Establishment of vitamin D receptor chromatin immunoprecipitation in CD8+ T cells to identify new vitamin D receptor binding sites.
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Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder of the central nervous system (CNS). Although, the cause of MS is still unknown, some genetical and environmental risk factor are known to be involved in the progression of MS. One of the environmental risk factors is low levels of vitamin D in serum. Vitamin D has many biological effects, and its action is mediated through its receptor vitamin D receptor (VDR, a nuclear receptor). This complex associates with retinoid X receptor (RXR), by which together will recognize specific DNA sequences, i.e. vitamin D response elements (VDREs), causing the regulation of genes that encode proteins involved in cell proliferation and differentiation of immune cells. CD8+ T cells, is likely to play a role in MS pathogenesis. More than 200 single nucleotide polymorphisms (SNPs) are identified to be associated with MS. We wanted therefore, to analyse if these MS risk variants can influence VDR binding site. But before we do so, the technique used to study binding of VDR to DNA (VDR chromatin immunoprecipitation) as well as activation of CD8+ T cell procedures must be optimized. CD8+ T cells were isolated from blood from healthy donors and were activated using 5 μg/ml anti-CD3 coated plate and 2 μg/ml soluble anti-CD28 antibodies for 40 hours to induce the expression of VDR. After 40 hours, VDR was highly expressed, and the cells were treated with either active form of vitamin D (calcitriol) or EtOH (vehicle control) for 3 hours. To verify the cells responsiveness to vitamin D, the expression of vitamin D responsiveness genes, TAGAP and CYP24A1 were measured. Furthermore, the ChIP procedure that includes (before immunoprecipitation) protein-DNA crosslinking (incubation temperature and time), cell lysis (one step or two step) and sonication to fragment chromatin (fragmentating for 10 or 20 min) was optimized prior to be performed in two healthy donors using anti-VDR to pull down DNA regions that are bound by VDR. Anti-IgG (isotype control) immunoprecipitation was performed as negative control. PCR of regions in the VDR, MYC and TAGAP was used to analyse the DNA fragments purified after ChIP. DNA regions that were pulled down by IgG were the same as VDR. So, given that our findings of ChIP are based on a limited number of experiments, the findings should be confirmed yet again doing more experiments. Therefore, immunoprecipitation part of the ChIP procedure needs to be optimized by including more materials and controls.
Master i biomedisin