LOT1 (PLAGL1/ZAC1), the Candidate Tumor Suppressor Gene at Chromosome 6q24–25, Is Epigenetically Regulated in Cancer

Abstract

LOT1 is a zinc-finger nuclear transcription factor, which possesses anti-proliferative effects and is frequently silenced in ovarian and breast cancer cells. TheLOT1 gene is localized at chromosome 6q24–25, a chromosomal region maternally imprinted and linked to growth retardation in several organs and progression of disease states such as transient neonatal diabetes mellitus. Toward understanding the molecular mechanism underlying the loss of LOT1 expression in cancer, we have characterized the genomic structure and analyzed its epigenetic regulation. Genome mapping of LOT1 in comparison with the other splice variants, namely ZAC1 andPLAGL1, revealed that its mRNA (∼4.7 kb; GenBankTM accession number U72621) is potentially spliced using six exons spanning at least 70 kb of the human genome. 5′-RACE (rapid amplification of cDNA ends) data indicate the presence of at least two transcription start sites. We found thatin vitro methylation of the LOT1 promoter causes a significant loss in its ability to drive luciferase transcription. To determine the nature of in vivomethylation of LOT1, we used bisulfite-sequencing strategies on genomic DNA. We show that in the ovarian and breast cancer cell lines and/or tumors the 5′-CpG island of LOT1is a differentially methylated region. In these cell lines the ratio of methylated to unmethylated CpG dinucleotides in this region ranged from 31 to 99% and the ovarian tumors have relatively higher cytosine methylation than normal tissues. Furthermore, we show that trichostatin A, a specific inhibitor of histone deacetylase, relieves transcriptional silencing of LOT1 mRNA in malignantly transformed cells. It appears that, unlike DNA methylation, histone deacetylation does not target the promoter, and rather it is indirect and may be elicited by a mechanism upstream of the LOT1regulatory pathway. Taken together, the data suggest that expression ofLOT1 is under the control of two epigenetic modifications and that, in the absence of loss of heterozygosity, the biallelic (two-hit) or maximal silencing of LOT1 requires both processes. LOT1 is a zinc-finger nuclear transcription factor, which possesses anti-proliferative effects and is frequently silenced in ovarian and breast cancer cells. TheLOT1 gene is localized at chromosome 6q24–25, a chromosomal region maternally imprinted and linked to growth retardation in several organs and progression of disease states such as transient neonatal diabetes mellitus. Toward understanding the molecular mechanism underlying the loss of LOT1 expression in cancer, we have characterized the genomic structure and analyzed its epigenetic regulation. Genome mapping of LOT1 in comparison with the other splice variants, namely ZAC1 andPLAGL1, revealed that its mRNA (∼4.7 kb; GenBankTM accession number U72621) is potentially spliced using six exons spanning at least 70 kb of the human genome. 5′-RACE (rapid amplification of cDNA ends) data indicate the presence of at least two transcription start sites. We found thatin vitro methylation of the LOT1 promoter causes a significant loss in its ability to drive luciferase transcription. To determine the nature of in vivomethylation of LOT1, we used bisulfite-sequencing strategies on genomic DNA. We show that in the ovarian and breast cancer cell lines and/or tumors the 5′-CpG island of LOT1is a differentially methylated region. In these cell lines the ratio of methylated to unmethylated CpG dinucleotides in this region ranged from 31 to 99% and the ovarian tumors have relatively higher cytosine methylation than normal tissues. Furthermore, we show that trichostatin A, a specific inhibitor of histone deacetylase, relieves transcriptional silencing of LOT1 mRNA in malignantly transformed cells. It appears that, unlike DNA methylation, histone deacetylation does not target the promoter, and rather it is indirect and may be elicited by a mechanism upstream of the LOT1regulatory pathway. Taken together, the data suggest that expression ofLOT1 is under the control of two epigenetic modifications and that, in the absence of loss of heterozygosity, the biallelic (two-hit) or maximal silencing of LOT1 requires both processes. LOT1 (lost-on-transformation 1) 1The abbreviations used are: LOT1, lost-on-transformation 1; TSA, trichostatin A; ROSE, rat ovarian surface epithelial; RACE, rapid amplification of cDNA ends; UTR, untranslated region; FACS, fluorescence-activated cell sorter; GFP, green fluorescence protein; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; TSS, transcription start site; ORF, open reading frame; HDAC, histone deacetylase is a growth suppressor gene (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar) localized on chromosome 6 at band q24–25, which is a frequent site for loss of heterozygosity in many solid tumors including ovarian cancer (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, 3Colitti C. Rodabaugh K. Welch W. Berkowitz R. Mok S. Oncogene. 