Functional Study Of CCDC152

CCDC152 (Coiled-coil Domain Containing Protein 152) belongs to the CCDC family. CCDC 152 gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, and frog. However, its function is unknown. The coiled-coil domains have important biological functions. It often acts as a transcription factor to regulate gene expression.Phyre2 3-D prediction showed CCDC 152 tertiary structure is significantly similar to GCN4 of yeast. PSI-blast sequence alignment showed CCDC152 might have the function of the methyl-transferase. GCN4 contains the basic region euine zipper (bZIP) and is the homologue of AP-1 transcription factor family, exists as a transcription activator. The bZIP dinner is a pair of continuous a helices that form a coiled coil to pass through the major groove of the DNA-binding site to promote its transcription. It is generally believed that, dimerization is the premise for yeast GCN4 specific recognition of DNA binding sites. The conserved binding site for GCN4 is: ATG ACT CAT, a common sequence in most amino acid biosynthase gene promoter region.To deal with nutrient deprivation, yeast cells have evolved complex mechanism to adjust nutrion level of cells. When facing lack of nutrition, cellular free tRNA will be increased, then eventually increased the phosphorylation of GCN4 via phosphorylated GCN2 and eIF2a. Phosphorylated GCN4 can up regulate GAAC(general amino acid control)pathway by binding sequence of amino acid biosynthesis gene promoter regions, in order to promote intake and biosynthesis of amino acids. In addition, autophagy degrade unnecessary cell contents, such as misfolded proteins and damaged organelles, this enables cells to survive. In this Autophagy process, the expression of p62 is down-regulated. It is generally suggested that ATF4 in mammals is the homologous to GCN4 of yeast, both of them are transcription factors to deal with amino acids starvation.Autophagy is a process for cells to survive when cells under stress condition, or dealing with damaged organelles or misfolded proteins, which involves a variety of autophagy-related genes. The occurrence of autophagy process is to form a double capsule membrane structure (a shift from LC3B I to LC3B II) to package the contents for degradation. Then its outer membrane and the lysosome membrane fused to form autolysosome, which can degrade the contents into small molecules. G protein coupled receptors (GPCR) signal-system is closely associated with autophagy, researches have shown that nutrient status can trigger the process of autophagy by affect the secretion of hormones and neurotransmitters to regulate the G protein coupled receptors. GPCR also can cause autophagy by direct sensing the alteration of extracellular nutrients.As bioinformatics study results indicated CCDC152 is structally similar to GCN4, we explored the relationship between CCDC152 function and cell autophagy, and also investigated if CCDC152 participated in histone modification; based on the transcriptomic sequencing information, we studied the relationship between CCDC152 system and GPCR signaling system. Collectively, we suggested CCDC152 played a role in mediating certain cellular activities by writing "histone code" and acting as a transcription factor.The research methods and main results are as follows:Methods:1. To verify CCDC152 is a real transcription factor:(1) To study its intracellular location of CCDC152 in different cells:Transfected red fluorescent fusion protein CCDC152-Red into 293T?MCF-7?Hela?HepG2?Caco-2 or HT22 cells, then observed its location. Co-transfected green fluorescent fusion protein CCDC152-GFP and CCDC152-Red into 293T cells, then observe their co-localization;(2) To study on the influence of CCDC152 on its own promoter: Luciferase reporter gene assay was used to testify its influence on its own promoter. CCDC152-ORF and control vector N3-pEGFP, respectively, to luciferase reporter gene PGL2-CCDC152 cotransfected into 293T cells. Then detected luciferase reporter gene activity. Transfect different concentrations of CCDC152-ORF into 293T cells, detected the change of luciferase reporter gene activity.2.To study CCDC152 response to amino acids deficiency:(1) The influence of amino acids deficiency to the expression of CCDC152 protein.293T cells were grown in EBSS (Earle's balanced salts) medium for different time, then the expression of CCDC152 was detected by WB;(2) Comparing the different expression patterns between CCDC152 and ATF4 after cells were cultured with EBSS for different time, the expression of CCDC152 and ATF4 was detected by WB;(3) To detect the intracellular translocation position of endogenous CCDC152 under the condition of nutrient deprivation:the nucleoproteins and cytoplasma proteins were extracted respectively, and then CCDC152 expression was detected by WB;(4) To study the influence of over-expression of CCDC152 on p62 expression. Transfect CCDC152-ORF or control vector N3-pEGFP into 293T cells, then detected the expression of p62 protein by WB.3. To study