Faculty Sponsor: Dr. Debbie Thurtle-Schmidt
All of the cells in the human body contain the same DNA. However, multicellular have many different cell types and functions, as different genes are expressed in different cells. Transcription factors, which are proteins that regulate gene expression by binding to DNA, play a critical role in making sure the right genes are turned on and off in specific cells at specific times. In fact, mutations in transcription factors have been linked to many different types of cancers. Despite their significance, little is known about how a transcription factor correctly regulates its target gene and not other nearby genes. To investigate this question, we studied NHR-25, an important transcription factor in the small roundworm C. elegans. To investigate transcription factor activity, one segment of DNA that NHR-25 binds to was deleted using CRISPR-Cas9. A second CRISPR-Cas9 edit was made just outside of an NHR-25 binding domain. mRNA was purified from the mutant and wild-type worms and sequencing libraries were created to measure gene expression by RNA-seq. The data indicates that our deletion in the NHR-25 binding domain caused no change in gene expression, while the deletion outside of the binding domain increased gene expression. This finding has been confirmed by qPCR data. To characterize how NHR-25 binds in the mutant strains of worms, we are optimizing a new protocol to profile transcription factor-DNA interactions called CUT & RUN for C. elegans. Initial results indicate promising recovery for an antibody targeted to a broadly expressed protein.