Altered epigenetic signals in human disease

12 downloads 52 Views 719KB Size Report
Polycomb group protein fail to complete gastrulation several cancers and correlate with increased proliferation and aggressiveness of breast cancer. SUZ12.
[Cancer Biology & Therapy 3:9, 831-837, September 2004]; ©2004 Landes Bioscience

Altered Epigenetic Signals in Human Disease

Focused Review: Epigenetics—Normal Control and Deregulation in Cancer

te

ABSTRACT

Luciano Di Croce1,2,* M. Buschbeck2 A. Gutierrez2 I. Joval2 L. Morey2 R. Villa2 S. Minucci3

2Center for Genomic Regulation; Barcelona, Spain 3European Institute of Oncology; Milan, Italy

*Correspondence to: Luciano Di Croce; Center for Genomic Regulation; Passeig Maritim 37-49; Barcelona 08003 Spain; Tel.: +34.93.224.09.32; Fax: +34.93.224.08.99; Email: [email protected] Received 07/22/04; Accepted 07/23/04

Bio

sci

KEY WORDS

Neoplastic transformation is characterized by inappropriate cell growth and a reduced capacity for differentiation. The altered transcription program in cancer cells is often related to aberrant epigenetic modifications, often on the level of DNA methylation and/or histone methylation. Epigenetics is defined as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence”.1 Since a few years, several chemical compounds have been used to try to “reroute” undifferentiated cancer cells into the correct differentiation program. A common mechanism of several of these compounds is the ability to interfere with the epigenetic modifications of the genome. In eukaryotic cells, genomic DNA is organized in chromatin, the structural unit of which is the nucleosome. Nucleosomes are composed of 147 bp of DNA wrapped around a core of eight histone proteins.2 This histone octamer consists of two copies each of the histones H2A, H2B, H3 and H4. Nucleosomal DNA is further condensed to a 30-nm diameter fiber, which, in turn, is further compacted into several higher order structures that are not yet fully understood.3 Chromatin structure is not uniform with respect to gene distribution and transcriptional activity. We can distinguish between euchromatin and heterochromatin, which have different structures, transcriptional activity and replication timing. Euchromatin is decondensed and transcriptionally active during interphase, whereas heterochromatin is defined as regions of chromatin that remain cytologically condensed and densely stained throughout the cell cycle. Large blocks of heterochromatin are found adjacent to functional chromosome structures such as centromeres and telomeres.4

en

Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=1103

INTRODUCTION

ce.

1ICREA and Center for Genomic Regulation; Barcelona, Spain

Do

No tD ist r

ibu

The genetic information of almost all eukaryotic cells is stored in chromatin. In cancer cells, alterations in chromatin organization or in its epigenetic marks occur frequently. Among these are changes in the patterns of DNA and histone methylation. Using Acute Promyelocytic Leukemia as model system we could demonstrate a direct correlation of epigenetic events induced by the driving oncogene product PML-RARα and cancer progression. Several of the enzymes ultimately responsible for these events can be inhibited by small compound inhibitors and thus can serve as targets in cancer therapy. In this article, we review the role of DNA methylation, histone methylation and chromatin alterations in human diseases. A picture is emerging in which these epigenetic signals “cross-talk” and are implicated in the physiological and pathological spreading of gene silencing.

ACKNOWLEDGEMENTS

es

epigenetics, chromatin, cancer, silencing, leukemia

©

20

04

La

nd

The work from our laboratory described in this review was supported by intramural funds from the Center for Genomic Regulation (CRG). We thank P.G. Pelicci, V. Raker and all members of the Di Croce laboratory for stimulating discussions. Apologies are extended to those investigators whose work is not directly cited due to space limitations.

www.landesbioscience.com

EPIGENETIC SIGNALS: DNA AND HISTONE METHYLATION

Epigenetic signals, key driving forces during embryo development and tissue specialization, regulate gene expression without changes in the DNA coding sequence and can be inherited through cell division.5 Recent findings have reshaped the way we think about epigenetics, especially how it is altered in cancers. Indeed modifications of the DNA and histone methylation patterns, which are considered to be the main mode for epigenetic transmission, have both been associated with cancer.6,7 Such changes affect virtually every step in tumor progression and are increasingly recognized as major mechanisms involved in silencing growth-controlling genes (such as cell cycle regulators, DNA repair enzymes, and other potential tumor suppressor genes).8,9 Epigenetic changes arise faster and occur at a higher frequency than genetic changes but are at least in part reversible upon treatment with pharmacological agents.10 The subcellular distribution of enzymes responsible for the epigenetic marking of the genome has also found to be altered in cancer cells (see below).

