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Valproic acid is a potential HDAC inhibitor which induces differentiation of transformed cells by reactivating the silenced genes and thus widely used in cancer treatment. HDAC inhibitors relieve the repression of transcription by binding to catalytic centres of HDACs and preventing access to substrates thereby decreases deacetylation. VPA was previously thought to have hyperacetylation activity but recent studies have shown that both increase and decrease of histone deacetylation occurs on different loci. VPA has no effect on histone methylation status. Our study has shown that both upregulation and downregulation of genes occur on VPA treated HepG2 cells.
The studies of inherited changes in gene functions that occur without any changes in the DNA sequence have lead to a novel path in carcinogenesis research. DNA methylation, histone modifications and RNA interference are the important epigenetic mechanisms that make the genes to function differently in tumor cells. Majority of histone modifications occur at the N-terminal end of lysine residues by either methylation or acetylation. Acetylation of histone proteins leads to efficient transcription and maintained by histone acetylases (HAT) and deacetylases (HDAC). There are HDAC inhibitors that act in a specific way and can cause both increase and decrease in expression of genes (Peart et al., 2005). Valproic acid (VPA), an antiepileptic drug is a potential inhibitor that binds to the catalytic centre of the HDACs to prevent the substrate access and inhibits the deacetylation activity thereby effects in the modulation of gene expression and activity (Göttlicher et al., 2001,). In this study we tried to determine the effect of VPA on methylation and acetylation status of six genes SRP14, UBE2D3, USP48, VPS37A, MEIS2 and EPHB4. Using ChIP, the principal technique used to validate the epigenetic marks of individual loci in the genome, we determined whether there was increase or decrease in H3K4me3 and the H3K9ac marks in the above genes when HepG2 liver cells were treated with Valproic acid (VPA).
Materials and Methods:
HepG2 cells grown in RPMI media were use to perform ChIP. Cell count in a flask was 10 million cells. 1 million cells contain 7µg of chromatin. ChIP was performed using HepG2 cells treated with Valproic acid (2mM) for one night and untreated cells. Highcell# ChIP kit was used and the protocol in the manual given with kit was followed.
Cell Harvesting and DNA-protein cross linking by HCHO:
Microscopic observation of HepG2 cell culture was done before cell collection. Cells from flasks were pippeted out into 15ml centrifuge tubes and trypsinisation was done. The cell suspension was washed twice using 5ml of PBS. To the pellet added 500µl of PBS, resuspended with pipette and then transferred to 1.5ml tube. Then 13.5µl of formaldehyde was added allow cross linking of DNA and protein and kept in a spinner (200rpm) at room temperature for 8 min. To stop fixation, 57µl glycine was added and gently vortexed for 5 min. Then cross linked cells was obtained as pellets after centrifugation at 1500rpm for 5 minutes at 4°C.
Cell lysis and chromatin shearing:
The harvested cross linked cells were washed twice using 1ml ice-cold PBS. The pellets were again resuspended and washed separately in 1 ml ice-cold lysis buffers L1 and L2 by incubating at 4°C for 10 min and by gentle mixing. Pellet was separated after spinning the sample at 1600 rpm for 5 minutes at 4°C. Pellet was resuspended in 600µl of shearing buffer S1 with protease inhibitor and divided equally into three 1.5ml tubes (3.3 million cells in each tube). To each tube 800µl of ChIP buffer C1 was added to dilute the sheared chromatin. The diluted sample was spinned at 12000rpm for 10 minutes and supernatant was collected.
Binding Antibodies to magnetic beads:
In three separate 1.5ml tubes, 28 µl of Protein A beads were resuspended and washed using 100 µl of ChIP buffer C1 (1.5 ml magnetic rack was used). The pellet was again resuspended using 110 µl of ChIP buffer C1 from which 100 µl was aliquoted into other tubes. Specific antibodies and control antibodies (from abcam) were added to respective tubes and incubated for atleast 2 hours at 4°C on a rotating wheel at 40rpm.
