Identification of the Ciona intestinalis FoxO DNA Binding Domain and Target Gene Sequences
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Faculty Mentor
Nicholas Grossoehme, Ph.D. and Heather Evans-Anderson, Ph.D.
Abstract
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.
Start Date
24-4-2015 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.
