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Epigenetic is the study of modification in the gene expression without any change in the DNA sequence.DNA methylation and the histone modification is the one of the process in epigenetic. Valproic acid is used to treated with HepG2 marked as treated cell and untreated cell and it mainly used to modify the histone acetylation in this cell. It inactive the gene from the post translation level so the gene is silence. ChIP analysis is used to identify the histone acetylation level in gene specifically H3K4me3 and H3K9ac1.Semi-quantitative PCR is used to amplified the chromatin sample to identify the modification level. Finally there given gene is contain same level of acetylation is occurred in the gene.
Gene expression level is modified without any changes in DNA sequence is only changes in the histone level otherwise called epigenetic .DNA hyper methylation in CpG Islands, Histone Code, Genomic Imprinting, and Chromatin this all are play the major role in gene expression. Histone acetyl transferase (HAT) and Histone deacetylase (HDAC) is mainly used in acetylation of histone study. Immunoprecipitation (chip) and microarray hybridization ChIP-chip method otherwise called microarray hybridization method is very useful for analysing the acetylation prototype of DNA bound histone. (Claes W .et.al. 2007)
Valproic acid otherwise called 2-propylvaleric acid (Dowdell KC ,et.al 2009) first isolated in 1882 and it also used for cancer treatment. Post- translational histone alterative changes play a major role in gene regulation and the expression of gene. Transcription commencement and histone acetylation like Histone H3 lysine 4 methylation (H3K4) and Histone H3 lysine 9 methylation is act the major role in gene silencing. (Emily W.Y., Louise M 2010). Histone deacetylase inhibitor (HDACIs) promotes hyper acetylation in histone change chromatin structure and expression level of the gene is also affected. This histone changes is affect or cause the disease like cancer, cell cycle changes etc., due to the modification of histone marker or in the histone tail .ChIP is used to study the histone mark changes and interaction between DNA -Protein.
Human hepatocellular liver carcinoma cell line (HepG2) were used to study the modification of histone and histone acetylation and methylation changes in the gene expression level with the help of Chromatin immunoprecipitation (ChIP) method. Here we considered gene are SRP14, MEIS2, UBE2D3, VPS37A_i1, EPHB4_i1 and USP48e20.HepG2 cells are grown in two different flask named as treated and untreated cells, treated with VPA for 12 hours cell. Histone H3 trimethylated at lysine 4 (H3K4me3) and histone H3 monoacetylation at lysine 4(H3K9ac1) modification changes observed by specific antibodies are used. Semi quantitative PCR was using to amplify from the shear chromatin with the help of specified design forward and reverses primer .finally the amplified sample is confirmed by using 2% agarose gel electrophoresis and it use for further study are ChIP-ChIP and microarray methods.
Figure:1 Functional genomics essentials being recognized from the ENCODE. (Wells, J et.al 2004)
Materials and Methods
Cell Culturing and harvesting
Human hepatocellular liver carcinoma cell line (HepG2) developed in RPMI medium contain 10% of fetal bovine serum and incubated with 5% of Co2 in 37°C. Culture was divided into two type treated cell and untreated cell. Treated cell contain 2mM Valproic acid (VPA) incubated for 12 hours long and the VPA was not contain in other cell culture flask otherwise called untreated cells. The medium was removed from the culture flask and the cells were washed with PBS and finally trypsinized the cell by trypsin.
The cell culture medium were centrifuged for 5 min at 1500 rpm in 4°C and removed the supernatant from the tube ,this step continued for twice and the pellet were washed and re-suspended with 500 µl of PBS transfer into separate tube. Formaldehyde was used to cross link the cell with 1% concentration with mild vortexed for 8 min at 37°C. After added of 140mM glycine for 5 min at 37°C, it blocked the cross linkage. Centrifuge the cross linked cell for 5 min at 1500 rpm in 4°C and removed the supernatant from the tube. Lysis buffer is used to lysed the cell and washed with the cold PBS. Pellet were washed with 1ml of cold PBS for twice, ice cold L1- lysis buffer 1ml were added and incubated for 10 min on ice box , centrifuged for 5 min at 1600rpm and removes the supernatant from the tube. After that added of 1ml ice cold L2- lysis buffer and again repeated the same step where used in L1 lysin buffer .shearing buffer S1 [Diagenode] 600µl was added to the pellet and incubated for 10 min in ice. Bioruptor® sonicator was used to shear the chromatin for 30 second ON and same time OFF in 15 cycles. Protease inhibitor 5µl was added with the C1 ChIP buffer 800µl in 3 different tubes. Spin the diluted chromatin for 10 min in 12,000 rpm and stored the supernatant in ice for further use to immunoprecipitation.