Magnetic Immunorecipitation and washes:
The tubes with antibody-coated beads were placed on the magnetic rack and supernatant was discarded. 950 µl of the sheared samples were pippeted out into respective antibody containing tubes and incubated for 2 hours at 4°C on a rotating wheel at 40rpm. 9.5 µl of sheared chromatin for input was stored at 4°C. The incubated samples were washed thrice using 1ml ice-cold ChIP buffer C1 by incubating for 5 minutes at 4°C on a rotating wheel at 40rpm during each wash. Final washing was done using 1 ml buffer W1 and beads were captured by removing the buffer W1 after placing the tubes in a magnetic rack.
The steps following were all carried out at room temperature. 100 µl of DIB added into each sample and resuspended. 90.5 µl of DIB was added to the input sample. 1.2 µl of Proteinase K was added to all the tubes and kept for incubation at 100°C for 15 minutes. The tubes were shortly spinned and placed on a magnetic rack to collect the supernatant. The supernatant collected is the required DNA sample. The DNA sample obtained must be stored at -20°C.
Semi-quantitative PCR and Data analysis:
Semi-quantitative PCR was performed using immuno-purified ChIP material. Specific primers for given six genes were used. PCR mix contained a total volume of 25µl (2.5 µl of 10X buffer, 1 µl of 10mM dNTP, 1 µl each of 10pM Forward and Reverse primer, 5 µl template, 0.15 µl enzyme, 14.35 sterile water). PCR was programmed as 5min at 95 for 1 cycle, followed by 33 cycles as 30 sec at 95°C, 40 sec at 55°C, 40 sec at 72°C and finished by 10 min at 72°C and 4°C for ∞. Finally agarose gel electrophoresis was run using the PCR amplified products.
Results and dicussion:
HepG2 liver cells were treated with 2mM Valproic acid for 12 hours to know the activity of valproic acid as HDAC inhibitor (Göttlicher et al., 2001). We performed ChIP analysis using antibodies against H3K4me3 and H3K9ac to determine the degree of histone methylation and acetylation in valproic acid treated and untreated cells. We initially verified the histone methylation and acetylation status of the above genes in ENCODE data of UCSC genome browser on human (NCBI36/hg 18) (march 2006). To verify the ChIP results, semi-quantitative PCR was performed using primers specific for six genes namely SRP14, UBE2D3, USP48, VPS37A, MEIS2 and EPHB4 and agarose gel was run. There was no change in methylation levels in any of the genes and this is similar to the results obtained in previous studies (Rada-Iglesias et al., 2007). The acetylation status varied between treated and untreated cells. VPA has induced Acetylation accumulation in three genes UBE2D3 (fig.1.b), USP48 (fig.1.c), VPS37A (Fig.1.d) and reduced the acetylation level in remaining three genes SRP14 (Fig.1.a), MEIS2 (Fig.1.e) and EPHB4 (fig.1.f) thus leading to both up-regultion and down-regulation of genes.
Fig.1: Validation of ChIP products by semi-quantitative PCR by agarose gel.
Daly and Shirazi-Beechey, 2006 has shown that equal number of genes will be up-regulated and down regulated when treated with HDAC inhibitors. Many recent studies have shown that there is downregulation in genes than being upregulated by HDAC inhibitors (Reid et al., 2005, Rada-Iglesias et al., 2007). . Peart et al., have also shown that genes were activated during initial hours of time on treatment with HDACi but repression of genes were seen with increase in treatment. This altered activity may be due to several other multiple mechanisms and factors influencing transcription (Reid et al., 2005). Thus our work has shown that HDAC inhibitor Valproic acid has both activation and repression activity on different loci. Wider studies must be undergone to know the exact mechanisms of the HDAC inhibitors and to use them as the therapeutic agents against cancer and in clinical development.