Discipline: Biological Sciences
Jessica Tolbert - Claflin University
Co-Author(s): Gayathri Raghupathy and Benjamin D. Ortiz, Hunter College CUNY, New York, NY
Locus control regions (LCR) are DNA regulatory elements composed of multiple DNAse I hypersensitive sites (HSs) that have varied functions, which collectively provide integration site-independence to, and spatiotemporally regulate the expression pattern of, a linked gene. We study the T cell receptor alpha (TCR) LCR which contains nine HSs. Recent findings in our lab show that the integration site-independence function of this LCR is mediated by the barrier insulation capacity of HS4 and HS6. Barrier insulators are DNA regulatory elements that prevent the spreading of inactive chromatin into active chromatin regions. Prior results of barrier insulation assays showed that LCR-linked reporter gene-bearing clones maintained reporter gene expression over a 90-day period. We seek to understand the factors that contribute to the barrier activity. Runx1 and RNA polymerase ll are two factors known to bind to the HS6 element. To assess the contributions of these factors to barrier function, we took two distinct loss-of-function approaches to interfere with the activity of these factors in LCR-linked reporter gene bearing T cells. Barrier insulation in vertebrates has not been explored much beyond the one known transcription factor (USF) found to functionally interact with vertebrate barrier elements. The data found shows the possible dosages needed to properly knock down the functionality of RNA polymerase ll phosphorylation. Our work aims to shed light on alternative mechanisms of barrier insulation capacity, and their relation to LCR activity.
Funder Acknowledgement(s): The Hunter College SPUR program is funded by NIH-NIDA (DA032520) and NIH-NIMHD (MD007599) and the Leadership Alliance.
Faculty Advisor: Benjamin D. Ortiz, Ortiz@genectr.hunter.cuny.edu
Role: I participated in bacterial colony prep to extract plasmids using a Midi Prep. I conducted a MTT Assay and Bradford Assay. Additionally, I cut desired plasmids from electrophoresis gel by identifying the size. I also performed a western blot to identify RNA Polymerase ll protein and maintained the cell culture of the mice T cells.