Cancer Biology & Therapy

831

ALTERED EPIGENETIC SIGNALS IN HUMAN DISEASE

Table 1

LINK BETWEEN EPIGENETIC ALTERATIONS

AND

DISEASE

Gene/Protein

Function

Knock-Out phenotype

Cancer and Disease-Related Observations

Refs.

DNMT1

Copy and maintenance of DNA methylation patterns; localizes to DNA replication fork

Global hypomethylation of the genome including satellite DNA, repetitive elements and imprinted genes, developmental failure at E8.5

Mice with reduced levels display genomic instability and develop aggressive T cell lymphomas at the age of 8 months

60,60 14,62

Required to maintain silencing of tumor suppressor genes

63

DNMT3a

De novo methylation

Defects in spermatogenesis and gut function, early postnatal death

DNMT3b

De novo methylation

Neural tube defects and embryonic lethality around E16.5

Contributes to transformation by activated Ras and large Tantigen; involved in cancer cell’ survival; cooperates with DNMT1 to silence genes in human cancers mutated in ICF syndrome

15,64 65–67 44

MECP2

Methyl-DNA binding

Complex neurological defects, tremors, ataxia, abnormal behavior

Mutated in Rett syndrome patients

68,69

MBD2

Methyl-DNA binding behavior

Abnormal maternal behavior

Required for efficient cell transformation in vitro and for intestine tumor formation in mice

70–72

MBD4

DNA methyl-binding, mismatch repair

Enhanced frequency of C-T transitions

Suppresses CpG mutability and tumorigenesis in animal models; deficient mice have accelerated formation of tumors

73

Reduced apoptotic response to DNA damaging agents

72

Mutated in several cancers

74–76

ESET

H3 K9 methylation

Peri-implantation lethality, null blastocysts display growth defects of the inner cell mass

Suv39H1/2

H3 K9 methylation

Reduced viability; chromosomal instability; defective chromosomepairing during spermatogenesis

Lsh

Chromatin remodelling

Genome-wide demethylation; postnatal lethality; reduced growth

ATRX

Chromatin remodelling

EZH2

H3 K27 methylation Polycomb group protein

SUZ12

Polycomb group protein

EED

Polycomb group protein

Peri-implantation lethality fail to complete gastrulation

Defective gastrulation; lack of X chromosome inactivation

DNA Methylation System. DNA methylation is essential for proper mammalian development and plays a role in transcriptional repression, modulation of chromatin structure, X-chromosome inactivation, genomic imprinting, and suppression of repetitive and parasitic DNA sequences.11 It has been known for more than 20 years that the patterns of DNA methylation are significantly altered 832

15,64

77

Deficient mice develop tumors at increased frequency

78

79 Mutated in the α-thalassaemia/ mental retardation, X-linked syndrome

80

Increased levels have been found in several cancers and correlate with increased proliferation and aggressiveness of breast cancer

53,81,82

Frequent translocated in endometrial stromal sarcomas

83 84,85

in tumor cells as compared to normal cells.12 Cancer cells display a global reduction of methylated cytosines throughout the genome. The genome-wide hypomethylation could contribute to tumorigenesis through oncogene activation, retrotransposon activation and chromosome instability. At the same time, a growing list of hypermethylated promoters has been documented in human tumors. The local

