Suppression of Metallothionein Gene Expression in a Rat Hepatoma Because of Promoter-specific DNA Methylation

Abstract

Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from −225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylatedMT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanningMT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors. Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from −225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylatedMT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanningMT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors. 5-azacytidine DNA methyl transferase metallothionein electrophoretic mobility shift assay glyceraldehyde-3-phosphate dehydrogenase polymerase chain reaction base pair(s) metal regulatory element ligation-mediated Methylation at the 5 position of cytosine is a unique modification of eukaryotic genome that occurs within CpG dinucleotides of some genes (for recent reviews see Refs. 1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar, 2Tajima S. Suetake I. J. Biochem. (Tokyo). 1998; 123: 993-999Crossref PubMed Scopus (60) Google Scholar, 3Jones P. Nat. Genet. 1999; 21: 163-167Crossref PubMed Scopus (2040) Google Scholar). This DNA modification has evolved as an epigenetic mechanism in vertebrates, plants, and fungi but not in yeast, flies, and nematodes. Although CpG dinucleotides are present within the coding region, clusters of this sequence known as CpG islands occur frequently in the promoter regions of some genes. The CpG regions are usually devoid of methylation, but in some cancer cells, these sites appear to be susceptible to methylation. In normal cells, only a few genes on the inactive X chromosome and foreign DNA,e.g. transposable elements and provirus are silenced by this mechanism (4Li E. Beard C. Forster A.C. Bestor T.H. Jaenisch R. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 297-305Crossref PubMed Scopus (85) Google Scholar, 5Jaenisch R. Beard C. Lee J. Marahrens Y. Panning B. Novartis Found. Symp. 1998; 214: 200-232PubMed Google Scholar, 6Walsh C.P. Bestor T.H. Genes Dev. 1998; 13: 26-34Crossref Scopus (348) Google Scholar). Many cancer cells have been shown to exhibit global hypomethylation of DNA compared with control cells (7Feinberg A.P. Gehrke C.W. Kuo K.C. Ehrlich M. ‘ Res. 1988; 48: 1159-1161Google Scholar, 8Gama-Sosa M.A. Slagel V.A. Trewyn R.W. Oxenhandler R. Kuo K.C. Gehrke C.W. Ehrlich M. Nucleic Acids Res. 1983; 11: 6883-6894Crossref PubMed Scopus (688) Google Scholar), which is consistent with the oncogenic potential of cells following treatment with 5-azacytidine (5-AzaC),1 a demethylating agent (9Santi D.V. Garrett C.E. Barr P.J. Cell. 1983; 33: 9-10Abstract Full Text PDF PubMed Scopus (340) Google Scholar). On the contrary, the promoters of some tumor suppressor genes are methylated, resulting in their inactivation (1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar). Mutation in the coding region of the tumor suppressor genes has been generally considered the major mechanism of inactivation of the tumor suppressor genes. Recently, aberrant DNA methylation of the CpG islands in the promoter region has emerged as an alternative mechanism for the silencing of these growth regulatory genes and may be one of the earliest events in the neoplastic transformation of cells (10Toyota M. Ho C. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8681-8686Crossref PubMed Scopus (2089) Google Scholar). Indeed, silencing of the tumor suppressor genes (e.g. p16, RB, e-cad, ER, VHL, APC, p53,WT1, and BRCA1) by promoter methylation is known to occur in many tumor types (1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar, 10Toyota M. Ho C. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. Proc. Natl. Acad. Sci. U. S. 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Symp. 1998; 214: Google Scholar). genes can be in in the absence of This that CpG methylation not by these sites to the basal transcriptional of the transcription with the has been in the molecular for the control of metallothionein expression Biochem. 1997; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar, Biochem. Res. 1997; PubMed Scopus Google Scholar, Y. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The of and The first are in all and are in the and the of M. 1997; PubMed Scopus Google Scholar, 33: PubMed Scopus Google Scholar). The of MT-I and in are but can be induced by a variety of agents that include heavy and agents that (for a see Refs. PubMed Scopus Google Scholar, Scholar, Nucleic Acids Res. PubMed Scopus Google Scholar). of in cells can to some of these agents A. 1988; PubMed Scopus Google Scholar, Y. A. M. 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The molecular for the suppression of induction in these cells has not been elucidated. the of expression in the parental cells (e.g. has not been is repression of induction is unique to cancer cells of and repression is in solid in which are the of The present was to this this we the expression of MT-I in a rat hepatoma in response to heavy metals and compared it with that in the of the The data show that the hepatoma is of MT-I following treatment of the rats with it is induced in the host liver. this has elucidated the mechanism for the repression of gene expression in the hepatoma is a tumor with a time of J. Biol. Full Text PDF PubMed Google Scholar). is by a of the solid tumor with a in the of rats The tumor on to within were the this heavy metal the rats were with the of the were a of the and the hepatoma were in for and the were for the of rats bearing the hepatoma were on with the in with of tumor tumor growth was of treatment the rats were with and to the and hepatoma DNA and were as of the on the was this of but the tumor growth was was from the and the hepatoma by P. N. Biochem. PubMed Scopus Google Scholar). of was by and to The was to MT-I N. PubMed Scopus Google Scholar), DNA A. R. J. Biol. 1988; PubMed Scopus Google Scholar), rat glyceraldehyde-3-phosphate dehydrogenase M. J. M. L. C. S. P. P. Nucleic Acids Res. PubMed Scopus Google as in following the The and the hepatoma were by in following the of M. U. Cell. Full Text PDF PubMed Scopus Google and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), The were for in to for assay of and for The were with from the following the of S. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar). in the was to with as The DNA of and were by and of the These were with of in the 5 and of Y. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. M. J. 1993; PubMed Scopus Google Scholar). the was with of the for on of the The reaction was on for and the were by in the reaction at the of the reaction was with of a from of The of the of the in the of for are as (for and (for in the that and were by of hepatoma and from the hepatoma bearing rats as in the In methylation of DNA by and the DNA were following the of P. C.E. 1997; 11: PubMed Scopus Google Scholar). the of control genomic DNA was from and hepatoma by was to the of and B. PubMed Scopus Google Scholar), with as by Full Text Full Text PDF PubMed Scopus Google Scholar). The elements and were one of and one of of the to the and the and the for are by S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Genomic DNA from the hepatoma and the from rats bearing hepatoma was DNA was in for at in of with of and in a at for and for for The DNA was DNA and in of The DNA was in the of at for The was by of to The DNA was as and in of and an was for The metallothionein promoter from to bp was of from the and hepatoma The is as of reaction and of DNA polymerase was in a the following and from the first of was to with a of the with the that the was at of the to the of rat from the DNA and the are S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the DNA was first with the that can only the DNA but not the The sites for these are only are to The of was by of the DNA with DNA was by by the DNA DNA The was sequencing with the and S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). 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The DNA of and were with from of and the were was in the hepatoma in response to this we hepatoma from the with for and the activity The are because their be with an of The was because its was by and the was MTF-1, because it be with of the of the be with of of the by showed that was at of in the hepatoma zinc treatment and These that the lack in the hepatoma is not due to absence of transcription the that the hepatoma MT-I promoter have a that not the to access their we genomic the sequence of the MT-I promoter the for the MT-I MT-I gene for that to and to bp to transcription of the This the of promoter in the hepatoma and zinc from the and hepatoma and treatment with zinc were to genomic DNA was and with and MT-I promoter was and for genomic DNA from and hepatoma was in a as and to the genomic a at a was as that are because of factor in with the control DNA In control footprinting was in the element and compared with the of and in of element in the from control footprinting of was in the as well as of was in control The footprinting in the was by of the in control as compared with footprinting of and were in as well and inducible footprinting at in response to zinc treatment in liver. heavy footprinting zinc treatment was at element in the some were with of factor of of the the element was in with footprinting of and The in of the in the and zinc treatment be with the of MT-I promoter of the footprinting in control was in hepatoma and and zinc treatment of the and This that the were in and hepatoma, MT-I promoter was to in the hepatoma unique footprinting in the hepatoma compared with the in the region from to bp. This be only a to a regulatory element of MT-I promoter in the hepatoma, a and as a factor be These data that MT-I promoter is to the present in the tumor, which is due to in the lack promoter in the hepatoma was due to in the we this region from the hepatoma DNA with of the that was in the tumor not growth suppressor genes in many cancer cells can be suppressed as a result of promoter methylation for we that MT-I promoter in the hepatoma in a a we the rats of tumor the growth of the tumor is with 5-AzaC. This cytosine DNA because of its DNA that in inactivation of the rats with for on the were to and for the and were and was by This was with tumor bearing and a is shown in The hepatoma from the with the cytosine showed significantly higher MT-I level compared with the with with which was zinc treatment compared with normal the host from the rats MT-I level The higher level in the livers of the rats is probably due to the induced by the tumor because is known to expression Y. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, 1999; Google Scholar). The host livers from the and rats MT-I level and was induction of MT-I in the the were with zinc of MT-I in the hepatoma treatment was not because this tumor higher activity and the gene was on as as its promoter was these were with MT-I in the tumor was to a higher level because of of These that hypermethylation of is responsible for the lack of MT-I induction in the tumor heavy metal of the genes that are because of methylation CpG islands in their promoters (1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar, 10Toyota M. Ho C. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8681-8686Crossref PubMed Scopus (2089) Google Scholar). of rat MT-I promoter sequence revealed it has and the CpG to is of genes CpG (1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar). MT-I promoter region from to +1 bp with to transcription CpG base The of CpG methylation of in the hepatoma and the was by genomic which one to the methylation of CpG in a DNA sequence M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S.J. J. M. Nucleic Acids Res. PubMed Scopus Google Scholar). The cytosine in the genomic DNA are to which are and as and The methylated treatment and are as Genomic DNA from and hepatoma were with and for and a of the MT-I promoter to was with gene to to the of the This region of the promoter for CpG base and of the that MT-I expression in response to of the genomic the DNA from was with and the sites of which are not present in this region of promoter and are only of to can be in and the from the and the hepatoma were by and the DNA from the and the hepatoma from were with these and of to the DNA from and hepatoma and treatment was to the and sites of in the region a of the sequence of the DNA from In the all the and were to in the hepatoma, only the cytosine were by the methylation of these CpG of the CpG in cytosine of were in and that the hepatoma these in the MT-I promoter in methylated and of the sequencing data revealed that of the 21 CpG dinucleotides present in promoter −225 and +1 all CpG dinucleotides were methylated in the hepatoma, and the methylation of the CpG in the treatment of the with in the hepatoma, all methylated one were as by their to was in CpG demethylation hepatoma with with of the corresponding cytosine was methylated in the from the animal, and was in in this tissue as is a of the methylation of CpG dinucleotides this These with of the hepatoma from that methylation of CpG islands of MT-I promoter is responsible for silencing of the gene in the probable for the higher level of MT-I promoter methylation in the hepatoma may be the activity of the because of expression of gene. this we first the activity of in the from the tumor and the host liver. This is known to at the 5 position of of CpG base The is to this activity because it not and is an of this activity was higher in the tumor that in the showed a significant in the level of in the hepatoma relative to the host revealed that the level in the hepatoma was at higher that in the liver. These showed that was in the hepatoma and that the of this in the hepatoma probably the hypermethylation of some genes as genes in this The present was to the molecular mechanism by which metallothionein gene is in a rat hepatoma. this is the first that the lack of induction in a tissue is due to hypermethylation of its Recently, we have shown that the suppression of induction in the cells, is due to hypermethylation of its and S. In the present we have shown that of MT-I gene induction in the hepatoma is not due to lack of of the transcription factor but due to methylation of the gene of the gene treatment that methylation was responsible for silencing the which was by genomic is not known only genes are in some cancer cells. Although global hypomethylation of genes can occur transformation of cells, hypermethylation of a few genes as tumor suppressor genes to be in the cells probably as a result of to higher level of DNA S.B. 1997; PubMed Scopus Google Scholar). that the of DNA A. J. Biol. 1998; PubMed Scopus Google and the of DNA S.B. 1997; PubMed Scopus Google may an in the genes that in many activity is significantly higher in the hepatoma. The higher level of that the in this is probably due to enhanced expression of its gene in the hepatoma. of cells following of the of this unique in this Baylin S.B. Issa J.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). these are in gene as a transcription factor and as a factor for a few genes that we can the that of of at to the of the promoter in hepatoma Genomic footprinting of the hepatoma MT-I promoter not a factor that and bp. the of of a at a of the The of to the as revealed by genomic that in the because of promoter hypermethylation is the probable mechanism for the silencing of gene in the hepatoma. This the lack of footprinting in the promoter region because that to CpG base be S. A.P. Cell. 58: Full Text PDF PubMed Scopus Google I. P. A. Cell. Full Text PDF PubMed Scopus Google Scholar). have shown the of at major methyl and that are in repression of transcription from the methylated promoter S. A. Novartis Found. Symp. 1998; 214: Google Scholar). The efficient transcription of many methylated genes in the absence of to the that the CpG methylation by not these sites to the basal transcriptional of the transcription with the is a well that is for P. A. Nat. 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The for M. 1997; PubMed Scopus Google and 33: PubMed Scopus Google are not in the tumor not is that the tumor suppressor p16, and are in the hepatoma not The expression of these genes that tumor suppressor may be in this In this it be that repression of tumor suppressor genes is the of the is potential in some types may in with known tumor suppressor to growth in in cancer cells. Indeed, of MT-I in has been shown to the from induced by 1999; PubMed Scopus Google Scholar). the lack of induction is in cancer cells and not in normal cells at of growth L. S. Res. 1998; Google Scholar, L. B. 1997; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). is to that the lack of expression may the growth of some tumors. is to that silencing of may be to at some highly cells. these are in of Bestor and for MTF-1, and