1998; 16: 555-559Google Scholar). The mouse ortholog of this gene was independently identified by Spengler et al. (4Spengler D. Villalba M. Hoffman A. Pantaloni C. Houssami S. Bockaert J. Journet L. EMBO J. 1997; 16: 2814-2825Google Scholar) and designated as Zac1, which is highly homologous to the hLOT1 and rLot1 genes. A splice variant of LOT1/ZAC1 was identified by Kas et al. (5Kas K. Voz M. Hensen K. Meyen E. Van de ven W. J. Biol. Chem. 1998; 273: 23026-23032Google Scholar) and named as PLAGL1 based on its homology with PLAG1 protein encoded by the genePLAG1 localized on chromosome 8q12 (6Kas K. Voz M. Roijer E. Astrom A.-K. Meyen E. Stenman G. Van de Ven W. Nat. Genet. 1997; 15: 170-174Google Scholar). The presence of other genes with high homology to LOT1/ZAC1/PLAGL1 indicates that this gene is a member of new family of zinc-finger proteins. Our studies and those of others have shown that LOT1 gene expression is frequently down-regulated in the ovarian and breast carcinoma cells (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, 7Bilanges B. Varrault A. Basyuk E. Rodriguez C. Mazumdar A. Pantaloni C. Bockaert J. Theillet C. Spengler D. Journot L. Oncogene. 1999; 18: 3979-3988Google Scholar). Functional analysis of LOT1 demonstrated that it may play a significant role as a transcription factor modulating growth suppression through mitogenic signaling pathways (8Abdollahi A. R. Hamilton T. Oncogene. 1999; 18: Scholar). It is that epigenetic of genomic DNA by methylation and/or histone deacetylation role in transcriptional silencing and loss of gene of suppressor genes. of DNA at cytosine CpG dinucleotides in higher P. S. Nat. Genet. Scholar, M. M. J. Biol. Scholar, A. Scholar). of gene expression by with the of transcription or by a of and histone P. G. P. D. S. J. A. Nat. Genet. 1998; Scholar, C. C. B. R. A. 1998; Scholar, B. C. B. P. D. A. Nat. Genet. 1999; Scholar, P. A. P. E. A. Nat. Genet. 1999; Scholar, P. A. D. 1999; Scholar). transcriptional through chromatin which be by In human methylation of CpG silencing the of genes that in the suppression of of CpG in cancer cells in the transcriptional of the genes T. T. T. B. J. Genet. Scholar, M. C. A. T. R. Van J. P. Cancer Res. Scholar, E. W. E. B. Genet. Scholar, J. A. L. R. J. D. S. Cancer Res. Scholar, J. J. A. S. Cancer Res. Scholar, J. S. Cancer Res. Scholar, J. M. B. S. D. D. J. W. S. S. A. Scholar, T. J. D. J. T. J. Genet. Scholar). CpG island methylation may play a significant role in the of imprinted genes D. Scholar) and genes on the chromosome S. D. K. T. B. S. A. Scholar, E. Nat. Genet. Scholar). the LOT1/ZAC1 at chromosome was identified as a maternally imprinted region (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, D. Villalba M. Hoffman A. Pantaloni C. Houssami S. Bockaert J. Journet L. EMBO J. 1997; 16: 2814-2825Google R. D. A. C. R. J. D. Genet. Scholar, M. M. M. S. T. K. K. R. D. Genet. Scholar, G. A. S. D. L. D. C. Biol. Scholar). studies have that the or loss of of the LOT1 is with transient neonatal diabetes imprinted disease characterized by growth retardation and is the molecular that expression of LOT1 in In this we have characterized the LOT1 gene structure with to splice and identified the promoter and a CpG Our indicate is to two epigenetic methylation of CpG and histone which may to the transcriptional silencing of the The ovarian cells in with and in a at (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar). rat ovarian surface cells from the of by A. Testa J. L. L. P. Hamilton T. J. Cancer Scholar, J. Getts L. L. Godwin A. Hamilton T. Cancer Res. Scholar). The cell was as (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar). The breast cancer cells and in with and To of the region of hLOT1 which we (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google a normal human cDNA in was A which to the hLOT1 of by with DNA and through The data analyzed using and DNA homology in the We the LOT1 transcription start site using cDNA The cDNA for 5′-RACE was from human and based on and K. S. Scholar, B. Scholar). The was using and The was from the of the LOT1 and at the of this and The the and in a of The amplification of at for of for for for by at for We analyzed the by using the The using both and the used in this by the Cancer DNA To the human LOT1 promoter, a human genomic DNA was using the in the accession number as a the open reading frame; a upstream of the LOT1 start and a to with to the start using both the and as as used in to of