Cancer Biology & Therapy

2004; Vol. 3 Issue 9

ALTERED EPIGENETIC SIGNALS IN HUMAN DISEASE

hypermethylation usually occurs in the context of CpG islands, a cluster of CpGs over a small stretch of DNA. Three DNA methyltransferases (DNMTs) are considered to be responsible for virtually all mammalian DNA methylation.13 These enzymes transfer a methyl group provided by S-adenosylmethionine to the 5’-carbon of the cytosine ring, to form methyl cytosine. This reaction occurs almost exclusively in the context of CpG dinucleotides. All three catalytically active DNMT enzymes (DNMT1, DNMT3a, and DNMT3b; Table 1) are essential for proper murine development.14,15 Biochemical studies and in vivo experiments suggest that the DNMT3a and DNMT3b act primarily as de novo methyltransferases to establish new methylation patterns for instance during embryogenesis.15 In contrast, DNMT1 is thought to maintain these methylation patterns during DNA replication. 16 Accordingly, DNMT1 is present at the DNA replication fork,17 and it has a 10to 40-fold preference for hemimethylated (i.e., methylated on one strand of DNA) versus unmethylated DNA.18 Recently, an alterative form of DNMT3 has been identified, termed DNMT3L, which acts as cofactor for both DNMT3a and DNMT3b.19 The presence of methylated CpGs (mCpGs) in a given promoter region has a dramatic influence on gene transcription, leading to reduction or even complete silencing of the gene. The molecular link between DNA methylation and gene repression is still not completely understood.20 The identification of a family of proteins that share a domain that enables them to recognize and bind to methylated DNA shed some light on the mechanism.21,22 The so-called methylCpG binding proteins (MBDs) are thus far the best candidates for interpreting the DNA methylation signal. This family consists of at least six members (reviewed in refs. 23,24). The presence of MBD proteins within methylated promoters could prevent gene activation by precluding binding of positive transcription factors. Recent data demonstrated that, in addition to this “passive” mechanism, MBD proteins act also by recruiting other repressive enzymes such as histone deacetylases (HDACs) and histone methyltransferases (HMTs) to hypermethylated promoters.25,26,27 Histone Tails Modification System. Posttranslational modification of the “N-terminal” tails of the histones seems to be a universal regulatory mechanism among eukaryotic organisms, conserved from yeast to human.28 Tail modifications (mainly of histone H3 and H4) include acetylation, phosphorylation, ADP-ribosylation, ubiquitination, and methylation. Increasing evidence suggests that combinations of these covalent modifications constitute a code that defines actual or potential transcriptional states. Such a “histone code”29 might act as scaffold for the recruitment of specific transcription factors and could also define distinct chromosomal domains. It was recently demonstrated that silencing through DNA methylation influences the acetylation and methylation state of the histones. For example, hypermethylated promoters are often assembled onto hypoacetylated nucleosomes.30,31 Histone hypoacetylation increases the avidity of DNA-histone interactions.32 This results in a “condensed” chromatin conformation that prevents access to other DNAbinding proteins. Histone acetylation/deacetylation is modulated by the interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs). As a further link between the malfunction of epigenetic mechanisms and cancer, the imbalance of the HAT/ HDAC interplay has been tightly linked to cancers.33 Finally, the discovery that HDACs can associate with both MBD proteins and DNMT enzymes provides important inside to the molecular mechanism underlying reduced histone acetylation at repressed loci. 26,27,34-36 www.landesbioscience.com

