Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Abnormal activation of the Wnt3a/ß-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

BACKGROUND: T-box transcription factor 3(TBX3) is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM's pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). METHODS AND RESULTS: We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (ICR) mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/ß-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/ß-catenin pathway as well as Anti-proliferation cell nuclear antigen( PCNA) and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/ß-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. CONCLUSION: These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/ß-catenin pathway. The activation of the Wnt3a/ß-catenin pathway in ADM promoted TBX3 expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM.

Research progress on activation transcription factor 3: A promising cardioprotective molecule.

Activation transcription factor 3 (ATF3), a member of the ATF/cyclic adenosine monophosphate response element binding family, can be induced by a variety of stresses. Numerous studies have indicated that ATF3 plays multiple roles in the development and progression of cardiovascular diseases, including atherosclerosis, hypertrophy, fibrosis, myocardial ischemia-reperfusion, cardiomyopathy, and other cardiac dysfunctions. In past decades, ATF3 has been demonstrated to be detrimental to some cardiac diseases. Current studies have indicated that ATF3 can function as a cardioprotective molecule in antioxidative stress, lipid metabolic metabolism, energy metabolic regulation, and cell death modulation. To unveil the potential therapeutic role of ATF3 in cardiovascular diseases, we organized this review to explore the protective effects and mechanisms of ATF3 on cardiac dysfunction, which might provide rational evidence for the prevention and cure of cardiovascular diseases.

Macrophage microvesicle-derived circ_YTHDF2 in methamphetamine-induced chronic lung injury.

Long-term abuse of methamphetamine (MA) can cause lung toxicity. Intercellular communication between macrophages and alveolar epithelial cells (AECs) is critical for maintaining lung homeostasis. Microvesicles (MVs) are an important medium of intercellular communication. However, the mechanism of macrophage MVs (MMVs) in MA-induced chronic lung injury remains unclear. This study aimed to investigate if MA can augment the activity of MMVs and if circ_YTHDF2 is a key factor in MMV-mediated macrophage-AEC communication, and to explore the mechanism of MMV-derived circ_YTHDF2 in MA-induced chronic lung injury. MA elevated peak velocity of the pulmonary artery and pulmonary artery accelerate time, reduced the number of alveolar sacs, thickened the alveolar septum, and accelerated the release of MMVs and the uptake of MMVs by AECs. Circ_YTHDF2 was downregulated in lung and MMVs induced by MA. The immune factors in MMVs were increased by si-circ_YTHDF. Circ_YTHDF2 knockdown in MMVs induced inflammation and remodelling in the internalised AECs by MMVs, which was reversed by circ_YTHDF2 overexpression in MMVs. Circ_YTHDF2 bound specifically to and sponged miRNA-145-5p. Runt-related transcription factor 3 (RUNX3) was identified as potential target of miR-145-5p. RUNX3 targeted zinc finger E-box-binding homeobox 1 (ZEB1)-related inflammation and EMT of AECs. In vivo, circ_YTHDF2 overexpression-MMVs attenuated MA-induced lung inflammation and remodelling by the circ_YTHDF2-miRNA-145-5p-RUNX3 axis. Therefore, MA abuse can induce pulmonary dysfunction and alveolus injury. The immunoactivity of MMVs is regulated by circ_YTHDF2. Circ_YTHDF2 in MMVs is the key to communication between macrophages and AECs. Circ_YTHDF2 sponges miR-145-5p targeting RUNX3 to participate in ZEB1-related inflammation and remodelling of AECs. MMV-derived circ_YTHDF2 would be an important therapeutic target for MA-induced chronic lung injury. KEY POINTS: Methamphetamine (MA) abuse induces pulmonary dysfunction and alveoli injury. The immunoactivity of macrophage microvesicles (MMVs) is regulated by circ_YTHDF2. Circ_YTHDF2 in MMVs is the key to MMV-mediated intercellular communication between macrophages and alveolar epithelial cells. Circ_YTHDF2 sponges miR-145-5p targeting runt-related transcription factor 3 (RUNX3) to participate in zinc finger E-box-binding homeobox 1 (ZEB1)-related inflammation and remodelling. MMV-derived circ_YTHDF2 would be an important therapeutic target for MA-induced chronic lung injury.

Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis.

