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The best studied examples for epigenetic changes are DNA methylation and histone modifications. Valproic acid is HDACi with should triggers epigenetically silenced genes by introducing global histone acetylation. As the previous studies have shown that they are both up regulation and down regulation of acetylation patterns upon drug treatment. We studied the acetylation pattern in HepG2 with or without VPA treatment and the levels of histone acetylation was analysed by using ChIP. The cultured cells with or without VPA are lysed and the particular marks of the histone H3K4me3, H3K9ac1 are pulled down by directing specific antibodies to these modified regions. The sonicated chromatin was amplified by using gene specific primers and the amplification showed the presence of these modifications. Here we found the equal numbers of genes showed increase and decrease acetylation patterns. There were no significant changes in methylation patterns in the given genes.
The heritable changes in the gene expressions that are not caused by altering DNA sequences are termed as epigenetics. The most common example of epigenetic changes are DNA methylation at CpG islands and modification of amino acids in histone tails1. The amino terminal of histone 3 (H3) and histone 4 (H4) tails are methylated and acetylated at lysine residues. This modification plays an important role in unwinding of chromatin and gene expression. The acetylation of histones are mainly regulated by balance activity of histone acetyl transferases (HAT) and histone deacetylase (HDAC)2. HAT leads to hyperacetylation of histones and causes relaxation of the nucleosome thereby enhancing the activity of gene transcription, whereas HDAC leads to condensation of nucleosome and represses the gene activity. Hence acetylation and deacetylation of histone proteins play an important role in the epigenetic regulation3. Thus the knowledge of HDAC and HAT provides deeper understanding in regulation of proteins that controls cell differentiation, transcription and cell death.
Valproic acid (VPA) is a histone deacetylase inhibitor (HDACi) theoretically which should block the activities of HDAC. HDACi are known to change the acetylation status of histone and non-histone proteins and there by altering the gene expression. These changes may be induction of apoptosis, cell cycle arrest, inhibition of angiogenesis and metastasis4. However HDACi drugs treated cells showed equal number of genes up and down regulated2. The changes in histones marks can be analysed using chromatin immunoprecipitation (ChIP) technique. This technique is based on the ability of cross-linker formaldehyde to bind to protein-DNA complexes5.
In this study, the effect of VPA on hepatocarcinoma HepG2 cell lines which changes the histone marks for the given set of genes SRP14, UBE2D3, USP48e20, VPS37A_i1, MEIS2 and EPHB4_i1 were studied using ChIP technique. We analysed the acetylation and methylation patterns of H3 in both un-treated and treated cells with VPA for 12 hrs. Specific antibodies for Histone H3 lysine 4, trimethylated (H3K4me3) and Histone H3 lysine 4, monoacetylation (H3K9ac1) are directed to detect these modifications. The sheared chromatin is amplified by semi-quantitative PCR using gene specific primers and was verified using agarose gel electrophoresis, as the amplification of DNA shows the presence of modified gene.
Materials and Methods
Cell Culturing and harvesting
HepG2 cells were grown in RPMI media supplemented with 10% foetal bovine serum, incubated at 370 C in 5% CO2. One set of culture was treated with 2mM Valproic acid (VPA) for 12 hrs and this was called as treated cells. The other flask was devoid of VPA and is termed as untreated cells. The cells were harvested by trypsination. Briefly, the medium from cultured flask was removed and cells were washed with Phosphate buffer saline (PBS), trypsinized using trypsin and re- suspended in medium.
Cell collection and Chromatin preparation
The medium containing cells were centrifuged at 1500 rpm for 5 minutes, supernatant was discarded and centrifugation step was repeated twice washing the pellets with PBS. The pellets were re-suspended in 500 µl PBS transferred into eppendorf tube. The cells were cross linked using 1% formaldehyde with gentle vortexing for 8 minutes at room temperature (RT). The cross linking was stopped by adding 140mM Glycine for 5 minutes at RT. The cross linked cells were spun down at 1500 rpm for 5 minutes using cooling centrifuge and the supernatant was aspirated6. The cells were lysed by using lysis buffer. Briefly, pellets were washed twice with 1ml cold PBS. Initially 1ml of ice-cold L1 lysis buffer (Diagenode) was added and incubated on ice for 10 minutes, centrifuged at 1600 rpm for 5 minutes and supernatant was discarded. Later 1ml of ice-cold L2 lysis buffer (Diagenode) was added and above steps was repeated. To the pellets 600 µl of shearing buffer S1 (Diagenode) was added and incubated on ice for 10 minutes. The chromatin was sheared by using Bioruptor® Sonicator for 15 cycles with 30 seconds ON and 30 seconds OFF. The sheared chromatin was split in 3 tubes, to each tube 800 µl of C1 ChIP buffer (Diagenode) and 5 µl of protease inhibitor mix was added. The solutions were spun down at 12000rpm for 10 minutes and supernatant was stored at -20°C to carry out IP.
