Role Of Enhancer In Escaping Noncoding Rna Mediated Silencing Biology

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ABSTRACT:

Genomic imprinting is the phenomenon where any one of the inherited alleles is expressed in the progeny. Imprinted domains are found clustered as domains. One of the important imprinted domains is the Kcnq1 domain in which imprinting control region regulates imprinting. Kcnq1 domain is located on mouse distal chromosome 7. A feature to be noted about this domain is that there are few genes present in between imprinted genes that escape imprinting. This lead to our study to find the mechanism by which these genes escape the RNA mediated silencing. Our prediction is that enhancers present in adjacent regions to this domain may impart their role in the evading mechanism. Previously published works helped us by providing the data about enhancer specific chromatin marks H3K27ac are present surrounding these bi-allelic expressed genes. We performed ChIP followed by qPCR to look for the enhancer specific histone modification and validated the enhancers presence in mouse placenta cells. Further to test for the function of enhancers on non imprinting genes, enhancer fragments cloning using luciferase bases vector must be carried out.

INTRODUCTION:

Mammalian imprinting regions found in clusters share their cis-acting elements including imprinting control region. Antisense RNA transcript 91 kb long has its crucial role in bidirectional silencing of the genes present in the Kcnq1 domain (Mancini-Dinardo et al, 2006, Pandey et al, 2008). An imprinting control region present in the domain aids the silencing by its differentially methylation pattern (Fitzpatrick et al, 2002).

Thakur et al, 2004 added that Kcnq1ot1 gene transcript regulated by unmethylated ICR is important in silencing of other genes. The ICR of Kcnq1 domain is methylated in maternal chromosome but unmethylated in paternal chromosome. This leads to the expression of antisense kcnq1ot1 RNA in paternal chromosome which in turn silences Ipl, Slc22a11, Cdkn1c, Kcnq1, Tssc4 and Ascl2 genes. Not all genes in this cluster are imprinted. The silencing pattern varies in different tissues as shown by Lewis et al, 2004 that in placenta cells the genes present near ICR are imprinted in placenta as well as embryo whereas the genes away from the control region are imprinted only in placenta. There are some genes like nap114 and cars 1 that escape the silencing mechanism (Pandey et al., 2008).

Enhancers are found in the adjacent regions to the imprinted clusters. Enhancers play a wide role in the regulation of Igf2 and H19 genes and this function is not restricted to these genes alone (Sasaki et al., 2000, Kandhuri et al., 2002). So we based our study towards the enhancers present surrounding the non imprinted genes in mouse cells. Based on the previous said data that enhancer specific acetylation and methylations marks are seen in the cluster (Mikkelsen et al, 2010), Chromatin Immuno Precipitation assay followed by Quantitative PCR was carried out to substantiate the enhancers presence.

MATERIALS AND METHODS:Culturing of MEFs:

Frozen MEF stock was initially immersed in 370C water bath for 3 minutes. The cells were transferred into centrifuge tube (15ml) and DMSO was added. The suspension was spinned for 5 minutes at 1500 rpm. The pellets were pipette out in to another 15m centrifuge tube using the MEF culture media after the previous aqueous phase was thrown. Pellets were diluted using MEM and spread evenly in ……..flask. Microscopic observation was done for the verification of uniform distribution of cells. Flask was incubated for and monitored for cell density. After 96 hours of incubation the cells remain attached to the surface. The media was aspirated and the flask was washed with PBS and aspirated. About 3 ml of trypsin-EDTA solution was added and kept for 5 minutes. Single cell suspension of cultured MEFs was prepared using PBS 1X.

Placenta cells:

…….female was cross linked with….to obtain embryo and placenta. Placenta cells were dissected out from 13.5 days dpc and grinded using PBS 1X buffer. The grinded cells were filtered using 40µm sized filter and made up to 20 ml using PBS to obtain single cell suspension.

Chromatin Immuno Precipitation Assay:

ChIP was performed as described by (Kandhuri et al., 2006).1%formaldehyde solution (270µl/10 ml cells) was added to the single cell suspensions prepared and incubated at room temperature for 10 minutes in shaker (50rpm) allowing the cross linking of chromatin and proteins. 2.5M glycine (68µl/ml cells) was added and kept for 5 minutes incubation in shaker to stop further cross linking. The cells were harvested after centrifugation at 1800 rpm for 5 minutes and washed twice using 10 ml cold PBS. Cells were resuspended in 500 µl ice cold cell lysis buffer for 5 minutes and spinned at 2500 rpm for 10 minutes. Obtained nuclei were resuspended in 3-4 ml lysis buffer. Transfer 1.0 ml into falcon tube and sonication was carried out (30 sec ON and 30 sec OFF cycle, power 24).MEF suspension was sonicated for 10 cycles and the placental cells for 30 cycles. Sonicated material was kept for centrifugation at 13000 rpm for 10 minutes and the supernatant was collected.

Input Preparation and DNA Quantification:

50 µl of the supernatant was taken and made up to 500 µl using nuclease free water. 20µl of 5M NaCl and 2 µl proteinase K were added and kept for incubation at 65°C overnight. Next day RNAse treatment was done for an hour at 37°C. Then DNA purification was done using Phenol: Chloroform method. DNA pellet obtained was washed using 80% alcohol. Quantification was done by nanodrop method. Gel electrophoresis was run to check the fragments size.

30 µg of cross linked chromatin for each antibody IgG, H3K4me1 and H3K27 act was taken and the volumes were made up to 550 µl using ChIP lysis buffer. Protein A sepharose beads saturated with bead buffer was added to pre-clear the sonicated chromatin keeping in shaker (250 RPM at 4°C) for 3 hours. Supernatant collected after centrifugation (2600 rpm for 1 minute) was incubated overnight with 2µg of antibodies raised against IgG, H3K4me1 and H3K27 on a shaker in low speed at 4°C. The next day after centrifugation (13000 rpm for 5 minutes at 4°C) 50µl of protein A sepharose beads was added to the supernatant and incubated at 4°C in a shaker(250 rpm) for 1.5 hours. Chromatin-antibody complex obtained after centrifugation at 2600 rpm for 1 minute at 4°C were washed using low salt buffer and high salt buffer. The beads were again washed twice using LiCl (beads were transferred to another tube before second wash). The beads were finally washed twice using TE buffer. The chromatin-antibody complex was eluted by adding 250µl elution buffer and incubating at 65°C for 10 minutes. To the eluent 20µl of 5M NaCl was added and incubated for 4 hours at 65°C to reverse the cross linking. 20µl of Tris pH.6.5, 10µl of 0.5M EDTA and 2.0 µl of proteinase K were added and incubated at 37°C overnight. Next day DNA was purified by phenol-chloroform extraction method and dissolved in TE buffer.

Quantitative PCR:

Quantitative PCR was performed using immuno-purified ChIP material in ……….system with SYBR green mix. PCR mix contained 7.5µl SYBR green, 4.5µl water, 0.15 µl dye (CxR), 0.5 µl forward primer , 0.5 µl reverse primer and 2 µl template DNA. The qPCR machine was programmed as:

Primer Designing:

Primer designing was done using UCSC genome browser and Primer 3 software. ChIP 12 was used as negative control.

Primers:

The primer sequences for

CD1 enhancer down 1: Forward primer:

Reverse Primer:

R74862 enhancer:




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