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Epigenetics is defined as a heritable change in gene expression without introducing any changes or mutations in its nucleotide bases. Now a day's many studies shown deregulation in epigenetic modifications will leads to many deadly human diseases especially cancer. [1,2] In cancer aberrant hypermethylation in the promoter region of tumour suppressor gene will be silenced or the N-terminal end of histone tails are deacetylated at lysine residues which leads to closed chromatin and there by transcription of gene is affected. In case of oncogene the promoter is hypomethylated or the N-terminal end of histone tails are acetylated at lysine residues which leads to chromatin open and the gene is transcribed all the time. The acetylation process is regulated by histone acetyl transferase (HAT) & histone deacetylase (HDAC) and methylation process is regulated by DNA methyl transferase (DNMT). [3, 4, 5]
In cancer cell lines the treatment of histone deacetylase inhibitor (HDACi) results in increased expression epigenetically silenced genes. The combined treatment of HDACi and DMTi will results more positive.  On other hand few genes are equally down-regulated by HDACi treatment. The most of the studies only shown and discuss about up-regulated genes, while the down-regulated genes are often hided or uninvestigated. [7, 8]
Valproic acid (VPA) is one of the histone deacetylase inhibitor. But the molecular mechanism behind VPA is poorly understood. Using chromatin immunoprecipitation and gene specific PCR we analyze the status of H3K4 tri methylation and H3K9 acetylation of six genes (UBE2D3, USP48e20, VPS37A_i1, SRP14, MEIS2, EPHB4_i1) in 2mM VPA treated (12h) and untreated HepG2 cells. We observed acetylation in three genes and deacetylation in another three genes.
MATERIALS & METHODS
CELL COLLECTION, DNA-PROTEIN CROSS LINKING, CHROMATIN SHEARING
HepG2 cells (approximately 1 x 107 cells) confluent of 50% was used for performing experiment. The media was removed from the flask and the cells were washed with 10ml of PBS (phosphate buffered saline) solution to remove dead cells and media. After 2 min PBS solution was discarded. Trypsin (1ml) was added to the flask and kept in 37oC for 2 min. This treatment detaches the adherent cells from the flask bottom. Once conform the cells were floating, The RPMI media was added to the flask to inhibit trypsinization process (2 min). The media with cells were pipetted out from the flask and poured it in a separate centrifuge tubes. The tubes were centrifuged at 1500 rpm for 3 min to remove media (supernatant). The pellet alone was collected and washed it with PBS (5ml) solution to remove trypsin. The tubes were centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the pellet was suspended with PBS (500Âµl) solution. The PBS solution contain cells was transferred to eppendorf tubes and kept in ice.
The DNA-Protein was cross linked after the samples were treated with 36.5% formaldehyde (13.5Âµl) in a shaker for 10 min. The 1.25M of glycine (57Âµl) was added and kept it in shaker for 5 min to quench the further cross linking process. The tubes were handled in ice (4oC) from this step onwards. The tubes were centrifuged at 1500 rpm for 5 min in 4oC room to remove supernatant. The cross linked cells were washed twice with ice cold PBS (1 ml). The supernatant was discarded by centrifuge the tubes at 1500 rpm for 5 min. The pellet was resuspended up and down with ice cold Lysis buffer L1 (1ml). The supernatant was aspirated by centrifugation (1600 rpm, 5 min). The pellet was treated with ice cold Lysis buffer L2 (1ml) and the above two steps were repeated.
The complete shearing buffer S1 (RT) was prepared in a fresh tube by adding protease inhibitor to it. The prepared complete shearing buffer S1 was added to the tubes with pellet of cells. The cells were resuspended by vortexing. The samples were sonicated to shear the chromatin by kept the tubes in BiorupterÂ® for two run of 10 cycles [30 sec "ON", 30 sec "OFF"] each. The tubes were spin and vortex between each run. The ChIP buffer C1 was prepared by adding protease inhibitor (5Âµl/ml) to it in a fresh tube. The tube contains sheared chromatin was treated with ChIP buffer C1 (200Âµl/800Âµl).