Ulcerative colitis (UC) results from a complex interplay between the environment, gut microbiota, host genetics, and immunity. Runt-related transcription factor 3 (RUNX3) regulates Th1/Th2 balance and, thus, the synthesis of cytokines and inflammation. We aimed to analyze the dependence of RUNX3 promoter 2 (P2) methylation level on: age, sex, body mass index (BMI), C-reactive protein (CRP), serum albumin, disease duration, Pediatric Ulcerative Colitis Activity Index (PUCAI), the Paris classification, and exposure to medications. This multicenter, cross-sectional study recruited hospitalized children with UC. Methylation of RUNX3 P2 was measured with methylation-sensitive restriction enzymes in the whole blood DNA. Sixty-four children were enrolled, with a mean age of 14.5 ± 2.8 years. Half of them were female (51.6%), and the average BMI Z-score was -0.44 ± 1.14. The mean methylation of RUNX3 P2 was 54.1 ± 13.3%. The methylation level of RUNX3 P2 did not correlate with age, sex, nutritional status, CRP, albumin, PUCAI, or the extent of colitis (Paris E1-E4). RUNX3 P2 methylation did not differ between patients recruited within two and a half months of diagnosis and children who had UC for at least a year. Current or past exposure to biologics, immunosuppressants, or steroids was not associated with RUNX3 P2 methylation. Methylation of RUNX3 promoter 2 in whole blood DNA does not seem to be associated with the characteristics of UC in children.

Long noncoding RNA Linc01612 represses hepatocellular carcinoma progression by regulating miR-494/ATF3/p53 axis and promoting ubiquitination of YBX1.

Long noncoding RNAs (lncRNAs) play an important role in the progression of hepatocellular carcinoma (HCC). Linc01612 is a novel lncRNA that function remains unknown in the progression of cancers, including HCC. In this study, we discovered that Linc01612 is significantly down-regulated in HCC tissues than in non-tumor tissues and correlated with poor prognosis. Linc01612 mainly localizes in the cytoplasm and functions as a tumor suppressor by repressing the growth and metastasis of hepatoma cells in vitro and in vivo. Mechanistically, in p53-expressing hepatoma cells, Linc01612 acts as a competitive endogenous RNA and promotes the expression of activation transcription factor 3 (ATF3) by sponging microRNA-494 (miR-494), which in turn inhibits MDM2-mediated ubiquitination of p53 and activates the p53 pathway. Furthermore, in p53-null hepatoma cells, Linc01612 exerts its biological functions by physically interacting with Y-box binding protein 1 protein (YBX1) and promoting the ubiquitin-mediated degradation of YBX1. Interestingly, the Linc01612-YBX1 signaling pathway is also present in p53-expressing hepatoma cells. In conclusion, our study indicated that Linc01612 is a functional lncRNA in HCC and Linc01612 may serve as a potential diagnostic biomarker and therapeutic target for HCC.

Transcriptional factor 3 binds to sirtuin 1 to activate the Wnt/ß-catenin signaling in cervical cancer.

Transcriptional factor 3 (TCF3, also termed E2A), first reported to exert crucial functions during lymphocyte development, has been revealed to participate in the pathogenesis of human cancers. The aim of this work was to investigate the function of TCF3 in cervical cancer (CC) and the molecular interactions. The bioinformatics prediction suggested that TCF3 was highly expressed in CC and linked to poor prognosis. Increased TCF3 expression was identified in CC cell lines, and its downregulation reduced proliferation and migration of CC cells in vitro as well as growth of xenograft tumors in vivo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the TCF-3-related genes and genes showed differential expression between CC and normal tissues were mainly enriched in the Wnt/ß-catenin pathway. TCF3 bound to sirtuin 1 (SIRT1) promoter for transcriptional activation, and SIRT1 promoted deacetylation and nuclear translocation of ß-catenin in CC. SIRT1 overexpression blocked the role of TCF3 silencing and restored cell proliferation in vitro and tumor growth in vivo. Treatment with XAV-939, a ß-catenin inhibitor, significantly suppressed the cell proliferation and tumor growth induced by SIRT1 overexpression. In conclusion, this study demonstrates that TCF3 augments progression of CC by activating SIRT1-mediated ß-catenin signaling.

Overexpression of transcription factor 3 drives hepatocarcinoma development by enhancing cell proliferation via activating Wnt signaling pathway.

