Methylation matters: a new spin on maspin
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news & views nature genetics • volume 31 • june 2002 123 Experimental results tend to be ambiguous, reflecting nature’s indifference to scientific theories. Rarely does one see a result that is so black-and-white as to be untainted by interpretation. On page 175 of this issue, Bernard Futscher and colleagues 1 report one of these rare instances. They address a two-decade–old theory, often cited but never proven, that methylation plays a role in tissue-specific gene expression. The the- ory strikes at the core of a fundamental bio- logical question—what determines the gene expression pattern that uniquely defines different tissues? Landmark publications in the 1970s expounded the growing suspicion that differential methylation of cytosines con- trols tissue-restricted gene expression during development and in differentiated adult tissues 2,3 . Although methylation patterns are unequivocally different from tissue to tissue, the methylation of tissue- specific genes has generally been in the wrong place or at the wrong time, or more often, there is a complete lack of correla- tion with gene expression. More recent studies of all CpG motifs (most methyla- tion occurs at cytosine residues within CpG dinucleotides) within defined pro- moters of tissue-specific genes have shown a similar lack of definitive correlation with gene expression 4 . The finding that tissue- specific genes previously suspected to be regulated by methylation are unaffected by demethylation in methyltransferase- deficient mouse embryos 5 seemed to drive the final nail in the coffin. However, absence of evidence is not necessarily evi- dence of absence, and the study by Futscher et al. 1 has now imbued this old theory with new life. Spotlight on SERPINB5 It is not trivial that this long-awaited exam- ple involves SERPINB5 (encoding maspin). The gene SERPINB5 is a member of the ser- ine protease inhibitor (serpin) gene cluster on chromosome 18q21.3 (refs 6,7). These proteins have a number of important func- tions, including the regulation of cell adhe- sion and differentiation. Maspin is unique in the serpin superfamily in that it is a serine protease substrate, rather than an inhibitor. Regulation of maspin is critical to normal development and the tissue-specific func- tions in the adult. For example, targeted overexpression of maspin in the mouse dis- rupts development of the mammary gland and inhibits that of lobular-alveolar struc- tures during pregnancy 8 . The implications of the study by Futscher et al. 1 extend beyond normal gene regulation into clinically relevant aspects of cancer. SERPINB5 was first dis- covered as a gene that is downregulated in breast-cancer cells 6 , and in mouse models, it is a potent tumor suppressor that inhibits cell motility, invasion, angiogene- sis and metastasis 7 . Loss of maspin expres- sion correlates with increased metastatic potential in breast-cancer and other human tumors 7 . Earlier work by Domann et al. 9 showed that silencing of SERPINB5 in breast cancer cells is associated with aberrant methyla- tion. The first clue that methylation might also be important in the normal expression of SERPINB5 stemmed from the observa- tion that ‘control’ peripheral blood lym- phocytes also have dense methylation of the SERPINB5 promoter 9 . Methylation matters: a new spin on maspin Joseph F. Costello 1 & Paula M. Vertino 2 1 Brain Tumor Research Center, Department of Neurological Surgery and UCSF Comprehensive Cancer Center, University of California, San Francisco, California, USA. 2 Department of Radiation Oncology and the Winship Cancer Institute, Emory University, Atlanta, Georgia, USA. e-mail: [email protected] and [email protected] The theory that DNA methylation controls tissue-specific gene expression has remained controversial despite being frequently cited for more than 25 years. A new study shows a pristine example of methylation and gene expression that is cell- type–restricted, although questions about causality persist. MeCP HDAC MeCP CH3 CH3 CH3 CH3 CH3 X CH3 co-activator p53 AP1 Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac fibroblast epithelium skin Model of the cell-type–specific control of SERPINB5 expression by methylation. The epithelial cells in skin have an unmethylated SERPINB5 promoter that is occupied by the transcriptional regulators AP1 and p53. In addition, the histones (blue) are acetylated (Ac), thus limiting histone–histone interactions and provid- ing an open chromatin structure that is required for SERPINB5 expression. In contrast, the promoter in skin fibroblasts is completely methylated (CH3), associated with hypoacetylated histones and adopts an inac- cessible transcriptionally inactive state. Methylation of DNA allows the binding of methyl CpG–binding proteins (MeCP), which can attract histone deactylases (HDAC) and chromatin remodelling complexes to direct a local change in chromatin organization. In this model, methylation is a primary impediment to SERPINB5 expression and thus determines the cell type–specificity. © 2002 Nature Publishing Group http://genetics.nature.com
Transcript of Methylation matters: a new spin on maspin
news & views
nature genetics • volume 31 • june 2002 123
Experimental results tend to be ambiguous,reflecting nature’s indifference to scientifictheories. Rarely does one see a result that isso black-and-white as to be untainted byinterpretation. On page 175 of this issue,Bernard Futscher and colleagues1 reportone of these rare instances. They address atwo-decade–old theory, often cited butnever proven, that methylation plays a rolein tissue-specific gene expression. The the-ory strikes at the core of a fundamental bio-logical question—what determines thegene expression pattern that uniquelydefines different tissues?
