Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.
We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.
Konduri
Santhi D
SD
Program of Cancer Biology, University of Illinois, Peoria, IL 61656, USA.
Srivenugopal
Kalkunte S
KS
Yanamandra
Niranjan
N
Dinh
Dzung H
DH
Olivero
William C
WC
Gujrati
Meena
M
Foster
Donald C
DC
Kisiel
Walter
W
Ali-Osman
Francis
F
Kondraganti
Shakuntala
S
Lakka
Sajani S
SS
Rao
Jasti S
JS
D001374:Azacitidine / Q000494:pharmacology; D001932:Brain Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing; D005909:Glioblastoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D005910:Glioma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D051783:Methyl-CpG-Binding Protein 2; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D012097:Repressor Proteins; C021240:tissue-factor-pathway inhibitor 2; C012589:trichostatin A; D004248:DNA (Cytosine-5-)-Methyltransferases; D006655:Histone Deacetylases; D001374:Azacitidine