the the role of CCDC152-ORF in cell autophagy:(1) Observe morphologic changes of 293T cells transfected CCDC152-ORF compared with control vector. Transfect CCDC152-ORF and control vector N3-pEGFP into 293T cells, then observe their influence on cellular morphology. As positive autophagy control, use RAPA to treat 293T cells and observe the cellular morphologic changes;(2) Detecting the transition of LC3B I to LC3B II to indicate the occurance of autophagy. Transfect CCDC152-ORF or control vector N3-pEGFP into 293T cells, as positive control, use CQ to deal with 293T cells. Then detect LC3B protein expression by WB;(3) Over-express CCDC152 then observe the amount of autolysosome. Transfect CCDC152-ORF or control vector N3-pEGFP into 293T?Panc-1 or HepG2 cells, then observe the autolysosome and record the amount of autolysosome by inverse fluorescent microscope.4. To study on the mechanism of autophagy induced by CCDC152:(1) Detect the autophagy-related genes that altered by CCDC152 with qPCR. Transfect CCDC152-ORF or control vector N3-pEGFP into 293T cells, Extract mRNA and reverse transcribed into cDNA as a template, then detect the expression of 84 autophagy genes;(2) Validate the expression of autophagy related genes in cells. Transfected CCDC152 or control vector by WB. Transfect CCDC152-ORF or control vector N3-pEGFP into 293T cells, then detect the expression of ULK1?Beclin1?GABARAP? ATG7?LC3A and PI3K proteins separately.5. Research the intracellular localization of CCDC152:Transfect CCDC152-ORF or its control vector PcDNA3.1 into 293T cells, then tested the positioning relationship between CCDC152-Red and Cajal body, PML body in nucleus by immunofluorescence.6. The transcriptomic sequencing-in-depth study of CCDC152 function:(1) Transfect CCDC152-ORF or its control vector N3-pEGFP into 293T, and extracted RNA for transcriptomic sequencing;(2) Test the alterations VCL and the F-actin in 293 T cells which over-expressed CCDC152 by immunofluorescence;(3) Test the expression of Caspase9 in CCDC152 over-expressed-cells by WB;(4)Test the modification of histone H3 at different lysine in CCDC152 over-expressed cells with H3 histone modification kit.Results:1. Red fluorescent fusion protein CCDC152-Red localized in the nucleus in 7 different celllines. The green fluorescent fusion protein CCDC152-GFP and CCDC152-Red are co-localized in 293T cells. These results indicated that CCDC152 is a homo-dimerized transcription factor;2. Luciferase reporter gene assay indicateds that over-expression of CCDC152 can obviously increase luciferase activity compared with the control group. In addition, luciferase activity was enhanced in turn along with increasing amount of CCDC152-ORF which transfected into'293T cells. This result showed that CCDC152 can promote the expression of its own promoter. It is also suggested that CCDC152 is a transcription factor;3. CCDC152 expression is increased to the highest level at 15min after amino acids starvation, while the expression of ATF4, which is now considered cognate GCN4 in yeast, reached the highest level at 30 min. It indicated CCDC152 responsed more earlier than ATF4 when facing amino acids deficiency;4. Amino acids starvation causes endogenous CCDC152 translocation from the cytoplasm to nucleus;5. Over-express CCDC152 could down regulate the expression of p62 protein. This was similar with GCN4 in yeast;6.293T cells transfected with CCDC152-ORF characterized morphologically s hrinkage compared with control. This is similar to positive control cells treated with RAPA;7. Over-expression of CCDC152, the ratio of membrance type LC3B? to plasma type LC3B? is significantly higher than the control and positive control. It shows that CCDC152 can induce cell autophagy;8. Over-expression of CCDC152 in 293T?Panc-1 or HepG2 cells, green fluorescent marker-autolysosomes are all increaseed significantly compared with control. The autophagy rate in these kinds of cells are 41.6%?45.2% and 56.5%, respectively, the rate of is about 10%;9. Over-expression of CCDC152 can up regulate 29 autophagy related genes such as ULK1?Beclin1?GABARAP?ATG7?LC3A, while down regulate 7 autophagy related genes, such as FARP1;10. WB verified the results of qPCR indicated that over-express CCDC152 can up regulate the expression of ULK1?Beclin1?GABARAP?ATG7?LC3A and PI3K proteins;11. In the nucleus, CCDC152 is co-localized with Cajal body and PML body;12. The transcriptomic sequencing showed that over expression of CCDC152 down-regulated GPCR and downstream signalling pathways;13. Over expression of CCDC152 significantly reduced the number of the VCL inside the cell, and affect the formation of focal adhension (FA);14. Over expression of CCDC152 can cause apoptosis in 293T and HepG2 cells, but not in Panc-1 cells;15. Over expression of CCDC152 could cause significantly methylation of histone H3 at different locus;Conclusion:CCDC152 may be as a transcription factors to participate in cell metabolism, via the methylation of histone H3 and regulation of gene transcription.