Landmark publications in the 1970sexpounded the growing suspicion thatdifferential methylation of cytosines con-trols tissue-restricted gene expressionduring development and in differentiatedadult tissues2,3. Although methylationpatterns are unequivocally different fromtissue to tissue, the methylation of tissue-specific genes has generally been in thewrong place or at the wrong time, or moreoften, there is a complete lack of correla-tion with gene expression. More recentstudies of all CpG motifs (most methyla-tion occurs at cytosine residues withinCpG dinucleotides) within defined pro-moters of tissue-specific genes have showna similar lack of definitive correlation withgene expression4. The finding that tissue-specific genes previously suspected to beregulated by methylation are unaffectedby demethylation in methyltransferase-deficient mouse embryos5 seemed to drivethe final nail in the coffin. However,absence of evidence is not necessarily evi-dence of absence, and the study byFutscher et al.1 has now imbued this oldtheory with new life.
Spotlight on SERPINB5It is not trivial that this long-awaited exam-ple involves SERPINB5 (encoding maspin).The gene SERPINB5 is a member of the ser-ine protease inhibitor (serpin) gene cluster
on chromosome 18q21.3 (refs 6,7). Theseproteins have a number of important func-tions, including the regulation of cell adhe-sion and differentiation. Maspin is uniquein the serpin superfamily in that it is a serineprotease substrate, rather than an inhibitor.Regulation of maspin is critical to normaldevelopment and the tissue-specific func-tions in the adult. For example, targetedoverexpression of maspin in the mouse dis-rupts development of the mammary glandand inhibits that of lobular-alveolar struc-tures during pregnancy8.
The implications of the study byFutscher et al.1 extend beyond normalgene regulation into clinically relevantaspects of cancer. SERPINB5 was first dis-
covered as a gene that is downregulated inbreast-cancer cells6, and in mouse models,it is a potent tumor suppressor thatinhibits cell motility, invasion, angiogene-sis and metastasis7. Loss of maspin expres-sion correlates with increased metastaticpotential in breast-cancer and otherhuman tumors7.
Earlier work by Domann et al.9 showedthat silencing of SERPINB5 in breast cancercells is associated with aberrant methyla-tion. The first clue that methylation mightalso be important in the normal expressionof SERPINB5 stemmed from the observa-tion that ‘control’ peripheral blood lym-phocytes also have dense methylation of theSERPINB5 promoter9.
Methylation matters:a new spin on maspin
Joseph F. Costello1 & Paula M. Vertino2
1Brain Tumor Research Center, Department of Neurological Surgery and UCSF Comprehensive Cancer Center, University of California, San Francisco,California, USA. 2Department of Radiation Oncology and the Winship Cancer Institute, Emory University, Atlanta, Georgia, USA.
e-mail: [email protected] and [email protected]
The theory that DNA methylation controls tissue-specific gene expression has remained controversial despite being frequentlycited for more than 25 years. A new study shows a pristine example of methylation and gene expression that is cell-type–restricted, although questions about causality persist.
MeCP
HDAC
MeCP
CH3CH3
CH3CH3
CH3
XCH3
co-activator
p53 AP1
Ac Ac
AcAc
Ac
Ac Ac
Ac
Ac Ac
AcAc
fibroblast
epithelium
skin
Model of the cell-type–specific control of SERPINB5 expression by methylation. The epithelial cells in skinhave an unmethylated SERPINB5 promoter that is occupied by the transcriptional regulators AP1 and p53.In addition, the histones (blue) are acetylated (Ac), thus limiting histone–histone interactions and provid-ing an open chromatin structure that is required for SERPINB5 expression. In contrast, the promoter in skinfibroblasts is completely methylated (CH3), associated with hypoacetylated histones and adopts an inac-cessible transcriptionally inactive state. Methylation of DNA allows the binding of methyl CpG–bindingproteins (MeCP), which can attract histone deactylases (HDAC) and chromatin remodelling complexes todirect a local change in chromatin organization. In this model, methylation is a primary impediment toSERPINB5 expression and thus determines the cell type–specificity.
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news & views
124 nature genetics • volume 31 • june 2002
Maspin is expressed in a cell-type–restricted manner and is expressedin epithelial cells of the airway, breast, skinand prostate, but not in skin fibroblasts,lymphocytes, bone marrow, heart andkidney1. At the transcriptional level,SERPINB5 is regulated by AP-1 and p53-binding sites, and can be negatively reg-ulated by a hormone-responsive elementrecognized by the androgen receptor7.Given that these proteins are ubiquitousin their expression, the question arises:what dictates the cell-type–specificity ofmaspin expression?