the and to the genomic structure of We a human which in of a new genomic designated as was by and used in promoter The and and used to of the LOT1 open reading using genomic DNA as luciferase gene under the control of promoter by and the of the LOT1 promoter at the as in using DNA from the or as a used to the region of the LOT1 promoter and The and linked to the on and the a site at its The with these designated as and designated as was using a by human normal ovarian genomic DNA as with the and The using a and the The DNA by the luciferase which was with or The used to cells and the by used for the using the The promoter was in vitro methylated by the or in the absence of The DNA was for at at DNA in and with LOT1 expression was as (8Abdollahi A. R. Hamilton T. Oncogene. 1999; 18: Scholar). the region was using and The was with to the LOT1 and the was of the promoter the site of cells in to the in and with of The cells and with a and using to drive the a and a to a The cells at using a which the analysis the cells by and in The cell or using a cells to cancer cells at a of in in to with the as using to the the cells with and with the DNA for in by for in was using the The cells with PBS, with the and and of the cell was to of the luciferase The luciferase was or of the using the was from the cells by the P. Scholar) or and was on The was to by and using a (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar). The for of rat and human LOT1 the as (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar). The for was from analysis was with from the cells on in with the with the using of and of protein from on by to a for in in and for with with in The with and for with the was from The protein by the and to DNA was by with by B. A. Scholar). by the of of and of the DNA was with and in and DNA from Godwin and and and at Cancer DNA was with and used for as M. L. D. C. G. P. C. S. A. Scholar, J. J. S. B. S. S. A. Scholar). the DNA in a of was by and with and at for DNA was using DNA was by for at The DNA was by and of and the with and in of The DNA was by under the and of in a The was with the of at for by the of of and of with a of at of was using of the and the and as from the DNA was methylated or unmethylated at CpG or the control region R. D. A. C. R. J. D. Genet. Scholar, M. M. M. S. T. K. K. R. D. Genet. Scholar). used for and used for the and by on a with and The amplification the using the to and The using both and The was on of genomic DNA The DNA was at with of or in a of both at sites. not the DNA the is The DNA was with and in and of the was to using the and and as The of at for by the of and of with a of at The the as was to the cells in with to The cells in and to at The cell in and on for The on with at for and The or chromatin from was with and was and used as The chromatin was for with and at with or The for at with with and with in the The and and with K. The DNA was by and used in The used for and as shown The amplification for the the for using and a analysis of the gene promoter and the presence of and of we to of the cDNA which we (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar). A normal human cDNA in the was used for and a to the hLOT1 and of the identified of the LOT1 gene was to the that is in the accession number We identified the transcription start site of 5′-RACE The cDNA for 5′-RACE was from human and based on and K. S. Scholar, B. Scholar) as in the cDNA We a band to in and that or of the shown with and The was and of and the others of the two transcription start and as shown in of the the at and six at of on chromosome accession number in of and number using the the at data suggest that the LOT1 is from these start at least in the of the revealed that the upstream from the the LOT1 splice variant at the of exons and as to the ZAC1 splice which from LOT1 at the of 1) In of the on the as shown to the and from the LOT1 splice variant at this upstream region. Taken together, the analysis and using of hLOT1 or rLot1 as not suggest that human LOT1 and rat number the for this gene in the human and (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, A. R. Hamilton T. Oncogene. 