BACKGROUND: Transcription factor 3 (TCF3) plays pivotal roles in embryonic development, stem cell maintenance and carcinogenesis. However, its role in hepatocellular carcinoma (HCC) remains largely unknown. This study aimed to analyze the correlation between TCF3 expression and clinicopathological features of HCC, and further explore the underlying mechanism in HCC progression. METHODS: The expression of TCF3 was collected from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) HCC datasets, and further confirmed by immunostaining and Western blotting assays. The correlation between TCF3 expression and the clinicopathological features was evaluated. Bioinformatical analysis and in vitro experiments were conducted to explore the potential role of TCF3 in HCC development. RESULTS: Both the mRNA and protein levels of TCF3 were significantly higher in HCC tumor tissues compared to tumor adjacent tissues (P < 0.001 and P < 0.01). Analysis based on TCGA datasets showed that TCF3 was positively correlated with tumor clinical stage and grade, and patients with high TCF3 expression had shorter overall survival (P = 0.012), disease-specific survival (P = 0.022) and progression-free survival (P = 0.013). Similarly, the immunostaining results revealed that the high expression of TCF3 was closely correlated with tumor size (P = 0.001) and TNM stage (P = 0.002), and TCF3 was an independent risk factor of HCC. In vitro study exhibited that TCF3 knockdown dramatically suppressed cancer cell proliferation, and the underlying mechanism might be that the silencing of TCF3 reduced the expression of critical regulating proteins towards cell cycle and proteins involved in Wnt signaling pathways. CONCLUSIONS: TCF3 expression is significantly elevated in HCC and positively associated with the tumor size and TNM stage, as well as poor prognosis of HCC patients. The mechanism might be that TCF3 promotes cancer cell proliferation via activating Wnt signaling pathway.

Rspo2 inhibits TCF3 phosphorylation to antagonize Wnt signaling during vertebrate anteroposterior axis specification.

The Wnt pathway activates target genes by controlling the ß-catenin-T-cell factor (TCF) transcriptional complex during embryonic development and cancer. This pathway can be potentiated by R-spondins, a family of proteins that bind RNF43/ZNRF3 E3 ubiquitin ligases and LGR4/5 receptors to prevent Frizzled degradation. Here we demonstrate that, during Xenopus anteroposterior axis specification, Rspo2 functions as a Wnt antagonist, both morphologically and at the level of gene targets and pathway mediators. Unexpectedly, the binding to RNF43/ZNRF3 and LGR4/5 was not required for the Wnt inhibitory activity. Moreover, Rspo2 did not influence Dishevelled phosphorylation in response to Wnt ligands, suggesting that Frizzled activity is not affected. Further analysis indicated that the Wnt antagonism is due to the inhibitory effect of Rspo2 on TCF3/TCF7L1 phosphorylation that normally leads to target gene activation. Consistent with this mechanism, Rspo2 anteriorizing activity has been rescued in TCF3-depleted embryos. These observations suggest that Rspo2 is a context-specific regulator of TCF3 phosphorylation and Wnt signaling.

E47 upregulates ΔNp63α to promote growth of squamous cell carcinoma.

Targeted therapy has greatly improved both survival and prognosis of cancer patients. However, while therapeutic treatment of adenocarcinoma has been advanced greatly, progress in treatment of squamous cell carcinoma (SCC) has been slow and ineffective. Therefore, it is of great importance to decipher mechanisms and identify new drug targets involved in squamous cell carcinoma development. In this study, we demonstrate that E47 plays the distinctive and opposite roles on cell proliferation in adenocarcinoma and squamous cell carcinoma. While E47 suppresses cell proliferation in adenocarcinoma cells, it functions as a oncoprotein to promote cell proliferation and tumor growth of squamous cell carcinoma. Mechanistically, we show that E47 can directly bind to the promoter and transactivate ΔNp63 gene expression in squamous cell carcinoma cells, resulting in upregulation of cyclins D1/E1 and downregulation of p21, and thereby promoting cell proliferation and tumor growth. We further show that expression of E2A (E12/E47) is positively correlated with p63 and that high expression of E2A is associated with poor outcomes in clinical samples of squamous cell carcinoma. These results highlight that the E47-ΔNp63α axis may be potential therapeutic targets for treatment of squamous cell carcinoma.

The transcription factor TAL1 and miR-17-92 create a regulatory loop in hematopoiesis.