Similar to the interplay between histone deacetylation and DNA methylation, there is also a “cross-talk” between DNA and histone lysine methylation. Methylation of lysine residues is known to occur on histone H3 (at positions K4, K9, K27, K36, and K79), histone H4 (K20) and histone H1 (K26). Several histone methyltransferases (HMTs) have been identified, namely one H3-K4 methyltransferase, five H3-K9 methyltransferases, Suv39h1 and Suv39h2, G9a, ESET/ SetDB1 and Eu-HMTase1, many of which contain a conserved SET domain.28,37 A link between DNA methylation and histone methylation has been suggested by recent studies in Neurospora and Arabidopsis. In Neurospora crassa, mutations of the DIM-5 (defective in methylation 5) gene, which encodes a SET-domain-containing H3-K9 HMT, resulted in a complete loss of genomic DNA methylation.38 Similarly, in Arabidopsis thaliana, which contains methylation predominantly at CpNpG motifs rather than CpG motifs, mutations in kryptonite (KYP)—also an H3-K9 HMT—led to a partial loss of methylation of CpNpG, but not CpG sites.39 Further analysis showed that histone methylation might regulate DNA methylation in Arabidopsis through the HP1β protein, which binds to the methylated H3-K940,41 and recruits the DNMT chromomethylase 3 (CMT3) to its target CpNpG sites. Cooperation between histone H3-K9 and DNA methylation is further supported by the fact that both modifications are generally associated with transcriptionally silent heterochromatin. However, whether histone methylation regulates CpG methylation in mammalians remains to be shown, since there are many differences in DNA methylation between lower organisms and mammalians. Interestingly, the chromodomain protein Polycomb (Pc) can also bind to histone tails methylated at H3-K27,42 suggesting that the assembly mechanisms proposed for HP1/HMT/DNMT may also apply to Polycomb mediated silencing. DNA Methylation and Human Disease. Alterations of the DNA methylation system are involved in some human disease. Point mutations in the DNMT3b gene are linked to a rare autosomal recessive disorder called ICF syndrome (for immunodeficiency, centromeric instability and facial anomalies).43 Although no ‘hotspot’ has been identified, and many of the mutations are heterozygous, all seem to affect the carboxy-terminal catalytic domain of DNMT3b. Patients affected by ICF syndrome present reduced level of methylation of pericentromeric DNA sequences. How these modified levels of DNA methylation are responsible for immunodeficiency and mental retardation is still unclear. Recently, the molecular defect in a large fraction of Rett syndrome patients was found to result from mutations in the MECP2 gene, located on human chromosome Xq28.44 This disease is characterized by a period of normal development, followed by progressive loss of the patients’ capability to speak or to use their hands. This regression continues until autism, loss of motor coordination (ataxia) and repetitive hand movements. How MeCP2 mutations lead to Rett syndrome is unclear. Able to bind directly to methylated DNA, MeCP2 is thought to act as a transcription repressor through the recruitment of the mSin3A/HDAC1-2 corepressor complex.20 However, it is not known which genes are controlled by MeCP2conatining complexes. Given the phenotypic consequences of MECP2 mutations, it is likely that one or more genes controlled by MeCP2 are critical for development and/or maintenance of neurons. Recent experiments suggest that the gene for brain-derived neurotrophic factor (BDNF), which is involved in normal development and is implicated in learning and memory (ref ), and Hairy2a, which

Cancer Biology & Therapy

833

ALTERED EPIGENETIC SIGNALS IN HUMAN DISEASE

A

B

C Figure 1. Mechanism of stepwise epigenetic silencing. Three possible models for heterochomatinization of an euchromatic and active gene locus are schematically depicted. (A) Heterochromatin formation via histone lysine methylation. Localization of HDAC and HMT enzymes to specific gene loci causes modifications of the amino-terminal tails of histones, creating docking site for the binding of HP1 proteins.40,41 The presence of HP1 proteins attract DNMTs,86 which can methylate CpG dinucleotides “de novo”. The further recruitment of MBDs proteins might be required for the stabilization and propagation of the newly inactive chromatin. (B) Heterochromatin formation via DNA methylation. The coordinate activities of HDACs and DNMT3a/b34-36 increases the content of DNA methylation over a given gene locus. The resulting methylation of CpG sites acts as a signal for MBD recruitment, which might not only stabilize the presence of HDACs26,27 but may also recruit HMTs.87 Subsequent methylation of histone lysines and recruitment of HP1 may help to condense the chromatin structure. (C) Aberrant heterochomatinization of PML-RARα target genes. In acute promyelocytic leukemia, binding of the oncogenic transcription factor PML-RARα to target genes leads to hypoacetylated histone tails via recruitment of HDACs.58 Additional interactions between PML-RARα and DNMTs enzymes leads to DNA methylation,51 creating docking sites for MBDs proteins. These in turn interact with HMTs. The presence of HMTs may help to condense the chromatin structure via methylation of histone lysines. Finally, HP1 might stabilize the newly formed heterochromatin by reinforcing the local concentration of DNMTs and HMTs. Both MBDs and HP1 might be required for spreading of the heterochromatin structure and for the silencing of genes involved in regulating the cell cycle and cell differentiation. In all proposed model, the newly methylated CpG are copied with high fidelity during replication and are thus transmitted to the next cell generation by the activity of DNMT1 enzymes, thus allowing the propagation of the epigenetic signals.

