Methyl-DNA Binding Protein Regulation and Function
Abstracts
Initial Award Abstract |
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Abnormal DNA methylation (that is an enzymatic modification of the building blocks of DNA) is found in many human cancers including lung and kidney cancers, both of which are much more frequently found in smokers than non-smokers. DNA methylation attracts specific methyl-DNA binding proteins which seem to act as a " molecular switch" to turn off the expression of genes. DNA methylation is one mechanism by which cancer cells switch off the expression of different tumor suppressor genes (genes that regulate growth and maturation in normal cells) thus allowing the cancer cells to escape normal growth control mechanisms. We have found that one of the main methyl-DNA binding proteins responsible for shutting off promoters of tumor suppressor genes, called PCM-1, interacts closely with an important signaling protein (G-kinase) which serves to carry signals from the cell membrane to the nucleus. It appears that G-kinase can inhibit the function of the methyl-DNA binding protein PCM-1. This is the first time that a methyl-DNA binding protein has been found to respond to signals other than those encoded in the methylated DNA. We hypothesize that the functions of PCM-1 which include binding to methylated DNA and preventing gene expression are inhibited by G-kinase and that this inhibition may allow us to reverse the effect of PCM-1 on certain tumor suppressor genes. We plan to study in detail the effect of G-kinase on PCM-1, both in the test tube and in intact cells. We will use kidney cancer cells in which a specific tumor suppressor gene is switched off by DNA methylation to examine the effect of G-kinase and PCM-1 on the expression of this gene. To test our hypothesis, we will lower the cellular content of PCM-1 and/or activate Gkinase to inhibit PCM-1 function in the cells and study the effect on expression of the tumor suppressor gene. Our work should lead to a better understanding of how methyl-DNA binding proteins lead to the silencing of tumor suppressor genes and how this effect may be reversible. Ultimately, this could lead to novel strategies for cancer therapy based on the re-activation of tumor suppressor genes. |
Final Report |
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To identify nuclear targets of G-kinase I, we performed a yeast two-hybrid screen with
G-kinase I(3 as bait. We found that G-kinase I(3, but not G-kinase la, interacted
specifically with TFII-I, an unusual transcriptional regulator which associates with
multiple proteins to modulate both basal and signal-induced transcription. Using purified
recombinant proteins, the interaction was mapped to the N-terminal 93 amino acids of G
kinase I(3, and one of six 95 amino acid repeats found in TFII-1. Treating intact cells with
cGMP analogs strongly enhanced co-immunoprecipitation of G-kinase I(3 and TFII-I, and
induced co-localization of both proteins in the nucleus. G-kinase phophorylated TFII-I in
vitro and in vivo on Sera" and See" outside of the interaction domain. G-kinase
phosphorylation did not alter DNA binding of TFII-I to an initiator element consensus
sequence, and did not modify TFII-I-mediated activation of an initiator element
containing promoter. However, G-kinase strongly enhanced TFII-I transactivation of a
serum response element-containing promoter, and this effect was lost when Sera" and
Ser'41 of TFII-I were mutated. Binding of G-kinase to TFII-I may position the kinase to
phosphorylate and regulate TFII-I and/or factors, which interact with TFII-I at the serum
response element. Further studies will focus on clarifying the exact mechanism involved.
Also, the identification of the amino acids responsible for the G-kinase Ip/TFII-1
interaction will be determined. |
