We study several different aspects of signaling pathways in mammalian cells, using many of the standard tools of molecular biology. We also use novel methods of generating mutant mammalian cells that are defective in signaling. The general objectives are to define mechanisms of signaling and its regulation and to identify hitherto unrecognized components of specific pathways. The major disease targets are cancer and inflammation. We have developed a valuable technique for insertional mutagenesis, in collaboration with Andrei Gudkov's laboratory. The vectors include the strong CMV promoter which, when inserted into the DNA of a cell near a gene, drives over-expression of the encoded protein. This method has very broad applications, including but not limited to the specific systems under investigation in our laboratory. We have used it extensively to identify novel mechanisms of resistance to chemotherapeutic drugs.
Activation of this transcription factor, which drives the expression of anti-apoptotic proteins, is vital for the survival of most cancer cells. We have used insertional mutagenesis to discover that post-translational methylation of specific lysine residues of NFkB is a novel mechanism of regulation of this key transcription factor. The essential modifications are carried out by histone lysine methyl transferases and occur when NFkB is bound to many but not all of the promoters that it activates. This work helps us to uncover aspects of the function of these enzymes as key regulators of both histone and transcription factor functions. More recent work has revealed a novel pathway of NFkB activation in cancers that are driven by mutationally activated growth factor receptors, involving the non-receptor tyrosine kinase FER.
STAT1, which is activated by tyrosine phosphorylation, is the major transcription factor in signaling in response to both major interferon types. The STAT1 gene is a transcriptional target of phospho-STAT1, so that the expression of STAT1 protein increases dramatically in response to interferons. The level of phospho-STAT1 is down-regulated quickly after exposure of cells to interferons, but the increased levels of total STAT1 persist for many days. In its unphosphorylated state, STAT1 is a transcription factor that drives the expression of a subset of the genes originally induced by phospho-STAT1. The encoded proteins prolong many antiviral responses in interferon-treated cells and also, intriguingly, are responsible for a phenotype of resistance to DNA damage in many different types of cancer.
In addition to its role as a transcription factor that is activated by tyrosine phosphorylation in response to several cytokines in the IL-6 family, STAT3 also drives gene expression in its unphosphorylated form. Unphosphorylated STAT3 is often over-expressed in cancer, and many of the genes that are regulated in this way participate in tumorigenesis (for example, Met and MRas). Furthermore, the long-term response to IL-6 relies not only on phospho-STAT3, which is present for a relatively short time, but also on increased expression of unphosphorylated STAT3, which drives a quite distinct set of genes. Similarly to NFkB, the phospho-STAT3 dimers that are bound to certain promoters are modified by lysine methylation through the action of a specific histone lysine methyl transferase, with important mechanistic consequences. We are exploring the hypothesis that mutation or over-expression of some of these lysine methyl transferases, and of the related demethylases, contributes to tumorigenesis not only through effects on histone methylation but also through effects on transcription factor methylation.
Lerner Research Institute
Cleveland Clinic, Mail Code NB21
9500 Euclid Avenue
Cleveland, Ohio 44195