is known to function in a key pathway in early neuronal development, are MeCP2 target genes.45-47 Misregulation of the expression of these genes may contribute to the Rett syndrome phenotype. More than 10 years ago, several reports identified the molecular defect responsible for fragile X syndrome—one of the most common forms of heritable mental retardation—by the cloning and characterization of the FMR1 gene. This syndrome occurs when a CGG repeat in the 5’ regulatory region of the FMR1 gene expands and becames methylated. These modifications lead not only to the silencing of the FMR1 gene but also to a “fragile” site at its locus on the X chromosome.48 Epigenetics and Cancer. Alteration in the DNA methylation pattern in cancer cells have been recognized for over two decades, and they include loss of methylation at normally methylated sequences (hypomethylation) and gain of methylated sequences at sites usually unmethylated (hypermethylation). The consequence of gain of methylation is the inactivation of genes that are essential for 834

the control of normal cell growth, differentiation or apoptosis.49 Numerous examples of promoter hypermethylation, resulting in gene silencing, have been described.12 Recent studies indicate that promoter hypermethylation is often an early event in tumor progression.50 The biological significance of DNA hypomethylation in cancer is less clear. The majority of hypomethylation events occur in repetitive elements localized in satellite sequences or centromeric regions. The alteration in the methylation status could cause the activation of normally silenced regions (such as inserted viral genes, imprinted genes, and genes on the inactive X chromosome) or could lead to genomic instability.5 The mechanisms responsible for aberrant methylation are not fully understood. Particularly intriguing is the fact that hypermethylation and hypomethylation occur concomitantly in the same cancer cells, suggesting a possible scenario in which a modified distribution of DNMTs could account for both disturbances. We have recently demonstrated that the oncogenic transcription factor PML-RARα

Cancer Biology & Therapy

2004; Vol. 3 Issue 9

ALTERED EPIGENETIC SIGNALS IN HUMAN DISEASE

affects the nuclear localization of DNMTs and induces epigenetic modifications, including DNA methylation.51 Most importantly, these epigenetic alterations were found to contribute directly to cancer progression. Several HMT-containing protein complexes have now been implicated in human cancers. For example, the human homolog of Drosophila trithorax, MLL/ALL-1/HRX, which displays specific H3-K4 HMT activity and facilitates transcriptional activation, is frequently rearranged in human leukemias.52 The Polycomb group (PcG) protein enhancer of zeste homolog 2 (EZH2), a SET-dependent HMT, was found to be overexpressed in metastatic prostate cancer.53 Thus, disturbance in the delicate equilibrium of epigenetic signals that lead to transcriptional memory might contribute or even cause development and the progression of cancer.

CHROMATIN REMODELING IN DISEASES

ATP-dependent chromatin remodelling multi-protein complexes (such as SWI/SNF, RSF, NuRD, hCHRAC, and hACF) use the energy of ATP hydrolysis to introduce transient changes in nucleosomal DNA that facilitate the binding of transcription factors. Detailed description of their properties have been presented elsewhere.54 The studies of two SNF2 family proteins, ATRX (α-thalassaemia/ mental retardation syndrome, X-linked) and Lsh (lymphoid-specific helicase), have revealed that proteins with chromatin-remodelling and DNA-helicase activities are also required for DNA methylation and histone modification.55 One possibility is that ATP-dependent chromatin-remodeling complexes may have to modify the higher order chromatin structure to allow proper targeting of DNMTs or HMTs. Alternatively, these complexes may be crucial for enabling access of the enzymes to nucleosomal DNA or to the histone tails.

MODELS OF SILENCING SPREADING

In mammalian cells, gene silencing relies often on both DNA methylation and histone modifications. As mentioned above, a “cross-talk” between these two pathways has been shown. Detailed studies on the Oct3/4 gene demonstrated that promoter inactivation occurs in a well-defined stepwise manner.56 The PcG EED–EZH2 complex acts together with a yet unidentified H3-K9 HMT to methylate histone H3 at lysine 27 and 9, respectively. Subsequent recruitment of HP1 may help to keep Oct3/4 stably silenced. Similarly, X chromosome inactivation depends on histone deacetylation, lysine methylation, DNA methylation, and Xist RNA coating. Remarkably, the methylated histone-binding HP1 protein in turn interacts with HMTs and DNMTs enzymes (as mentioned above). Thus, this network of interactions might account for the propagation of the heterochromatin over a gene locus (Fig. 1), since every methylation site on DNA or histones creates a binding site for proteins capable of recruiting further DNMTs and HMTs. In this model, the status of the covalent modification of histone H3 plays a critical role in the initiation of the heterochromatin formation, while DNA methylation serves to “lock in” and maintain the repression. An alternative scenario can also been envisioned, in which methyl-CpG binding proteins, such as MeCP2, drive spreading of silencing via direct interaction and recruitment of HMTs to methylated promoters (Fig. 1). Interestingly, two HMT proteins, ESET and CLLD8, contain a putative MBD domain.28 CLLD8 was initially characterized as a candidate gene for leukemogenesis and was found to contain a methyl-CpG binding domain, in addition to a preSET and a SET www.landesbioscience.com