1999; 18: Scholar). To the genomic structure of hLOT1 we the open reading of in the region and used to normal genomic DNA. The and a that the of the The data not of from zinc-finger to the of the not a DNA that the to and the is of the analysis of and presence of in zinc-finger two and identified In we a human genomic with the in the of LOT1, and a number was identified the using or from this and We the genomic by the LOT1 cDNA with genomic DNA and by using specific to cDNA as as of in the data to determine the of the LOT1 gene which the M. P. Res. 15: Scholar). in the gene of six exons and at least 70 The from to kb in The of was by of the transcription start exons and from other by a of kb in A CpG island is in and the region of the which does not a The LOT1 is encoded by exons and in a The is at the from the of and the is at from the of 6 by kb of including the analysis of the region in cancer cell lines and by not of the LOT1 gene (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar) have identified and in the as A. E. B. A. Pantaloni C. Bockaert J. S. A. 1998; Scholar) and PLAGL1 (5Kas K. Voz M. Hensen K. Meyen E. Van de ven W. J. Biol. Chem. 1998; 273: 23026-23032Google Scholar). to the genomic structure of LOT1 in with the other variants, we these The LOT1 cDNA was with the cDNA from ZAC1 and PLAGL1 (5Kas K. Voz M. Hensen K. Meyen E. Van de ven W. J. Biol. Chem. 1998; 273: 23026-23032Google Scholar, A. E. B. A. Pantaloni C. Bockaert J. S. A. 1998; Scholar, T. R. K. J. A. M. M. Genet. Scholar, A. B. D. Basyuk E. B. C. D. Bockaert J. Journot L. J. Biol. Chem. Scholar) and with the genomic in the The data shown in which the and of have a on 1; the exons differentially in and exons and LOT1 and ZAC1 have open reading the open reading of PLAGL1 the upstream of the in the LOT1 and ZAC1 (5Kas K. Voz M. Hensen K. Meyen E. Van de ven W. J. Biol. Chem. 1998; 273: 23026-23032Google Scholar). We the of the LOT1 gene the upstream of the luciferase gene to determine this region possesses transcription The of the shown in A, and the of a from at least two using these in B. is shown in of the human LOT1 genomic to with to the of LOT1 in accession number U72621) designated as in a to in the of the luciferase gene in the ovarian cancer cell The transcription was the of the promoter, as is shown for to a promoter with for was with the region to as is from the presence of at least two potentially promoter region in the LOT1 We the promoter in other cell lines including the human ovarian cell and the cell of these cells with the expression and a of the transcription was in the cells than cells. of the promoter in cells was we found the hLOT1 promoter to be highly homologous to the promoter to the by of the hLOT1 promoter with that of the mouse in number not It is that methylation of in the promoter of specific suppressor genes is with transcriptional silencing in human we in vitro of promoter by is of transcriptional shown in the methylated promoter The region of the LOT1 gene a CpG island that the of on chromosome accession number The and or the region on and of the have the of CpG dinucleotides and we the methylation of specific this CpG island or the control region R. D. A. C. R. J. D. Genet. Scholar, M. M. M. S. T. K. K. R. D. Genet. Scholar) the transcription start site by The genomic DNA from cancer cell lines and which unmethylated not methylated cytosine as is shown in The DNA was to and a and of the was by the of the cytosine that not We the ratio of methylated unmethylated and the data as shown in A, the ovarian cancer cell lines cell 31 and methylation, and the cell 99% methylation of the CpG on In the two breast cancer cell and the methylation and in these ovarian and breast cancer cell lines the or methylated CpG dinucleotides in the differentially methylated region ranged from to It is that we the DNA from cells to analysis and found that the DNA was the We have shown that the ovarian cancer cell lines of LOT1 (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar). we not to a the expression data and the methylation in these cell Furthermore, we and cancer cell lines with the to determine the expression of LOT1/ZAC1 be The that in these cell lines LOT1 is to not suggest that other than methylation may be in the of In other we that in of LOT1 may be to upstream in the and that of CpG dinucleotides may not be to the normal expression of this of DNA for the methylated A, of the used for methylation analysis the not with and the The indicate the of CpG the of methylated that the that the the other and to analysis of the CpG island of the LOT1 gene in ovarian and breast DNA was from the cell lines or tumors and analyzed for methylation by and by The ratio of methylated to unmethylated was and as indicates a CpG site in the DNA and of analysis of a The of the CpG site at the and to the in and unmethylated and methylated CpG ovarian cancer cell breast cancer cell and ovarian We the methylation in and ovarian and found that in the DNA higher methylation than in the normal DNA and in the was We the methylation of normal DNA with The DNA was with or both at sites. does not the DNA the is and the DNA of methylation The DNA was to using the and spanning the in the genomic on chromosome accession number A was for the not for the that is methylated in normal DNA not and the of methylated in normal DNA suggest that or methylation of the is in