Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall A
Jalen Wilcher - Delaware State University
Co-Author(s): Holly Miller, Delaware State University, Dover, DE
The CD44 gene encodes a cell membrane receptor which undergoes proteolytic cleavage within the cell membrane to generate a 74 residues peptide known as the CD44 intracytoplasmic domain (CD44-ICD). This peptide can be translocated into the nucleus where it has the ability to regulate transcription (1,2). This transcriptional regulatory mechanism is not well understood but published chromatin immunoprecipitation (ChIP) data demonstrated that a GFP-tagged CD44-ICD is present in a complex with Runx2 in the MMP-9 gene promoter region (3). We hypothesize that non-tagged wild-type (wt) CD44-ICD, similar to the GFP-tagged CD44-ICD, interacts with Runx2 in the MMP-9 gene promoter region. To test this hypothesis, we carried out ChIP assays treating MCF-7/CD44 cells with formaldehyde (a protein-DNA crosslinker) and DSG (a protein-protein crosslinker). The ChIP DNA was first amplified in its totality using a whole genome amplification kit (Sigma) and then PCR-amplified with primers flanking the CD44-ICD response element (CIRE) in the promoter region of the MMP-9 gene. The PCR results were not conclusive since the expected band is not consistently present or multiple non-specific bands were generated. We have concluded that the crosslinking process in the ChIP assay might be affecting the availability of the CD44-ICD epitope since in Proximity Ligation Assays (PLA) in which no crosslinkers are used, we were able to detect the hypothesized wt CD44-ICD/Runx2 protein-protein interaction in the same cell line using the same anti-CD44-ICD antibody. Additional ChIP assays are in progress in which DSG is not included.
Funder Acknowledgement(s): This project was supported in part by the Delaware INBRE program, with a grant from the NIH National Institute of General Medical Sciences ??NIGMS (P20 GM103446) and the state of Delaware, the National Science Foundation HBCU-UP Research Initiation Award Grant No. 1700228 (K.M.) and the Delaware Economic Development Office Grant No. 103 (K.M.).
Faculty Advisor: Karl Miletti, firstname.lastname@example.org
Role: The entire project