MAGNETIC BEADS PREPARATION & IMMUNOPRECIPITATION
Magnetic beads of 28Âµl were added in to three separate eppendorf tubes. Beads were washed and resuspended twice by adding ChIP buffer C1 in to each tube. After each wash the solution alone was discarded by using 1.5ml of magnetic rack. The ChIP buffer C1 (110Âµl) was added to each tube containing washed beads. Aliquot of 100Âµl of washed beads were transferred in to fresh tubes. The specific antibodies (H3k4me3, H3K9ac, IgG) were added to each tubes in the ratio of 10Âµg of chromatin/1Âµg of ab. The tubes were kept in shaking incubator for 2H at 4oC. After incubation the solution was discarded and diluted sheared chromatin (950Âµl) was added per IP tube (ab bind magnetic beads). The 1% of sheared chromatin (9.5Âµl) was kept separately as input sample. The tubes were kept in shaking incubator for 3H at 4oC. The incubated tubes were washed three times using ice cold ChIP buffer C1 (1ml) for 5 min at 4oC on a rotating wheel (40 rpm). After each wash the solution alone was discarded by using magnetic rack. The magnetic beads was washed with buffer W1 (1ml) and incubated for 5 min at 4oC on a rotating wheel (40 rpm).
DE-CROSS LINKING, DNA ISOLATION & VERIFICATION
Complete DNA isolation buffer (DIB) was prepared by adding Proteinase K with DIB (1Âµl/100Âµl) in a fresh tube. The solution in the IP tubes was aspirated by using magnetic rack. Prepared complete DIB (100Âµl) was added to each IP tube and 90.5Âµl of complete DIB was added to 9.5Âµl of input DNA sample. The beads were resuspended and the suspension was transferred in to fresh separate tubes. Transferred tubes were incubated at 55oC (15 min) and 100oC (15 min) respectively. To get down the liquid present in the lid the tubes were spin down. The tubes were placed in the magnetic rack for 1 min and the supernatant alone was transferred into new labeled tubes.
To analyze the results the DNA was amplified using appropriate primers in semi quantitative PCR and verified by using 2D gel electrophoresis.
RESULTS & DISCUSSION:
HepG2 cells were untreated and treated (12h) with valproic acid (VPA) separately to analyze the effect of VPA on the status of H3K4 tri methylation and H3K9 acetylation. Using ENCODE data, six different loci's (SRP14, UBE2D3, USP48e20, VPS37A_i1, MEIS2, and EPHB4_i1) were selected to study the effect of VPA. Upon, analysis of VPA treated and untreated ChIP data with gene specific PCR, we observed an increase in H3K9 acetylation [Fig 1] in three genes (UBE2D3, USP48e20, VPS37A_i1) and loss of acetylation [Fig 2] (H3K9) on remaining 3 genes (SRP14, MEIS2, EPHB4_i1 genes) with VPA treatment. There was also no change observed in H3K4me3 pattern with any of the six genes.
Figure 1 & 2: Validation of ChIP results using semi quantitative PCR for given six genes. ChIPs were prepared from 2mM VPA treated (12h) and untreated HepG2 cells. Fig 1: Valproic acid treated (VPA [T]) HepG2 cells shows increase in H3k9ac for UBE2D3, USP48e20, VPS37A_i1genes. Fig 2: SRP14, MEIS2, EPHB4_i1 genes were acetylated at H3K9ac in VPA untreated (UT) cells after Valproic acid treatment (VPA [T]) HepG2 cells shows decrease in H3k9ac for same three genes.
Theoretically, treating a cell with HDACi results in hyperacetylation of histones and thereby believed to be upregulation of gene expression. Practical assessment of HDACi treatment on cells here reveals both hyper and hypo acetylation of histones. Hence the actual molecular mechanism behind HDACi is not clear and this could be known better if the cells for study are subjected to HDACi treatment with varying time interval at different concentrations. Also, analyzing the results with still more loci's could give us better understanding of the link between acetylation patterns and HDACi treatment.