this and in breast or other studies have revealed that histone deacetylation is a mechanism of DNA silencing to its on and of chromatin structure K. S. G. A. EMBO J. 1997; 16: Scholar). we the of TSA, a and specific inhibitor of histone deacetylase M. S. T. on LOT1 gene We have shown that the cancer cell or expression in comparison with its normal cells (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar) we deacetylation mechanism a role in the of in this malignantly transformed cell and found that the gene silencing The transcription is in with the inhibitor suggest that a significant at least in in it appears that of the gene by is of We of LOT1 by the of histone deacetylation is to of the We the LOT1 promoter and found that it was not to and the histone deacetylase to of transcription from this promoter not In we of chromatin from the control and cells with histone and the LOT1 The data as shown in that the of histone with the promoter is not in to we that the may target the LOT1 to indirect upstream of the To this we the LOT1 cDNA of the green gene and used the to ovarian cancer cells. The data in show that the number of cells protein is the of HDAC, as by fluorescence with the cell which or expression of the gene not analysis that the protein with is in the cells with The of of histone deacetylase in and was by analysis and cell of the cells. is shown in the number of cells was than that of cells and that the number the with Taken together, these data and the analysis show the transcriptional of gene to TSA, and The suppressor gene LOT1 a nuclear transcription factor and is by the of the growth factor signaling (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, A. R. Hamilton T. Oncogene. 1999; 18: Scholar). In this we have of the characterized the genomic and analyzed its epigenetic regulation. We have the splice of LOT1 and its splice variants, namely ZAC1 and TheLOT1 gene of six exons and at least 70 The from to kb in The LOT1 is encoded by exons and in a A island is in and the region of the which does not a We the of LOT1 using the 5′-RACE of the using cDNA from normal two transcription start and the absence of a the ofLOT1 that the of transcription may be We have identified a promoter region kb of of the in a to of the luciferase gene in the ovarian cancer cell a promoter was with the region the presence of at least two potentially promoter region in We that the LOT1 gene expression may be by that may histone DNA methylation, and/or mRNA (8Abdollahi A. R. Hamilton T. Oncogene. 1999; 18: Scholar). We have identified CpG in the upstream of and the promoter of LOT1, which suggest that DNA methylation of CpG may play a role in silencing the gene In is of a maternally imprinted chromosomal region R. D. A. C. R. J. D. Genet. Scholar, M. M. M. S. T. K. K. R. D. Genet. Scholar, G. A. S. D. L. D. C. Biol. Scholar). we have analyzed the based on methylation of the and found that DNA from both normal and ovarian cancer cell lines methylation of the cytosine It appears that ovarian tumors and cancer cells relatively of methylation in CpG The breast cancer cell lines high of The presence of methylated in normal DNA that of the of the LOT1 gene is in this and in breast or other based on these it is to that the LOT1 gene may be to or silencing by loss of heterozygosity and Furthermore, in this we have for the role of histone deacetylation in the LOT1 gene of the cells with trichostatin A, a inhibitor of histone deacetylase, the silencing in and ovarian cancer cell suggest that a at least in in of with the and or may in deacetylation of these and of gene transcription. of found to the genomic of the promoter to the transcription K. S. G. A. EMBO J. 1997; 16: Scholar). this is by DNA methylation, which may histone deacetylation and/or may in methylated protein to the of DNA by the S. T. A. 1998; Scholar, M. 1997; Scholar, A. Scholar, A. EMBO J. 1998; Scholar). in this we found that does not the in vitro methylated LOT1 promoter and does not the promoter histone that a histone for gene silencing of this by DNA methylation, as for other R. S. T. S. G. J. Biol. Chem. Scholar). with it that of mouse with does not in the expression of the gene A. G. C. J. Biol. Chem. Scholar). is by other that in cancer the genes such not be with E. K. J. S. Nat. Genet. 1999; Scholar). it is that ofLOT1 by the of histone deacetylase may be to the on the unmethylated or of the gene and be elicited by a upstream in pathway. We that other than or methylation may play in the expression to be expression from the or from the may be of cell and for and in a and of to in the mechanism of LOT1 gene by and epigenetic which may be in the role of this gene in cancer and/or We and for the and and for and We T. and for fluorescence and analysis and for DNA