| The mammalian BTG/Tob family comprises six proteins (BTG1, BTG2PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2) in human cells, which regulate cell cycle progression, cell growth and differentiation, DNA damage repair,tumourigenesis and cancer suppression in a variety of cell types. Down regulation or impaired expression of BTG/Tob is observed in several tumors, including prostate cancer, clear renal cell carcinomas and breast cancer, mice lacking BTG/Tob are prone to spontaneous carcinogenesis with high frequencies of tumors found in liver, lung and lymph nodes, indicating the deregulation of BTG/Tob early at the tumorigenic process may also be an important step in the development of mammary tumors and BTG/Tob as new tumor suppressor is demonstrated by more and more evidence.Several members of the BTG/Tob family is shown to be implicated in transcription in the nucleus and cytoplasmic mRNA deadenylation and turnover in recent studies, moreover, BTG/Tob factors impact deadenylases through the interaction with DNA-binding transcription factors as well as the paralogues CNOT7 (human Caf1/Caf1a) and CNOT8 (human Pop2/Calif/Caf1b), two deadenylase subunits of the CCR4-NOT complex.The CCR4–NOT complex conserved from yeast to human is composed of nine subunits, CNOT1, CNOT2, CNOT3, CNOT6/hCcr4a, CNOT6L/hCcr4b, CNOT7/hCaf1, CNOT8/hPop2, CNOT9 and CNOT10, among which CNOT6, 6L, 7, 8 possess exonuclease domain, and are implicated in several aspects of mRNA metabolism, and that both factors have a predominant role in mRNA poly(A) tail shortening in vivo.Except for BTG4, all BTG/Tob proteins can interact with the highly similar paralogues CNOT7 and/or CNOT8,whose amino acid sequences are 76% identical (89% similar).Studies on crystal structures of the conserved BTG/Tob domain of BTG2 and Tob1 revealed that it is composed of five?-helices and four?-strands, and this characteristic structure contacts with CNOT7 surface.Moreover, key residues from both box A and box B motifs of Tob1 are involved in the Tob1–CNOT7 interaction, an interaction between Tob1 and the CNOT1 subunit of the large CCR4–NOT assembly was also described recently. These observations suggested that BTG/Tob proteins might direct CNOT7 to its mRNA substrates in controlling mRNA deadenylation. More importantly, the association between Tob and CNOT7 appears to be crucial for the exertion of a strong anti-proliferative activity.We previously showed that depletion of CNOT6L or CNOT7 subunit of the complex impaired cell growth or spermatogenesis in mice, respectively, suggesting that the CCR4-NOT complex has non-housekeeping functions despite its fundamental cellular function in mRNA metabolism. However, the functions and underlying mechanism of action of most subunits remain largely unknown. To analyze their biological significance, we performed extensive analysis of their expression in mouse central nervous system to establish the foundation for further study of CCR4-NOT complex.Objective1 Study extensively on the expression of CCR4-NOT complex in mouse brain tissues and different cell types, and to reveal its new functions in central nervous system.2 Study the functions and underlying mechanisms of CCR4-NOT complex on cell differentiation by applying the Neuro-2a mouse neuroblastoma cell line as model system.Materials and Methods1 Expression of BTG2 and CCR4-NOT complex in various mouse organs: Northern Blot and Western Blot were used for analysis of expression pattern.2 Expression of CCR4-NOT complex in different regions and cells of brain: Western Blot and?-Gal Staining were used for analysis of expression pattern. Primary culture of neural stem cells (NSCs) and oligodendrocyte progenitor cells (OPCs) were induced by retinoic acid (RA) and removal of cytokines. Western Blot?Immunostaining?siRNA were applied for study on the expression and functions of CCR4-NOT complex.3 The functions of CCR4-NOT complex in Neuro-2a cells: The retrovirus vectors pSIREN-CNOT6 , pSIREN-CNOT8 for knockdown and pMX-EGFP-CNOT6 ,pMX-EGFP-CNOT8 for over expression were constructed. The effect of CCR4-NOT complex on Neuro-2a differentiation was studied after CNOT6 or CNOT8 knockdown and over expression. Results1 Expression of BTG2 and CCR4-NOT complex in various mouse organs: BTG2 was expressed abundantly in kidney, lowly in the lung, brain, liver and heart, not expressed in muscle, thymus and spleen.CCR4-NOT complex subunits were expressed in most of mouse tissues, being especially high in the brain, lung, small intestine, testis, thymus, ovary, and spleen.2 Expression of CCR4-NOT complex in different regions and cells of brain: We further found that all CCR4-NOT subunits were highly expressed in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of mouse brains, moreover, they were highly expressed at early developmental stages. The brain consists of three major types of cells: astrocytes, neurons, and oligodendrocytes. All CCR4-NOT subunits were expressed in these three cell types, being particularly high in oligodendrocytes. The expression of several subunits of the CCR4-NOT complex were down-regulated in primary cultured neurons compared with in neural stem cells. The expression of CCR4-NOT complex were the same in OPCs and oligodendrycytes(OLs), but cell proliferation of OLs were inhibited after CCR4-NOT complex knock down.3 The functions of CCR4-NOT complex in Neuro-2a cells: CONT6 or CNOT8 deletion promotes neurite outgrowth of Neuro-2a, on contrast, CONT6 or CNOT8 over expression inhibits neurite outgrowth, furthermore, over expression rescues the phenotype of CONT6 or CNOT8 knock down Neuro-2a cells,?-catenin was also unregulated in CONT6 or CNOT8 knock down cells.Conclusions1 CCR4–NOT complex may be a pivotal factor involved in the regulation of cell proliferation and differentiation, demonstrated by the expression pattern in the mouse brain.2 CNOT2, CNOT3, CNOT6, CNOT8, CNOT9 were down-regulated in primary cultured neurons compared with in NSCs, indicating CCR4–NOT complex may be required for maintaining the undifferentiated state of neural stem cells and involved in early neuronal differentiation or proliferation of NSCs.3 CCR4–NOT complex was not required in the differentiation of OPCs to OLs, but it play important role in regulating the proliferation of OLs.4 The inhibitory effect of CCR4–NOT complex on neurite outgrowth of Neuro-2a indicates that it may function during neuronal maturation through?-catenin inactivation. |
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