A network of gene regulatory factors such as transcription factors and microRNAs establish and maintain gene expression patterns during hematopoiesis. In this network, transcription factors regulate each other and are involved in regulatory loops with microRNAs. The microRNA cluster miR-17-92 is located within the MIR17HG gene and encodes six mature microRNAs. It is important for hematopoietic differentiation and plays a central role in malignant disease. However, the transcription factors downstream of miR-17-92 are largely elusive and the transcriptional regulation of miR-17-92 is not fully understood. Here we show that miR-17-92 forms a regulatory loop with the transcription factor TAL1. The miR-17-92 cluster inhibits expression of TAL1 and indirectly leads to decreased stability of the TAL1 transcriptional complex. We found that TAL1 and its heterodimerization partner E47 regulate miR-17-92 transcriptionally. Furthermore, miR-17-92 negatively influences erythroid differentiation, a process that depends on gene activation by the TAL1 complex. Our data give example of how transcription factor activity is fine-tuned during normal hematopoiesis. We postulate that disturbance of the regulatory loop between TAL1 and the miR-17-92 cluster could be an important step in cancer development and progression.

TERT promoter alterations could provide a solution for Peto's paradox in rodents.

Cancer is a genetic disease caused by changes in gene expression resulting from somatic mutations and epigenetic changes. Although the probability of mutations is proportional with cell number and replication cycles, large bodied species do not develop cancer more frequently than smaller ones. This notion is known as Peto's paradox, and assumes stronger tumor suppression in larger animals. One of the possible tumor suppressor mechanisms involved could be replicative senescence caused by telomere shortening in the absence of telomerase activity. We analysed telomerase promoter activity and transcription factor binding in mammals to identify the key element of telomerase gene inactivation. We found that the GABPA transcription factor plays a key role in TERT regulation in somatic cells of small rodents, but its binding site is absent in larger beavers. Protein binding and reporter gene assays verify different use of this site in different species. The presence or absence of the GABPA TF site in TERT promoters of rodents correlates with TERT promoter activity; thus it could determine whether replicative senescence plays a tumor suppressor role in these species, which could be in direct relation with body mass. The GABPA TF binding sites that contribute to TERT activity in somatic cells of rodents are analogous to those mutated in human tumors, which activate telomerase by a non-ALT mechanism.

ID3 Promotes Stem Cell Features and Predicts Chemotherapeutic Response of Intrahepatic Cholangiocarcinoma.

Cancer stem cells contribute to a high rate of recurrence and chemotherapeutic resistance in many types of cancer, including intrahepatic cholangiocarcinoma (ICC). Inhibitor of differentiation 3 (ID3) has been reported to promote cancer stem cells, but its role in ICC is obscure. In this study, we identified that ID3 is highly expressed in human ICC tissues compared with matched normal tissues and correlates with poor prognosis. Functional studies demonstrate that ID3 is required for stemness maintenance in cholangiocarcinoma both in vitro and in vivo. Consistent with the regulation of cancer stem cell features by ID3, transgenic expression of ID3 enhances chemoresistance of cholangiocarcinoma cells. Moreover, we found that ICC patients with low ID3 levels benefited from postoperative transarterial chemoembolization, whereas patients with high ID3 levels did not, indicating the significance of ID3 in individualized ICC therapy. Mechanistically, ID3 could interact with E47 and block E47 recruitment to the promoter of ß-catenin, which leads to activation of Wnt/ß-catenin signaling. Conclusion: Our results show that ID3 could promote the stemness of ICC by increasing the transcriptional activity of ß-catenin and could serve as a biomarker in predicting ICC patients' response to adjuvant chemotherapeutics.

Hepatitis B virus surface protein-induced hPIAS1 transcription requires TAL1, E47, MYOG, NFI, and MAPK signal pathways.

The protein inhibitor of activated STAT1 (PIAS1) plays important roles in regulating virus-induced chronic hepatitis, but the interaction between hepatitis B virus (HBV) and hPIAS1 is not clear. Our aim was to verify if HBV encoding proteins enhance the transcription of hPIAS1 and which cis-elements and transcription factors were involved in the mechanism. In order to do, so a series of molecular biological methods, along with functional and histological studies, were performed. We found that the HBV surface protein (HBs) enhanced hPIAS1 transcription through the activities of TAL1, E47, myogenin (MYOG), and NFI, dependent on the activation of p38MAPK and ERK signaling pathways in vitro, which might contribute to the ineffectiveness of treatment in CHB patients. Furthermore, liver samples from patients with high HBsAg levels and HBV DNA displayed increased hPIAS1 expression and high levels of TAL1, E47, MYOG, and NFI, compared to those patients with low HBsAg levels and HBV DNA, and healthy controls. These findings suggest that the HBs protein-induced hPIAS1 transcription requires TAL1, E47, MYOG, NFI, and MAPK signal pathways. It provides new potential targets for antiviral therapeutic strategies for controlling HBV-associated diseases.

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences.

The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.

The Heterodimeric TWIST1-E12 Complex Drives the Oncogenic Potential of TWIST1 in Human Mammary Epithelial Cells.