Preparation of conjugated antibody
Conjugated antibody coated in the magnetic beads, protein- A added 28µl of to three different tube and marked separately and cold ChIP buffer 100µl was used to wash the tube. The three tube was marked as IgG (Diagenode), H3K4me3 and H3K9ac1 (Abcam) and the concentration of antibody is precise on tube. Added chromatin 10µg and 1µg of antibody and tube was incubated at 4°C for 2 hrs in shaker.
Magnetic immunoprecipitation was contained magnetic beads coated with the antibody. Centrifuged tube was kept on the magnetic rack for minimum of 1min to attach the magnetic beads to the magnetic tray. After the supernatant was leftover and added 950µl of shear chromatin on three tubes and incubated at 4°C for two and half hours. From the total volume 1% of sample was used for input for immunoprecipitation. After the time period of incubation the antibody was washed with cold L1 ChIP buffer for three times and added once with W1 buffer. Incubated the tube for five minutes at 4°C in shaker and finally after that step remove the supernatant from the tube with the help of magnetic rack.
Isolation of DNA
Complete DNA isolation buffer volume of 100µl was added on the final washed tubes for immunoprecipitation and input sample volume is 90.5µl.incubated the tube at two different temperatures in the first time 55°C for 15min and second time 100°C for other 15 min it like continue time without any delay. Finally the tube was stored at low temperature like -20°C for PCR amplification.
Amplification of Quantitative PCR
Semi-quantitative PCR was used for DNA amplification from the isolated sample and the master mixture contain 10% of 10X buffer,4mM dNTP and two different primer forward and reverse design for the specific gene with Taq enzyme total volume prepared for marked tube with amount of 20µl. Labelled the positive and negative control of the sample and the parameter value was just for this specific primer on the PCR thermal cycler for 33 cycles. First and second denaturation time period for template DNA is 95°C for five minutes and 95°C for 30 seconds, annealing is 55°C for 40 seconds and extension for 40seconds at 72°C.finaly the cycler temperature is increase to 72°C for 10 minutes and after finished all cycle the temperature is decrease to 4°C. After the PCR amplified sample was confirmed by 2% of agrose gel electrophoresis at 130V for 20 minutes.
Results and Discussion
The alternation changes occur in DNA by methylation is one of the epigenetic mechanisms is change the gene expression or inactive the gene functions. The DNA sequence is not modify or changes but only the gene function is change due to the Histone acetylation or deactivation and histone methylation method. Chromatin Immunoprecipitation (ChIP) was used to study the changes in histone acetylation and methylation in histone3 tail. It is normally occur in all cell type specifically in human cell. So here Human hepatocellular liver carcinoma cell line (HepG2) was used to study the histone modification on the cell line.HepG2 cell line were grown in RPMI medium with two different flask one flask is treated with VPA fig 1(a) and other is untreated fig 1 (b) (Luciana Dini, et.al., 2003),, the split chromatin were IP used with extract antibodies in opposition to H3K9ac1 and H3K4me3.specific primer was used to the amplified DNA. Histone acetylation was increased in the half part of the gene from the full gene and the other part of the gene is pragmatic reduce acetylation altitude in treated HepG2 cell. Histone3 trimethylation at lysine 4 is the universal methylation prototype in all type of genes.
Figure:1 HepG2 cell were grown in RPMI medium, fig 1(a) untreated treated cell and fig1(b) cell were treat with valproic acid (VPA).
Figure:2 The treated and untreated of HepG2 cell with valproic acid and it effect in Histone H3 lysine 9 is clearly visible in treated cell UBE2D3, USP48e20 and VPS37A_i1.
Figure 3: Level of acetylation is decreased in SRP14, MEIS2 and EPHB4_i1 gene from the HepG2 treated and untreated cell with the valproic acid and the IgG is used for control
Histone acetylation increased in USP48e20, UBE2D3 and VPS37A_i genes (fig2) and the other gene SP14, MEIS2 and EPHB4 methylation prototype of H3K4me3 (fig3) with the untreated cell contain without Valproic acid. Histone Deacetylase Inhibitors treatment is also have the activity of both deactelylation and acetylation in gene expression after the treatment of HDAC. For the detail study Encyclopaedia of DNA Elements (ENCODE) is used to analyse the gene expression and to specify the activity of histone methylation and acetylation of gene. ChIP-ChIP microarray and chip sequencing is used to study the genome level otherwise called long study of gene. It is used to understand the DNA- Protein interaction. CITED1 gene is best example for study the methylation in ENCODE database.