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1.
Life Sci ; 232: 116649, 2019 Sep 01.
Article in English | MEDLINE | ID: mdl-31301415

ABSTRACT

AIMS: To investigate the potential biological role of E2F6 and its underlying molecular mechanism in gastric carcinoma (GC) progression. MAIN METHODS: The expressions of cancer susceptibility candidate 2 (CASC2), E2F6 and matrix metalloprotein-2 (MMP-2) were measured by quantitative real-time polymerase chain reaction and western blotting. The inhibitory effect of E2F6 on CASC2 was evaluated using luciferase reporter assay. Cell growth was assessed by colony formation assay and cell counting kit-8. Cell invasion and apoptosis were measured by transwell assay and flow cytometry assay, respectively. In vivo tumorigenicity was assessed by tumor xenografts in nude mice. KEY FINDINGS: Our data revealed that CASC2 was downregulated while E2F6 was upregulated in GC tissues and cell lines. Remarkably, lower expression of CASC2 was associated with poor survival in GC patients. E2F6 inhibited the expression of CASC2. Subsequently, reliable data showed that downregulation of E2F6 suppressed the proliferation and invasion, and promoted the apoptosis of GC cells. Furthermore, downregulation of E2F6 decreased the expression of MMP-2 and increased the activity of caspase-3. However, these changes triggered by E2F6 knockdown could be reversed by inhibition of CASC2. Moreover, we also proved that downregulation of CASC2 reverses the effect of E2F6 knockdown on tumor growth in vivo. SIGNIFICANCE: Our data demonstrated that E2F6 could regulate the proliferation, invasion and apoptosis of GC cells via inhibiting the expression of CASC2, suggesting that E2F6/CASC2 axis is another regulator of GC progression.


Subject(s)
Down-Regulation/physiology , E2F6 Transcription Factor/physiology , RNA, Long Noncoding/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Tumor Suppressor Proteins/genetics , Animals , Blotting, Western , Cell Line, Tumor , Disease Progression , Heterografts , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Prognosis , Real-Time Polymerase Chain Reaction , Up-Regulation
2.
Biochem Biophys Res Commun ; 513(3): 560-566, 2019 06 04.
Article in English | MEDLINE | ID: mdl-30981507

ABSTRACT

Both type 1 and type 2 diabetes are associated with loss of functional beta cell mass, and strategies to restore beta cells are urgently needed. We reported previously that overexpression of the nuclear receptor TLX induces beta cell proliferation, but the underlying molecular mechanism has not been defined. Here, we identified direct targets of TLX in beta cells at the genome-wide level by ChIP-Seq. These targets include a cadre of regulators that are known to be critical for proliferation. Among these ChIP targets, E2F6 was tightly associated with the cell cycle modules, and thus, we further analyzed E2F6 expression and function in beta cells. We showed that E2F6 is strongly downregulated by TLX, and its expression inhibits beta cell proliferation. Moreover, coexpression of E2F6 with TLX partially abrogated the proliferative effects of TLX. These results strongly suggest that TLX acts through E2F6 to regulate beta cell proliferation. Together, the results of this study reveal a direct interaction between TLX and E2F6 and suggest new targets for the expansion of functional beta cell mass.


Subject(s)
E2F6 Transcription Factor/metabolism , Insulin-Secreting Cells/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Cell Line , Cell Proliferation , E2F6 Transcription Factor/genetics , E2F6 Transcription Factor/physiology , Gene Expression Regulation , Genome , Insulin-Secreting Cells/cytology , Mice , Promoter Regions, Genetic
3.
PLoS Genet ; 14(1): e1007193, 2018 01.
Article in English | MEDLINE | ID: mdl-29381691

ABSTRACT

Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , DNA/metabolism , E2F6 Transcription Factor/physiology , Nuclear Proteins/physiology , Polycomb Repressive Complex 1/metabolism , Transcription Factors/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cells, Cultured , E2F6 Transcription Factor/genetics , Gene Expression Regulation , Gene Knockdown Techniques , HEK293 Cells , Human Embryonic Stem Cells/physiology , Humans , Mice , Mouse Embryonic Stem Cells/physiology , Nuclear Proteins/genetics , Protein Binding/genetics , Transcription Factors/genetics
4.
Curr Biol ; 23(17): 1629-37, 2013 Sep 09.
Article in English | MEDLINE | ID: mdl-23954429