Methylation does itFutscher et al.1 use primary human cul-tures to show that cell-type–specificexpression of maspin is inversely corre-lated with methylation of the SERPINB5promoter. Not only are the critical AP1and p53 promoter elements differentiallymethylated, but so is the entire CpG-richisland surrounding the promoter andfirst exon of the gene, and it is strictly all-or-none (see figure). They further showthat SERPINB5 expression can be reacti-vated by 5-aza-2´-deoxycytidine treat-ment of immortalized fibroblasts,suggesting that methylation is the onlyimpediment to SERPINB5 expression ina non-expressing tissue. Taken together,these results satisfy two criteria for a rolefor methylation in tissue-specific generepression—its promoter is denselymethylated in non-expressing tissues andinduced demethylation results in ectopicactivation of the gene.
One issue raised by this study is the pre-sumed methylation status of CpG islandsin normal tissues. CpG islands are regionsof DNA characterized by an unusuallyhigh C+G content and CpG frequency rel-ative to the remainder of the genome10,11.Most CpG islands encompass the pro-moter and first exons of genes and arefound in nearly all housekeeping genes
and more than 50% of tissue-restrictedgenes. There is a widely held assumptionthat CpG islands are unmethylated in allnormal tissues, regardless of expressionstatus. Well-documented exceptionsinclude genes on the inactive X chromo-some and those subject to parentalimprinting. In these cases, CpG islandmethylation is a developmentally regu-lated event that occurs in the early embryoand is necessary to maintain repression indifferentiated cells of the adult12,13.
Is it possible that CpG island methyla-tion has a similar role in restricting theexpression of some tissue-specific genes?Albeit smaller and less CpG-rich than someCpG islands, the promoter of the maspingene qualifies as a CpG island based on theestablished definitions11,14; yet, it is heavilymethylated in normal human cell typesthat lack maspin expression. And, as withother genes that undergo permanentmethylation-dependent repression (forexample, genes on the inactive X chro-mosome, imprinted genes, and aber-rantly methylated genes in cancer),Futscher et al. show that, when methy-lated, the SERPINB5 promoter is in atranscriptionally repressive chromatinconformation (see figure)1. The SER-PINB5 methylation pattern is not entirelywithout precedent. De Smet et al.15 haveshown that the MAGE genes, which alsohave promoters of intermediate CpG-richness, are unmethylated and expressedin male germ cells but are heavily methy-lated and silent in somatic tissues. Impor-tantly, ectopic activation of MAGE occursin human melanomas and is associatedwith hypomethylation of the promoter.
To be continuedThe maspin example underscores thenecessity of distinguishing between normalmethylation cues and abnormal ones.Although SERPINB5 expression is maskedby methylation in many normal cell types,
the promoter is unmethylated in breastepithelial cells. Thus, the methylation andsilencing of maspin in human breast can-cers, which are primarily derived fromepithelial cells, would clearly be an aberrantevent. Perhaps cancer cells have inappropri-ately activated a silencing mechanism that isotherwise operative only during a particu-lar stage in cellular differentiation.
Cautious scientists must agree that thereis much work to be done before we willcome to know the full impact of this study.Because it necessarily relied on primaryhuman cells in short-term culture, theresults must be confirmed in vivo. It willalso be of interest to determine at whatdevelopmental stage the cell-type–specificmethylation of SERPINB5 is set, andwhether it establishes silencing or ensuresthe propagation of a stably repressed state.Finally, is SERPINB5 unique or are thereother genes that rely on methylation as ameans of restricting expression to differenttissues or cell types? If such studies supportthe present findings, the publication byFutscher et al.1 will be inextricablylinked across two decades to one of themost influential and tenacious theoriesin the field. �1. Futscher, B.W. et al. Nature Genet. 31, 175–179
(2002).2. Riggs, A.D. Cytogenet. Cell Genet. 14, 9–25 (1975).3. Holliday, R. & Pugh, J.E. Science 187, 226–232
(1975).4. Warnecke, P.M. & Clark, S.J. Mol. Cell. Biol. 19,
164–172 (1999).5. Walsh, C.P. & Bestor, T.H. Genes Dev. 13, 26–34
(1999).6. Zou, Z.Q. et al. Science 263, 526–529 (1994).7. Hendrix, M.J.C. Nature Med. 6, 374–376 (2000).8. Zhang, M. et al. Dev. Biol. 215, 278–287 (1999).9. Domann, F.E., Rice, J.C., Hendrix, M.J.C. & Futscher,
B.W. Int. J. Cancer 85, 805–810 (2000).10. Bird, A.P. Nature 321, 209–213 (1986).11. Gardiner-Garden, M. & Frommer, M. J. Mol. Biol.
196, 261–282 (1987).12. Li, E., Beard, C. & Jaenisch, R. Nature 366, 362–365
(1993).13. Panning, B. & Jaenisch, R. Genes Dev. 10, 1991–2002
(1996).14. Takai, D. & Jones, P.A. Proc. Natl Acad. Sci. USA 99,
3740–3745 (2002).15. De Smet, C., Lurquin, C., Lethe, B., Martelange, B. &
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