Event Title

Identification of the Ciona intestinalis FoxO DNA Binding Domain and Target Gene Sequences

Presenter Information

Lucas Boncorddo, Winthrop University

Faculty Mentor

Nicholas Grossoehme, Ph.D. and Heather Evans-Anderson, Ph.D.

College

College of Arts and Sciences

Department

Chemistry, Physics and Geology and Department of Biology

Location

DiGiorgio Campus Center, Room 221

Start Date

24-4-2015 1:35 PM

Description

Ciona intestinalis is a useful model for studies examining heart development. FoxO1 is a highly conserved gene as well as an important transcription factor that regulates myocardial development. Previous in vitro experiments in our lab have characterized the interaction between the forkhead DNA binding domain of FoxO from C. intestinalis and target sequences of complementary Human and Drosophila melanogaster DNA. These studies confirmed that the C. intestinalis FoxO DNA Binding Domain (DBD) is able to bind to target sequences from other organisms. Our next step is to determine which regions of the genome FoxO binds to in C. intestinalis. This will help to identify which genes are targeted and regulated by FoxO in C. intestinalis, which will help us to understand the function of FoxO during heart development. We will utilize Chromatin Immunoprecipitation (ChIP)-Seq assay to identify sequences of the Ciona intestinalis genome that FoxO binds to, as well as the potential target genes. A hexahistidine-tagged FoxO DBD sequence was inserted into a pCes vector containing a H2B-cherry fluorescent tag to produce an expression plasmid (FoxO DBD-H). Once confirmed by sequencing, the FoxO DBD-H expression plasmids were electroporated into Ciona intestinalis embryos prior to the first cell cleavage. The ultimate goal of this project is to generate transgenic Ciona intestinalis embryos that express the FoxO DBD-H sequence so that it will interact with chromatin in vivo. The resulting embryos will be collected and their chromatin isolated in order to perform a ChIP-Seq assay that will identify the FoxO1 target DNA sequences.

Comments

Presented at the South Carolina Academy of Science Annual Meeting, April 2015

Supported by a grant from the National Institutes of Health and a South Carolina EPSCoR/IDeA Science Affiliate Network grant

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Apr 24th, 1:35 PM

Identification of the Ciona intestinalis FoxO DNA Binding Domain and Target Gene Sequences

DiGiorgio Campus Center, Room 221

Ciona intestinalis is a useful model for studies examining heart development. FoxO1 is a highly conserved gene as well as an important transcription factor that regulates myocardial development. Previous in vitro experiments in our lab have characterized the interaction between the forkhead DNA binding domain of FoxO from C. intestinalis and target sequences of complementary Human and Drosophila melanogaster DNA. These studies confirmed that the C. intestinalis FoxO DNA Binding Domain (DBD) is able to bind to target sequences from other organisms. Our next step is to determine which regions of the genome FoxO binds to in C. intestinalis. This will help to identify which genes are targeted and regulated by FoxO in C. intestinalis, which will help us to understand the function of FoxO during heart development. We will utilize Chromatin Immunoprecipitation (ChIP)-Seq assay to identify sequences of the Ciona intestinalis genome that FoxO binds to, as well as the potential target genes. A hexahistidine-tagged FoxO DBD sequence was inserted into a pCes vector containing a H2B-cherry fluorescent tag to produce an expression plasmid (FoxO DBD-H). Once confirmed by sequencing, the FoxO DBD-H expression plasmids were electroporated into Ciona intestinalis embryos prior to the first cell cleavage. The ultimate goal of this project is to generate transgenic Ciona intestinalis embryos that express the FoxO DBD-H sequence so that it will interact with chromatin in vivo. The resulting embryos will be collected and their chromatin isolated in order to perform a ChIP-Seq assay that will identify the FoxO1 target DNA sequences.