ABSTRACT

BACKGROUND: In eukaryotic cells, detection of replication stress results in the activation of the DNA replication checkpoint, a signaling cascade whose central players are the kinases ATR and Chk1. The checkpoint response prevents the accumulation of DNA damage and ensures cell viability by delaying progression into mitosis. However, the role and mechanism of the replication checkpoint transcriptional response in human cells, which is p53 independent, is largely unknown. RESULTS: We show that, in response to DNA replication stress, the regular E2F-dependent cell-cycle transcriptional program is maintained at high levels, and we establish the mechanisms governing such transcriptional upregulation. E2F6, a repressor of E2F-dependent G1/S transcription, replaces the activating E2Fs at promoters to repress transcription in cells progressing into S phase in unperturbed conditions. After replication stress, the checkpoint kinase Chk1 phosphorylates E2F6, leading to its dissociation from promoters. This promotes E2F-dependent transcription, which mediates cell survival by preventing DNA damage and cell death. CONCLUSIONS: This work reveals, for the first time, that the regular cell-cycle transcriptional program is part of the DNA replication checkpoint response in human cells and establishes the molecular mechanism involved. We show that maintaining high levels of G1/S cell-cycle transcription in response to replication stress contributes to two key functions of the DNA replication checkpoint response, namely, preventing genomic instability and cell death. Given the critical role of replication stress in oncogene transformation, a detailed understanding of the molecular mechanisms involved in the checkpoint response will contribute to a better insight into cancer development.


Subject(s)
Cell Cycle/genetics , DNA Replication , E2F6 Transcription Factor/physiology , Protein Kinases/physiology , Transcription, Genetic/physiology , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , Humans , Promoter Regions, Genetic
5.
FASEB J ; 26(6): 2569-79, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22403008

ABSTRACT

The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P<0.05) in left ventricular end-diastolic diameter and reduced ejection fraction (<40%, P<0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F-responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P<0.01) decrease in gap junction protein connexin-43, resulting in abnormal electrocardiogram including a 24% (P<0.05) increase in PR interval. Further, mir-206, which targets connexin-43, was up-regulated 10-fold (P<0.05) in Tg myocardium. The mitogen-activated protein kinase pathway, which regulates the levels of miR-206 and connexin-43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post-transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.


Subject(s)
Cardiomyopathy, Dilated/etiology , E2F6 Transcription Factor/physiology , Animals , Connexin 43/biosynthesis , Down-Regulation , Gene Expression/drug effects , Heart Failure/physiopathology , Mice , Mice, Transgenic , Myocardium/metabolism
6.
Cell Death Dis ; 1: e105, 2010 Dec 09.
Article in English | MEDLINE | ID: mdl-21368878

ABSTRACT

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2'-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer.


Subject(s)
Apoptosis/drug effects , Drug Resistance, Neoplasm/genetics , MicroRNAs/genetics , Prostatic Neoplasms/drug therapy , Animals , Antimetabolites, Antineoplastic/therapeutic use , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Apoptosis Regulatory Proteins/physiology , Azacitidine/analogs & derivatives , Azacitidine/therapeutic use , Cell Line, Tumor , DNA Methylation , DNA Modification Methylases/antagonists & inhibitors , Decitabine , Down-Regulation , E2F6 Transcription Factor/genetics , E2F6 Transcription Factor/metabolism , E2F6 Transcription Factor/physiology , Humans , Male , Mice , MicroRNAs/metabolism , Promoter Regions, Genetic , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism
7.
Stem Cells ; 25(10): 2439-47, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17600109

ABSTRACT

E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34(+) human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34(-) bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F-6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Apoptosis/drug effects , E2F6 Transcription Factor/physiology , Hematopoietic Stem Cells/drug effects , Apoptotic Protease-Activating Factor 1/biosynthesis , Apoptotic Protease-Activating Factor 1/genetics , Binding, Competitive , Cell Differentiation/drug effects , Cells, Cultured/cytology , Cells, Cultured/drug effects , Colony-Forming Units Assay , Culture Media, Serum-Free/pharmacology , Cytokines/pharmacology , E2F Transcription Factors/biosynthesis , E2F Transcription Factors/genetics , E2F1 Transcription Factor/antagonists & inhibitors , E2F1 Transcription Factor/pharmacology , Hematopoietic Stem Cells/cytology , Humans , K562 Cells/cytology , K562 Cells/drug effects , Promoter Regions, Genetic/drug effects , Recombinant Fusion Proteins/physiology , Transduction, Genetic , Up-Regulation
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