Cell Signaling Research

Heintz Bio

Function of E2F in Transcriptional Regulation of S Phase Genes

Our laboratory studies the control of transcription and replication during the eukaryotic cell cycle. A primary focus of the lab is the role of the E2F family of transcription factors in regulating genes required for S phase entry. Recently we have discovered that regulation of E2F-6, a member of the E2F family that functions as a repressor of transcription, is regulated in a redox-dependent manner. We are presently engaged in determining the role of reactive cysteine residues in the E2F-6 dimerization domain in control of E2F-dependent transcription under conditions of oxidative stress. Chromatin binding assays and studies on the subcellular localization of E2F family members indicate that low levels of oxidative stress blocks cell cycle progression by perturbing the temporal events required for expression of cyclin D1, a regulator of the E2F pathway.

Effect of Environmental Agents on Cell Cycle Control

In a related series of studies, we are examining control of cell cycle progression after exposure of cells to environmental agents, such as reactive oxygen and nitrogen species (ROS/RNS). These studies focus on delineating the cell signaling pathways that regulate E2F-dependent gene expression after environmental insults, both in cells in culture and in mice. A primary target of RNS and ROS during cell cycle re-entry appears to be trafficking of transcription factors in and out of the nucleus, a dynamic process that is required for expressing genes required for S phase entry. We also wish to correlate changes in cell cycle progression and induction of apoptsosis with alterations in p53-dependent cell checkpoint mechanisms. Our studies in environmental pathology are pursued in collaboration with Dr. Brooke Mossman and Dr. Yvonne Janssen-Heininger.

Protein-Mediated DNA Looping in Gene Transfer

In a third project, we are refining a novel method for introducing large DNA molecules into cells in culture and in vivo. Using an unusual DNA binding protein that is able to mediate DNA looping, we are able to condense large molecules (<150 kb) into small protein-DNA complexes that are readily internalized by cells in culture. This method allows us to introduce entire genes and their flanking sequences into cells for studies on gene expression, DNA replication, and chromatin organization. Recently, we have used this method to study alterations in chromosome structure after random integration of centromeric DNA and putative origins of replication into mammalian chromosomes. The method is also being explored in mice as a delivery vehicle for gene therapy.

Summary

In summary, our laboratory uses contemporary methods in molecular biology, cell biology, cell imaging and DNA delivery, to assess the role of specific genes, stuctural elements, and gene products in the control of cell growth. Our long term aim is to understand the relationship between changes in the regulation of E2F-dependent transcription and DNA replication in the pathogenesis of human malignancies. Individuals interested in participating in our research efforts should contact Dr. Nicholas Heintz by e-mail at nicholas.heintz@uvm.edu or by phone at 802-656-0372.