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Valproic Acid, a histone deacetylase inhibitor is an antiepileptic drug used for the treatment of seizures, bipolar disorders and migraine. HDACi usually reactivates the inactive cells by epigenetic modifications, mainly blocking the activity of class I and II HDACs. VPA has the potential to arrest tumor cell growth and regulates the expression of differentiating apoptotic genes and hence is used as an anti-cancer drug. But recent studies show contradictory results that many genes are silenced offlate, when they are exposed to HDACi. Hence to investigate these results we treated HepG2 cells with Valproic Acid and analyzed the pattern of H3K4Me3 and H3K9Ac of the given six genes using Chromatin Immunoprecipitation technique followed by Semi- quantitative PCR.
Epigenetics, a study of the inherited changes in the cellular information independent of the underlying DNA sequences has emerged as a crucial factor in determining the fate of complex diseases, especially cancer cells. Hence determining the association of epigenetic alterations in carcinogenesis is indispensable for the discovery of a suitable therapeutic drug. Epigenetic modifications can be classified into three types namely, DNA methylation, histone modification and nucleosome positioning (6). The N-terminal tails of histone is free to be modified by different enzymes and the type of modification determines the chromatin state, resulting in regulation of gene expression (2).These modifications are dynamically regulated by the activity of two enzymes namely, histone acetyl transferase (HAT) and histone deacetylase (HDAC). Histone deacetylase inhibitors (HDACi), which hinders the activity of HDAC by binding to the catalytic centre of the substrate, has several properties such as exhibiting hyperacetylation, by activating the inactive cells, inducing growth arrest, causes apoptosis and thus is used as a promising drug in the treatment of cancer (4). Valproic acid (2-propylpentanoic acid), a short chained fatty acid is a potent HDAC inhibitor when subjected to high millimolar concentration (in vitro) and is already used to treat epilepsy and bipolar disorders
(1).It has been reported that VPA regulates the intracellular enzyme activity (MAPks, PKC, GSK-3Î²) consequently leading to transcriptional activation (3). Hence to investigate the inhibitory effect of Valproic acid, we analyzed the interaction of proteins with specific genomic regions by assessing the status of H3K4Me3 and H3K9Ac of SRP14, UBE-2D3, USP48, VPS37A, MEIS2, EPHB-i1 genes in the liver cells (with and without treatment of Valproic acid) using chromatin immunoprecipitation (ChIP) technique.
Materials and Methods
HePG2 cells which were grown in RPMI medium was treated with 2mM Valproic Acid (VPA) for 12 hours. The cell morphology was observed under microscope and the cells were collected using trysinisation method. The cell-suspension was washed twice with PBS and we followed the protocol of High Cell # ChIP kit.
Cross linking of DNA-protein
Cross-linking was done by adding 13.5Âµl of 36.5% HCHO per 500Âµl of sample ( final sample-approx 1%) to fix the protein-DNA interactions. The tubes were gently vortexed followed by an incubation for 8 minutes at room temperature. Glycine (57Âµl) to a final concentration of 1.25M was added to the sample to stop the fixation. The tube was gently vortexed for quenching and was incubated for 5 minutes at room temperature. All the following steps were performed on ice at 4°C. Centrifugation was carried out at 1,500 rpm for 5 minutes using a swing-out rotor for gradual deceleration. The supernatant was discarded leaving behind 30 Âµl, by slow and careful decanting. Thus the obtained cross-linked cells are now prepared for chromatin shearing and the DNA pellet was kept undisturbed.
Cell lysis and Sonication
The cells were washed with 1ml of PBS and was resuspended with a pipette. The tube was centrifuged at 1,500 rpm for 5 minutes and the supernatant was removed carefully. The above step was repeated. The supernatant was removed leaving behind 10 to 20Âµl. Ice- cold lysis buffer L1 (1 ml) was added to the DNA pellet and cells were resuspended by pipetting several times followed by a 10 minute incubation with gentle mixing. Centrifugation was carried out at 1,600 rpm for 5 minutes and supernatant was removed carefully without losing DNA pellet. Then ice-cold lysis buffer L2 (1ml) was pipetted into the pellet followed by resuspension and a 10 minute incubation. The contents were centrifuged for 5 minutes at 1,600 rpm and supernatant was discarded without dislarging DNA pellet. Meanwhile, 3Âµl of protease inhibitor was added to the shearing buffer (S1) and was stored at room temperature. Complete shearing buffer S1 (600Âµl) was added to the cells and the contents were vortexed until it was resuspended followed by a 10 minute incubation on ice. The 600Âµl solution was separated equally (200Âµl per tube containing 3.3 million cells approximately) into three tubes labelled as IgG, H3K4Me3 and H3K9Ac.The sample was transferred to TPX- tubes and was subjected to sonication to shear the chromatin for 2 run of 10 cycles (30 sec ON & 30 sec OFF cycle) using Bioruptor. The tube was spinned and vortexed between 2 runs. In the meantime, 5 Âµl of protease inhibitor was added per ml of chip buffer C1. Later, 800 Âµl of chip buffer C1 was added to 3 tubes containing 200 Âµl of sheared chromatin.