The TWIST1 embryonic transcription factor displays biphasic functions during the course of carcinogenesis. It facilitates the escape of cells from oncogene-induced fail-safe programs (senescence, apoptosis) and their consequent neoplastic transformation. Additionally, it promotes the epithelial-to-mesenchymal transition and the initiation of the metastatic spread of cancer cells. Interestingly, cancer cells recurrently remain dependent on TWIST1 for their survival and/or proliferation, making TWIST1 their Achilles' heel. TWIST1 has been reported to form either homodimeric or heterodimeric complexes mainly in association with the E bHLH class I proteins. These complexes display distinct, sometimes even antagonistic, functions during development and unequal prometastatic functions in prostate cancer cells. Using a tethered dimer strategy, we successively assessed the ability of TWIST1 dimers to cooperate with an activated version of RAS in human mammary epithelial cell transformation, to provide mice with the ability to spontaneously develop breast tumors, and lastly to maintain a senescence program at a latent state in several breast cancer cell lines. We demonstrate that the TWIST1-E12 complex, unlike the homodimer, is an oncogenic form of TWIST1 in mammary epithelial cells and that efficient binding of both partners is a prerequisite for its activity. The detection of the heterodimer in human premalignant lesions by a proximity ligation assay, at a stage preceding the initiation of the metastatic cascade, is coherent with such an oncogenic function. TWIST1-E protein heterodimeric complexes may thus constitute the main active forms of TWIST1 with regard to senescence inhibition over the time course of breast tumorigenesis.

Combined Id1 and Id3 Deletion Leads to Severe Erythropoietic Disturbances.

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of ß-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.

E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding.

Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.

Hepatitis B virus X protein induces epithelial-mesenchymal transition by repressing E-cadherin expression via upregulation of E12/E47.

Previous reports have demonstrated that hepatitis B virus (HBV) X protein (HBx) represses E-cadherin expression to induce epithelial-mesenchymal transition (EMT), an essential component of cancer progression to more aggressive phenotypes characterized by tumour invasion, migration and metastasis; however, the underlying mechanism for this phenomenon is still unclear. In this study, we found that ectopic expression of HBx in human hepatocytes using overexpression and 1.2-mer WT HBV replicon systems upregulated levels of the transcriptional repressors E12 and E47, resulting in inactivation of the E-cadherin promoter, containing three E-box motifs, and subsequent repression of its expression. E12/E47 knockdown using a specific small interfering RNA almost completely abolished the potential of HBx to repress E-cadherin expression. HBx inhibited the ubiquitin-dependent proteasomal degradation of E12/E47 without affecting their expression at the transcriptional level. Upregulation of E12/E47 by HBx ultimately led to EMT in human hepatocytes, as demonstrated by morphological changes, altered protein levels of EMT markers, including E-cadherin, plakoglobin, fibronectin, vimentin and N-cadherin, and increased capacity for cell detachment and migration.

PAK5-mediated E47 phosphorylation promotes epithelial-mesenchymal transition and metastasis of colon cancer.

The p21-activated kinase 5 (PAK5) is overexpressed in advanced cancer and the transcription factor E47 is a direct repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). However, the relationship between PAK5 and E47 has not been explored. In this study, we found that PAK5-mediated E47 phosphorylation promoted EMT in advanced colon cancer. PAK5 interacted with E47 and phosphorylated E47 on Ser39 under hepatocyte growth factor (HGF) stimulation, which decreased cell-cell cohesion, increased cell migration and invasion in vitro and promoted metastasis in a xenograft model. Furthermore, phosphorylation of E47 facilitated its accumulating in nucleus in an importin α-dependent manner, and enhanced E47 binding to E-cadherin promoter directly, leading to inhibition of E-cadherin transcription. In contrast, PAK5-knockdown resulted in blockage of HGF-induced E47 phosphorylation, attenuated association of E47 with importin α and decreased E47 binding to E-cadherin promoter. In addition, we demonstrated a close correlation between PAK5 and phospho-Ser39 E47 expression in colon cancer specimens. More importantly, high expression of phospho-E47 was associated with an aggressive phenotype of colon cancer and nuclear phospho-E47 staining was found in certain cases of colon cancer with metastasis. Collectively, E47 is a novel substrate of PAK5, and PAK5-mediated phosphorylation of E47 promotes EMT and metastasis of colon cancer, suggesting that phosphorylated E47 on Ser39 may be a potential therapeutic target in progressive colon cancer.