- Molecular Mechanisms of Pancreatic Morphogenesis and Cancer
- Zinc Finger Transcription Factors
- Transcriptional Repression
- Modular Structure of Transcription Factor Proteins
Significance of Research
The study of the molecular mechanisms of transcriptional regulation is currently one of the most active areas of research in cell and molecular biology. Research in this field has been fuelled by an increased awareness of the crucial role of transcription factors in normal cell physiology and disease. For instance, transcription factors are involved in the regulation of many vital cellular functions including cell growth, differentiation, apoptosis, metabolism, and secretion. In addition, alterations in transcription factors are increasingly being identified as causes of human diseases including cancer. Consequently, the knowledge derived from the studies of these proteins have profound implications in medicine. Our laboratory has pioneered studies on the role of pancreatic zinc finger transcription factors in the regulation of cell growth. These studies are beginning to fill a gap in the existing knowledge by characterizing those pancreatic zinc finger proteins which participate in growth factor-induced cell proliferation, cell cycle arrest, death, and the modulation of neoplastic transformation. The importance of this information is further emphasized by the fact that several therapeutical protocols currently exist which take advantage of controlling abnormal cell growth by interfering with growth factor signaling pathways. Thus, it is likely that zinc finger transcription factors that interfere with neoplastic cell growth will be used in gene transfer experiments for the therapy of cancer.
Role of Novel Sp1-like Zinc Finger Proteins Pancreatic Cell Growth
Sp1-like zinc finger transcription factors are involved in the regulation of cell growth and differentiation in many cell populations. The discovery and functional characterization of the founding member of this family, Sp1, as a GC-rich binding zinc finger protein provided a useful paradigm for understanding mechanisms mediating transcriptional activation in eukaryotic cells. This early paradigm suggested that promoters carrying GC-rich sequences are activated by Sp1 through its interaction with proteins from the basal transcriptional machinery to up-regulate gene expression. Since this seminal work, studies from several laboratories, including ours, have led to the discovery of many Sp1-like transcription factors containing highly homologous DNA binding motifs that bind to similar sequences. Consequently, this knowledge poses many important questions regarding whether these related proteins have similar or antagonistic biochemical and functional properties to Sp1. The goal of our laboratory has been to define the current repertoire and functionally characterize of Sp1-like zinc finger transcription factors in pancreatic epithelial cells. Our studies have led to the identification of novel Sp1-like transcription factors known as TIEG proteins. In addition, we have demonstrated that these proteins share a role in morphogenetic pathways (see below). Together, this information is aimed at better understanding how this growing family of transcription factors work to regulate gene expression and morphogenesis.
- TIEG1 and Epithelial Cell Apoptosis. Members of the TGF? family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGF? peptides is necessary for better understanding normal morphogenesis as well as carcinogenesis of the pancreas. We have reported the cloning, expression, and function in exocrine pancreatic epithelial cells of TIEG1, a TGF?-inducible early gene. This Sp1-like zinc finger transcription factor encoding gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we have shown that the expression of TIEG1 is regulated by TGF?1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG1 in the TGF?-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG1 in linking TGF?-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.
- TIEG2 and Epithelial Cell Growth. Recent experimental evidence demonstrating that mammalian cells express novel, yet uncharacterized, Sp1-like proteins has stimulated a search for novel members of this family. In these studies, we have identified and characterized the nuclear localization, DNA binding activity, and growth inhibitory effects of TIEG2, a novel TGF?-inducible gene structurally and functionally related to TIEG1. TIEG2 is ubiquitously expressed in human tissues, with an enrichment in pancreas and muscle. Sequence analysis of TIEG1 and TIEG2 reveals that these proteins share 91% homology within the zinc finger region and 44% homology within the N-terminus. Biochemical characterization demonstrates that TIEG2 is a nuclear protein which, as predicted from the primary structure, specifically binds to the Sp1 consensus sequence in vitro. In addition, transcriptional regulatory assays have revealed the presence of 3 novel domain within this protein that are required for transcriptional repression. Functional studies using 3H-thymidine uptake and MTS assays demonstrate that the overexpression of TIEG2 in Chinese hamster ovary epithelial cells inhibits cell proliferation. Thus, these studies reveal that TIEG2, together with TIEG1, defines a new subfamily of TGF?-inducible Sp1-like proteins involved in the regulation of cell growth.
Role of Zinc Finger Transcription Factors in the Modulation of Neoplastic Cell Growth
- KRAB-independent Suppression of Neoplastic Cell Growth by the Novel Zinc Finger Transcription Factor KS1. The study of zinc finger proteins has revealed their potential to act as oncogenes or tumor suppressors. We have recently reported the molecular, biochemical, and functional characterization of KS1 (KRAB/zinc finger suppressor protein 1), a novel, ubiquitously expressed zinc finger gene initially isolated from a rat pancreas library. KS1 contains 10 C2H2 zinc fingers, a KRAB-A/B motif, and an ID sequence that has been shown previously to participate in growth factor-regulated gene expression. Northern blot analysis using pancreatic cell lines demonstrates that KS1 mRNA is inducible by serum and EGF, suggesting a role for this gene in cell growth regulation. Biochemical analysis reveals that KS1 is a nuclear protein containing two transcriptional repressor domains, R1 and R2. R1 corresponds to the KRAB-A motif, whereas R2 represents a novel sequence. Transformation assays using NIH3T3 cells demonstrate that KS1 suppresses transformation by the potent oncogenes Ha-ras, G?12, and G?13. Interestingly, deletion of the R1/KRAB-A domain does not modify the transformation suppressive activity of KS1, whereas deletion of R2 abolishes this function. Thus, KS1 is a novel growth factor-inducible zinc finger transcriptional repressor protein with the potential to protect against neoplastic transformation induced by several oncogenes.
- Protein purification
- Gene cloning, sequencing, and site directed mutagenesis
- SDS-PAGE, western blot, northern blot, and Southern blot
- Plasmid and viral-based gene transfer methodology
- Transgenic mouse technology
- Antisense-mediated gene knock-out
- Electrophoretic mobility shift assays (EMSA)
- Reporter and Gal4-based transcriptional assays
- Light, immunofluorescence, video, and electron microscopy
- Computer-based molecular modeling