Presentation Title

Activation Capabilities of SETD5 Truncated Variants

Presenter's Name(s)

Nathan Patrick EhrenreichFollow

Abstract

The SETD5 gene is a functionally uncharacterized gene indicated in chromatin regulation and the development of autism spectrum disorders (ASD). Previous papers have indicated that the SETD5 protein has downstream effects on a variety of genes indicated in neuronal signaling and synaptic function (NS/SF genes), such as EGR2, FOSB, and NR4A1. Additionally, the mutations that influence SETD5 function were found to largely be nonsense mutations and splice site deletions, rather than point mutations. The six identified nonsense mutations used in this study are S1258, R1001, E720, T552, K399, and R308. The purpose of this project is to understand the function of SETD5 in an attempt to shed light on the link between SETD5 and ASD. We will first perform a Gal4 assay in HEK293 T-REx cells to determine whether SETD5 is a transcriptional activator or repressor in reference to the NS/SF genes. Next, we will assay NS/SF gene expression in primary cortical neurons using PCR-generated SETD5 truncations, as well as a dCas9 knockout using a KRAB repressor. Coupling these assays with qRT-PR will allow us to determine how much NS/SF mRNA is produced in the presence of the SETD5 wild-type protein, each of the SETD5 truncations, and the SETD5 knockout. Finally, we will analyze the recovered NS/SF RNA values per truncation to determine where the most important domain within the SETD5 gene might be located.

Primary Faculty Mentor Name

James Stafford

Status

Undergraduate

Student College

College of Agriculture and Life Sciences

Program/Major

Molecular Genetics

Primary Research Category

Biological Sciences

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Activation Capabilities of SETD5 Truncated Variants

The SETD5 gene is a functionally uncharacterized gene indicated in chromatin regulation and the development of autism spectrum disorders (ASD). Previous papers have indicated that the SETD5 protein has downstream effects on a variety of genes indicated in neuronal signaling and synaptic function (NS/SF genes), such as EGR2, FOSB, and NR4A1. Additionally, the mutations that influence SETD5 function were found to largely be nonsense mutations and splice site deletions, rather than point mutations. The six identified nonsense mutations used in this study are S1258, R1001, E720, T552, K399, and R308. The purpose of this project is to understand the function of SETD5 in an attempt to shed light on the link between SETD5 and ASD. We will first perform a Gal4 assay in HEK293 T-REx cells to determine whether SETD5 is a transcriptional activator or repressor in reference to the NS/SF genes. Next, we will assay NS/SF gene expression in primary cortical neurons using PCR-generated SETD5 truncations, as well as a dCas9 knockout using a KRAB repressor. Coupling these assays with qRT-PR will allow us to determine how much NS/SF mRNA is produced in the presence of the SETD5 wild-type protein, each of the SETD5 truncations, and the SETD5 knockout. Finally, we will analyze the recovered NS/SF RNA values per truncation to determine where the most important domain within the SETD5 gene might be located.