domain, that can potentially act as a histone H3-specific methyltransferase. A similar conjunction of MBD and SET domains has been found in the ESET protein. Although the functional importance of having these two domains present in one polypeptide is still poorly characterized, it is possible that it could direct the lysine methylase activity to DNA methylated promoters. In conclusion, these studies underline the complexity of the interplay between DNA methylation and histone modifications, arguing against a simple and unidirectional signal flow and while supporting the existence of a reinforcing feedback loop. The exact relationship between HP1 proteins, MBDs, DNMTs, and HMTs remains unclear and is currently the subject of intensive research. Perhaps DNA methylation has the unique role of a real epigenetic signal in terms that it is copied with high fidelity during replication and thus transmitted to the next cell generation. Understanding how histone modifications are transmitted during cell division is one of the most challenging goals of the epigenetic research community today.

A CONVENIENT MODEL SYSTEM: PML-RARα

Repressive chromatin appears to be self-maintaining through multiple protein-protein interactions, but the mechanism of de novo repression of particular genes in normal and cancer cells is much less understood. The molecules that target DNA methyltransferases and histone methyltransferases to specific genomic loci also remain to be discovered. Perhaps paradoxically, among the most informative studies on this issue derive from the analysis of acute promyelocytic leukemia (APL), a form of acute myeloid leukemia caused by the fusion protein PML-RARα, in which the retinoic acid receptor (RAR) gene is fused to the PML gene.57 In this disease, leukemic blasts are arrested at the promyelocytic stage of myeloid differentiation: clinical treatment of APLs with RA induces differentiation of leukemic blasts and disease remission. At the molecular level, the basic mechanism underlying all of the subsequent alterations is the conversion—in the context of PML-RARα—of RARα from a monomeric to an oligomeric form (due to its fusion to the oligomerization domain of PML).58 In turn, this causes the fusion protein to interact aberrantly with chromatin modifiers. Physiologically, RARs behave as transcription regulators of RA-target genes: they repress transcription in the absence of ligand, due to recruitment of HDACs and other corepressors. RA dissociates the HDAC complex and leads to recruitment of HATs and other coactivators, thus resulting in transcriptional activation.59 PML-RARα retains the ability of RAR to interact with corepressors. RA treatment can dissociate corepressors from both RAR and PML-RARα complexes, but substantially higher concentrations are required for PML-RARα, providing a biochemical explanation for the efficacy of pharmacological doses of RA in the treatment of the disease. HDAC inhibitors, however, are unable to fully revert the transcriptional repression of RAR target genes by PML-RARα, implying that other phenomena must occur. Recently, a further link has been established between DNA methylation and histone deacetylation: PML-RARα is able to recruit DNA methyltransferases (DNMTs), directing DNA methylation and histone deacetylation for the establishment of stable transcriptional silencing to the promoter of target genes (Fig. 1).51 This hints to a possible mechanism through which aberrant patterns of DNA methylation may be established and maintained in cancer cells: oncogenic transcription factors may directly recruit DNMTs to specific genomic regions (in the case of PML-RARα, to RAR binding sites) and thereby establish a locally altered DNA methylation status.

Cancer Biology & Therapy

835

ALTERED EPIGENETIC SIGNALS IN HUMAN DISEASE

In contrast to the results obtained with HDAC inhibitors, demethylating agents “relieve” the block of transcription and maturation imposed by the fusion protein. Thus, a complex process of coordinated epigenetic changes starts upon PML-RARα targeting, enabling the establishment of a fully repressive chromatin structure that will lead to stable, inheritable silencing of target genes.

CONCLUDING REMARKS: TOWARDS AN “EPIGENETIC” THERAPY?