Preparation of magnetic bead conjugated antibodies
Initially 3 separate tubes were taken and labelled appropriately, to each tube 28 µl of Protein A-coated (Diagenode) was added and washed twice with 100 µl of ice cold ChIP buffer. Magnetic rack was used to discard the supernatant. Specific antibodies IgG (Diagenode kit), H3K4me3 and H3K9ac1 (Abcam) were used and to each tube specific antibodies of concentration, for every 10 µg of Chromatin used 1 µg of antibodies was added. The solution was incubated on shaker for 2 hours at 4 0 C.
After incubation, the tubes were briefly centrifuged and placed on magnetic rack for 1 minute. The supernatant was discarded and to each tube 950 µl of sheared chromatin was added and incubated on a rotator for 2.5 hours at 4 0 C. To normalize IP, 1% of total volume was used as input (9.5 µl). After incubation, the solution containing antibodies was washed thrice using 1ml of ice-cold C1 ChIP buffer and once with 1ml of Buffer W1. Washing was done by incubating for 5 minutes on shaker at 4° C and 1 minute on magnetic rack to remove the supernatant.
After final wash, 100µl of complete DNA isolation buffer (DBI) was added to each IP tubes and 90.5µl to input sample. The tubes were incubated at 55° C for 15 minutes followed by 100° C for another 15 minutes. Briefly the tubes were spun down and stored at - 20° C for further PCR analysis.
PCR Amplification and Verification
The isolated DNA was amplified using semi quantitive-PCR and to each tube 20 µl of prepared Master mix was added. The master mix contained 10% of 10X buffer, 10mM dNTP's, 10 pm of forward and reverse primers of specific gene and dream Taq enzyme. The labelling was done based on template DNA and primer used. The negative control which is devoid of template DNA was prepared to check contamination in master mix. The samples were placed in Thermal cycler block, by adjusting following parameters: The cycle was programmed for 33 cycles, with initial denaturation for 10 min, following by denaturation at 95°C for 30 sec, annealing at 55° C for 40 sec and extension at 72° C for 45 sec. After the completion of 33 cycles, the cycler was adjusted to 72° C for 10 min and holding program was at 4° C forever. The presence of amplified DNA was verified by using 2% agarose gel electrophoresis using 1X TAE buffer and electrophoretic conditions was at 130 V for 20 min.
Results and Discussion
ChIP assay was performed to examine the changes of methylation and acetylation patterns in histone (H3) tail. HepG2 cells were cultured with or without VPA and the fragmented chromatin were immunoprecipitated using specific antibodies against H3K4me3 and H3K9ac1. DNA was amplified using gene specific primers. We found that half of the genes showed increase in histone acetylation, whereas in the remaining genes we observed a decrease in acetylation patterns in treated cells. The global methylation pattern of H3K4me3 remained the same in all genes irrespective of VPA treated or un-treated cells. The genes UBE2D3, USP48e20 and VPS37A_i1 showed increase in H3 acetylation (fig 1). In the remaining genes SRP14, MEIS2 and EPHB4_i1 the acetylation patteren was completely abolished in VPA treated cells over the un-treated ones. The methylation pattern of H3K4me3 remained the same (fig 2). IgG is used as non-specific control.
As mentioned in previous report (Rada-Iglesias A et al., 2007), even our studies have shown that HDACi treatment can induce both acetylation and deactelylation in certain genes. Further understanding is required for analysing the specific modifications. As the Chromatin immunoprecipitation (ChIP) following by genomic microarray hybridization (ChIP-chip) or parallel sequencing (ChIP-seq) provides in-depth knowledge in genome-wide identification and characterization of protein-DNA interactions.
Figure 1. Effect of VPA on the acetylation of H3K9 of HepG2 cells cultured with or without VPA. The genes UBE2D3, USP48e20 and VPS37A_i1 showed a clear increase in acetylation pattern over H3K9 in treated cells over un-treated ones. The methylation of H3K4 remained the same in both treated and untreated cells. IgG is used as non-specific control.
Figure 2. Effect of VPA on the acetylation of H3K9 of HepG2 cells cultured with or without VPA. Tthe genes SRP14, MEIS2 and EPHB4_i1 showed a clear decrease in acetylation pattern over H3K9 in treated cells over un-treated ones. The methylation of H3K4 remained the same in both treated and untreated cells. IgG is used as non-specific control.