To 28Âµl of protein A coated magnetic beads, 100 Âµl of ice-cold chip buffer C1 was added in order to wash the beads with ice-cold chip buffer C1. The tubes were placed in magnetic rack and the supernatant was removed. The beads were again resuspended in 110Âµl of chip buffer C1. the washed beads (100 Âµl) was transferred to a new tube. The immunoprecipitated tubes were incubated by placing it on a shaker for atleast 2 hours at 4°C. Since we have used 23.2Âµg of chromatin, approximately 3Âµl concentration of specific antibody was added to the tube. The diluted chromatin was centrifuged at 12,000 rpm for 10 minutes and the supernatant was collected. The tubes containing antibody- coated beads were placed on the ice- cold magnetic rack for 1 minute and the liquid part was removed. Diluted sheared chromatin (950Âµl) was added
to the pellet of antibody- coated beads (each IP tubes). 1% of diluted sheared chromatin- 9.5Âµl was kept as input sample at 4°C. The tubes were incubated in 4°C at 40 rpm for 2 hours. The
tubes were placed on magnetic rack and the supernatant was removed. Ice- cold chip buffer C1 (1 ml) was added to the tubes and it undergoes 5 minute incubation at 4°C on shaker. The above step was repeated 2 times and the tubes were spinned each time before removal of supernatant. Wash buffer W1 (1 ml) was added to the beads followed by a 5 minute incubation at 4°C on shaker. The tubes were spinned and then was kept on magnetic rack to capture the beads and wash buffer W1 was discarded.
The following steps were performed on both input DNA samples and immunoprecipitated samples simultaneously. DNA isolation buffer (100 Âµl) and 1.2 Âµl of proteinase K was added to the immunoprecipitated DNA sample and the contents were resuspended. DNA isolation buffer (90.5 Âµl) and 1.2 Âµl of proteinase K was added to the input DNA sample which contains 9.5 Âµl of chromatin. Both immunoprecipitated DNA sample and input DNA sample were incubated for 15 minutes at 15°C followed by an incubation for 15 minutes at 100°C. The contents were spinned for two times and the tubes were placed on ice- cold magnetic rack and was left for 1 minute. The supernatant which contains DNA was finally transferred to new tubes for qPCR analysis and was stored at -20°C.
The qPCR cocktail containing 2.5Âµl of 10x buffer, 1Âµl of 10 mM dNTP, 1Âµl of 10pm forward primer and 1Âµl of 10pm reverse primer, 5Âµl of DNA template, 0.15 Âµl of enzyme, 14.35 Âµl sterile water was prepared for each reaction. qPCR was programmed as 95°C for 5 minutes- 1st cycle which was followed by 33 cycles (95°C for 30sec, 55°C for 40sec, 72°C for 40sec, 72°C for 10 sec and finally 4°C -âˆž). The results were analyzed by running the PCR products on a 2% agarose gel electrophoresis.
Results and Discussion
HepG2 cells were treated with Valproic acid (VPA) for 12 hours to determine the inhibitory activity of histone deacetyase inhibitor (HDACi). Chromatin immunoprecipitation (ChIP) assay was performed to determine the changes in specific epigenetic signatures- H3K4Me3 and H3K9Ac, on VPA treated liver cells. Chip analysis was also carried out on HepG2 cells which were not treated with VPA, in order to compare the results. Semi quantitative PCR was performed using the specific primers of the 6 genes (SRP14, UBE-2D3, USP48, VPS37A, MEIS2, EPHB-i1) which were used to ascertain whether the above mentioned genes were bound by the protein of interest.We observed no changes in the methylation pattern (H3K4Me3) of the VPA-treated liver cells when compared with the untreated liver cells. This agrees with the previously published data where H3K4Me3 levels were unaltered after 12 hours of butyrate treatment on HepG2 cells and butyrate which resembles VPA in structure is also a histone deacetylase inhibitor (HDACi) (7).
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
(e) MEIS2 (f) EPHB-i1
Fig-1: Validation of ChIP results and effects of H3K4Me3 and H3K9Ac on valproic acid-treated HepG2 cells and untreated HepG2 cells. Here, Lanes 1,2,3,4 contains the untreated HepG2 cells (U.T) and Lanes 5,6,7,8 contains VPA-treated HepG2 cells (T).
Lane 1- IgG (U.T) Lane 2- H3K4Me3 (U.T) Lane 3- H3K9Ac (U.T) Lane 4- Input (U.T)
Lane 5- IgG (T) Lane 6- H3K4Me3 (T) Lane 7- H3K9Ac (T) Lane 8- Input (T).
HDACi generally activate the silenced genes resulting in an increase in acetylation. The same result was reproduced in a recent study where there was an increase in the acetylation pattern of butyrate treated HepG2 cells (7).Our results correlates with the known data, and we have also observed a clear increase in the acetylation pattern (H3K9Ac) of 3 genes (UBE-2D3, USP48, VPS37A) upon Valproic acid treatment. Interestingly, recent studies have shown that some genes are downregulated by the activity of HDAC inhibitors. Decrease in acetylation, RNA polymerase II elongation and gene expression was observed in genomic regions that is proximally located to transcription start sites (TSS) in HepG2 cells after treatment with HDACi such as butyrate and trichostatin A (TSA) (7).The same result was emphasized by another study which mentions that treatment with VPA and trichostatin A leads to deacetylation of multiple genes, resulting in antagonistic effects in women (8). Similarly, a decrease in acetylation mark (H3K9Ac) was observed in remaining 3 genes (SRP14, MEIS2, EPHB-i1) in our experiment replicating the previously mentioned results. Peart et al., has observed that genes when subjected to treatment with HDACi, initially upregulate but as time progresses, deacetylation takes place and it was also found that more genes are silenced rather than getting activated. We have also checked the level (increase or decrease) of specific histone modification- H3K4Me3 and H3K9Ac of the six genes in ENCODE database and the results of our experiments is in concordance with the ENCODE data. Since several recent studies are contradictory to the earlier published data, the definite mechanism of HDACi should be elucidated which is a plausible drug for anticancer therapy.