We are in an exciting period of epigenetic research. The rapid progress within the past few years towards elucidating the molecular mechanisms controlling DNA and histone methylation, with respect to chromatin structure and heterochromatin formation, has been fundamental to set the basis for our understanding of many human diseases. We have learned that disruption of the epigenetic equilibrium within cells can cause severe diseases, including mental retardation and cancer. Although much has been learned, there are still several unanswered questions regarding how DNA methylation and histone modification influence each other, how the “histone code” affects promoter activity, how the transcriptional silencing spreads across a given region, how transcriptional memory is propagated through cell division, and, most importantly, how to reset the epigenetic equilibrium in tumor cells. The availability of molecules able to interfere with the action of several chromatin modifiers (at this stage, mainly HDAC inhibitors and demethylating agents), some of which are already in clinical trials, will help to exploit to the highest degree the new knowledge acquired in basic research. Understanding how epigenetic states are established and maintained and developing strategies to modify them therapeutically will therefore be an area of intense future research. References 1. Wolffe AP, Matzke MA. Epigenetics: Regulation through repression. Science 1999; 286:481-6. 2. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 a resolution. Nature 1997; 389:251-60. 3. Kornberg RD, Lorch Y. Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 1999; 98:285-94. 4. Ridgway P, Almouzni G. Chromatin assembly and organization. J Cell Sci 2001; 114:2711-2. 5. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16:6-21. 6. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349:2042-54. 7. Pasini D, Bracken AP, Helin K. Polycomb group proteins in cell cycle progression and cancer. Cell Cycle 2004; 3:396-400. 8. Esteller M. Cancer epigenetics: DNA methylation and chromatin alterations in human cancer. Adv Exp Med Biol 2003; 532:39-49. 9. Laird PW. The power and the promise of DNA methylation markers. Nat Rev Cancer 2003; 3:253-66. 10. Felsher DW. Cancer revoked: Oncogenes as therapeutic targets. Nat Rev Cancer 2003; 3:375-80. 11. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer 2004; 4:143-53. 12. Plass C. Cancer epigenomics. Hum Mol Genet 2002; 11:2479-88. 13. Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet 2000; 9:2395-402. 14. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992; 69:915-26. 15. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases dnmt3a and dnmt3b are essential for de novo methylation and mammalian development. Cell 1999; 99:247-57. 16. Leonhardt H, Page AW, Weier HU, Bestor TH. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 1992; 71:865-73. 17. Chuang LS, Ian HI, Koh TW, Ng HH, Xu G, Li BF. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 1997; 277:1996-2000. 18. Fatemi M, Hermann A, Pradhan S, Jeltsch A. The activity of the murine DNA methyltransferase dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA. J Mol Biol 2001; 309:1189-99. 19. Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the establishment of maternal genomic imprints. Science 2001; 294:2536-9. 20. Bird AP, Wolffe AP. Methylation-induced repression—belts, braces, and chromatin. Cell 1999; 99:451-4.

