BACKGROUND: Cbln1 is a gene whose expression is negatively correlated to seizures.
Krishnan et al. has recently shown that seizures synergize with transcriptional co-regulator Ube3a to repress Cbln1 expression, which ultimately manifests as ASD associated behavioral discrepancies. (Krishnan et al., 2017) Seizures increase the expression of REST and the Cbln1 gene contains an intronic RE-1 binding site previously shown to interact with REST. This could therefore be a point of convergence for the transcriptional downregulation of Cbln1.
OBJECTIVE: To determine if Cbln1’s RE-1 sequences confers gene repression to minimal promoter reporter system by REST and Ube3a.
METHODS: Desired RE-1 sequences from Cbln1 were subcloned into a pGL3-basic vector using specific restriction enzymes that flanked each DNA region. Specific oligonucleotide target sequences were annealed together and ligated into the plasmid vector before transfecting into live HEK293T cells. A minimal luciferase promoter with just enough sequence for the polymerase to sit was also ligated into the cassette. A luciferase assay was then conducted on the plated cells under exposure to separate testing conditions such as excess Ube3a, REST, dnREST, and various combinations of these factors to determine the effect of these TFs on gene expression controlled by Cbln1’s RE-1 site.
RESULTS: REST strongly, and Ube3a weakly, repressed the minimal promoter reporter construct when Cbln1’s RE-1 sequences were added. REST occluded the repressive effects produced by Ube3a indicating that their effects are not additive or synergistic.
CONCLUSION: Both REST, that is increased by seizures (Krishnan et al., 2017), and Ube3a (more weakly) can repress gene expression when Cbln1’s RE-1 binding sequences are added. These repressive effects may help explain how seizures and Ube3a can decrease Cbln1 expression that ultimately leads to reduced sociability. / 2019-10-31
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/23772 |
| Date | 12 July 2017 |
| Creators | Cruz, Tristan |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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