Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Jalen Wilcher - Delaware State University
Co-Author(s): Holly Miller,Delaware State University, Dover, DE; Karl Miletti-Gonzalez,Delaware State University, Dover, DE
CD44 is a cell membrane receptor which undergoes a proteolytic cleavage within the cell membrane to produce 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. This transcriptional regulatory mechanism is not completely 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 (Miletti-Gonzalez, et al., 2012). We are interested in validating this data by analyzing the wild-type (wt) CD44-ICD peptide and thus we hypothesize that the wt CD44-ICD interacts with Runx2 in the nucleus. To test this hypothesis, we carried out ChIP assays treating the MCF-7/CD44 cells with formaldehyde (a protein-DNA crosslinker) as well as with DSG (a protein-protein crosslinker). The ChIP DNA was 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. 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 CD44-ICD/Runx2 protein-protein interaction in the same cell line using the same anti-CD44-ICD antibody.
Funder Acknowledgement(s): NSF
Faculty Advisor: Dr. Karl Miletti, firstname.lastname@example.org
Role: All of the steps including cell culture maintenance