carcinomas, and melanomas. In summary, diffuse moderate-to-strong ETV4 expression is present in most CIC-rearranged sarcomas and unclassified round cell sarcomas. More limited expression is seen in small subsets of various other round cell neoplasms. Nuclear WT1 expression is also present in most CIC-rearranged sarcomas and unclassified round cell sarcomas, along with Wilms tumors and desmoplastic small round cell tumors, and subsets of various histologic mimics. The sensitivity and specificity of diffuse ETV4 expression for CIC-rearranged sarcomas are 90% and 95%, respectively, whereas the sensitivity and specificity of WT1 are 95% and 81%, respectively. Diffuse ETV4 along with at least focal WT1 expression is helpful to distinguish CIC-rearranged sarcoma from Ewing sarcoma and other histologic mimics.

Phenamil enhances the adipogenic differentiation of hen preadipocytes.

A study was conducted to examine the effect of phenamil on adipogenic differentiation and expression of key adipogenic transcripts in hen preadipocytes. Preadipocytes were isolated from 20-week old Single Comb White Leghorn hens (Gallas gallus, Lohman strain). The experiment lasted for 48 h and had six treatments. Non-treated control (C) cells, cells treated with dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and oleic acid (DMIOA) (T1), DMIOA + 15 μM phenamil (T2), DMIOA + 30 μM phenamil (T3), 15 μM phenamil alone (T4), and 30 μM phenamil alone (T5). Neutral lipid accumulation and the mRNA expression of key adipogenic transcripts were measured in all treatments and compared. Lipid accumulation was detected in T1, T2, and T3 only. Expression of peroxisome proliferator receptor-activator gamma 2 (PPARγ2), the core enhancer binding protein α (C/EBPα), C/EBPβ, fatty acid binding protein 4 (FABP4), and lipoprotein lipase (LPL) as well as ETS variant 4 (ETV4) and 5 was higher (P < 0.05) in T2, T3, T4, and T5 compared to C. Expression of these transcripts was higher (P < 0.05) in T2 and T3 compared to T4 and T5. The core enhancer binding protein α, C/EBPβ, and FABP4 were highly expressed (P < 0.05) in T1 compared to C. However, the expression of PPARγ2, LPL, and ETV4 and ETV5 was not significantly different. Expression of C/EBPα, C/EBPβ, and FABP4 was higher (P < 0.05) in T2 and T3 compared to T1. Expression of sterol regulatory element binding protein 1 (SREBP1) and leptin receptor (LEPR) was not significantly different among the treatments. In conclusion, phenamil enhances DMIOA-induced adipogenic differentiation of hen preadipocytes but does not induce adipogenesis by itself.

SOX9 regulates expression of the male fertility gene Ets variant factor 5 (ETV5) during mammalian sex development

In humans, dysregulation of the sex determining gene SRY-box 9 (SOX9) leads to disorders of sex development (DSD). In mice, knock-out of Sox9 prior to sex determination leads to XY sex reversal, while Sox9 inactivation after sex determination leads to spermatogenesis defects. SOX9 specifies the differentiation and function of Sertoli cells from somatic cell precursors, which then orchestrate the development and maintenance of other testicular cell types, largely through unknown mechanisms. Here, we describe a novel testicular target gene of SOX9, Ets variant factor 5 (ETV5), a transcription factor responsible for maintaining the spermatogonial stem cell niche. Etv5 was highly expressed in wild-type XY but not XX mouse fetal gonads, with ETV5 protein localized in the Sertoli cells, interstitial cells and germ cells of the testis. In XY Sox9 knock-out gonads, Etv5 expression was strongly down-regulated. Similarly, knock-down of SOX9 in the human Sertoli-like cell line NT2/D1 caused a decrease in ETV5 gene expression. Transcriptomic analysis of NT2/D1 cells over-expressing SOX9 showed that ETV5 expression was increased in response to SOX9. Moreover, chromatin immunoprecipitation of these cells, as well as of embryonic mouse gonads, showed direct binding of SOX9 to ETV5 regulatory regions. We demonstrate that SOX9 was able to activate ETV5 expression via a conserved SOX site in the 5′ regulatory region, mutation of which led to loss of activation. In conclusion, we present a novel target gene of SOX9 in the testis, and suggest that SOX9 regulation of ETV5 contributes to the control of male fertility.