Date of Award
5-2009
Document Type
Thesis
Degree Name
Master of Science (MS)
College/School
College of Science and Mathematics
Department/Program
Biology
Thesis Sponsor/Dissertation Chair/Project Chair
Carlos A. Molina
Committee Member
Johannes Schelvis
Committee Member
Kirsten Monsen
Abstract
Inducible cAMP Early Repressor (ICER) is a regulator of cAMP signaling in the cell. ICER can be found in normal cells, but it is not present in tumor cells. The growth of these tumor cells is hampered when ICER protein is artificially reintroduced in these cells. Therefore, it is hypothesized that ICER manipulation could potentially be used as a new therapy for the treatment of cancer. Although much is known about the physiological role of ICER, little is known about the regulation of the gene. In this study, the negative autoregulation of ICER by binding to its own regulatory sequences, is being examined.
ICER is an iso form o f the cyclic AMP Responsive Element Modulator (CREM), binding to regulatory sequences on the promoter of specific genes. These regulatory DNA sequences are termed cAMP Responsive elements (CRE). The ICER promoter has been shown to contain four of these CRE sites, termed cAMP autoregulatory elements (CARE) 1 -4, whereas most other promoters only contain one. Therefore, it was the goal of this research to determine if the multiple CARE sites act independently of each other when binding ICER, or if some form of cooperative regulation occurs between two or more of the CARE sites.
Oligonucleotides that represent CARE1, CARE2, and CARE1&2 with fluorescent and quencher molecules attached were obtained to monitor the binding of ICER by using fluorescence resonance energy transfer (FRET). An electro-mobility shift assay (EMSA) was also performed to verify that the binding effects seen in the FRET analysis were in fact due to ICER binding to the CARE sites constructs.
The results show that CARE 1 and CARE 2 follow the pattern of independent binding of ICER with binding constants of 0.12 ± 0.04 uM and 0.16 ± 0.03 uM, respectively. The construct for CARE1&2 clearly shows the characteristics of cooperative binding, rather than independent, and has a binding constant of 0.59 ±0.12 uM and a Hill coefficient for cooperativity of n = 3.6 ± 1.6.
ICER’s binding to the CARE constructs have already provided novel information about its interaction with its promoter and may lead to new information about its role as a tumor suppressor.
File Format
Recommended Citation
Queenan, Craig Stephen, "Binding of ICER to Its Own Promoter as a Mode of Cooperative Regulation" (2009). Theses, Dissertations and Culminating Projects. 1239.
https://digitalcommons.montclair.edu/etd/1239