836

21. Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol 1998; 18:6538-47. 22. Meehan RR, Lewis JD, McKay S, Kleiner EL, Bird AP. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 1989; 58:499-507. 23. Robertson KD. DNA methylation and chromatin - unraveling the tangled web. Oncogene 2002; 21:5361-79. 24. Wade PA. Methyl CpG-binding proteins and transcriptional repression. Bioessays 2001; 23:1131-7. 25. Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T. The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem 2002. 26. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 1998; 19:187-91. 27. Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 1998; 393:386-9. 28. Sims IIIrd RJ, Nishioka K, Reinberg D. Histone lysine methylation: A signature for chromatin function. Trends Genet 2003; 19:629-39. 29. Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293:1074-80. 30. Meehan RR. DNA methylation in animal development. Semin Cell Dev Biol 2003; 14:53-65. 31. Csankovszki G, Nagy A, Jaenisch R. Synergism of xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation. J Cell Biol 2001; 153:773-84. 32. Yang XJ, Seto E. Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression. Curr Opin Genet Dev 2003; 13:143-53. 33. Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. Histone deacetylases and cancer: Causes and therapies. Nat Rev Cancer 2001; 1:194-202. 34. Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new corepressor, DMAP1, to form a complex at replication foci. Nat Genet 2000; 25:269-77. 35. Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet 2000; 25:338-42. 36. Fuks F, Burgers WA, Brehm A, Hughes-Davies L, Kouzarides T. DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat Genet 2000; 24:88-91. 37. Lachner M, O’Sullivan RJ, Jenuwein T. An epigenetic road map for histone lysine methylation. J Cell Sci 2003; 116:2117-24. 38. Tamaru H, Selker EU. A histone H3 methyltransferase controls DNA methylation in neurospora crassa. Nature 2001; 414:277-83. 39. Jackson JP, Lindroth AM, Cao X, Jacobsen SE. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 2002; 416:556-60. 40. Bannister AJ, Zegerman P, Partridge JF, Miska EA, Thomas JO, Allshire RC, et al. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 2001; 410:120-4. 41. Lachner M, O’Carroll D, Rea S, Mechtler K, Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 2001; 410:116-20. 42. Fischle W, Wang Y, Jacobs SA, Kim Y, Allis CD, Khorasanizadeh S. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev 2003; 17:1870-81. 43. Xu GL, Bestor TH, Bourc’his D, Hsieh CL, Tommerup N, Bugge M, et al. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 1999; 402:187-91. 44. Amir R, Dahle EJ, Toriolo D, Zoghbi HY. Candidate gene analysis in rett syndrome and the identification of 21 SNPs in Xq. Am J Med Genet 2000; 90:69-71. 45. Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, et al. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 2003; 302:885-9. 46. Martinowich K, Hattori D, Wu H, Fouse S, He F, Hu Y, et al. DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science 2003; 302:890-3. 47. Stancheva I, Collins AL, Van den Veyver IB, Zoghbi H, Meehan RR. A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in xenopus embryos. Mol Cell 2003; 12:425-35. 48. El-Osta A. FMR1 silencing and the signals to chromatin: A unified model of transcriptional regulation. Biochem Biophys Res Commun 2002; 295:575-81. 49. El-Osta A. DNMT cooperativity—the developing links between methylation, chromatin structure and cancer. Bioessays 2003; 25:1071-84. 50. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 1999; 96:8681-6. 51. Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 2002; 295:1079-82. 52. Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD, et al. MLL targets SET domain methyltransferase activity to hox gene promoters. Mol Cell 2002; 10:1107-17. 53. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002; 419:624-9.

Cancer Biology & Therapy

2004; Vol. 3 Issue 9

ALTERED EPIGENETIC SIGNALS IN HUMAN DISEASE

54. Neely KE, Workman JL. The complexity of chromatin remodeling and its links to cancer. Biochim Biophys Acta 2002; 1603:19-29. 55. Cho KS, Elizondo LI, Boerkoel CF. Advances in chromatin remodeling and human disease. Curr Opin Genet Dev 2004; 14:308-15. 56. Erhardt S, Su IH, Schneider R, Barton S, Bannister AJ, Perez-Burgos L, et al. Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development. Development 2003; 130:4235-48. 57. Villa R, De Santis F, Gutierrez A, Minucci S, Pelicci PG, Di Croce L. Epigenetic gene silencing in acute promyelocytic leukemia. Biochem Pharmacol 2004; In press. 58. Minucci S, Maccarana M, Cioce M, De Luca P, Gelmetti V, Segalla S, et al. Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation. Mol Cell 2000; 5:811-20. 59. Di Croce L, Okret S, Kersten S, Gustafsson JA, Parker M, Wahli W, et al. Steroid and nuclear receptors. EMBO J 1999; 18:6201-10. 60. Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003; 300:455. 61. Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, et al. Induction of tumors in mice by genomic hypomethylation. Science 2003; 300:489-92. 62. Lei H, Oh SP, Okano M, Juttermann R, Goss KA, Jaenisch R, et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 1996; 122:3195-205. 63. Robert MF, Morin S, Beaulieu N, Gauthier F, Chute IC, Barsalou A, et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet 2003; 33:61-5. 64. Hata K, Okano M, Lei H, Li E. Dnmt3L cooperates with the dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 2002; 129:1983-93. 65. Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 2002; 416:552-6. 66. Soejima K, Fang W, Rollins BJ. DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated ras. Oncogene 2003; 22:4723-33. 67. Beaulieu N, Morin S, Chute IC, Robert MF, Nguyen H, MacLeod AR. An essential role for DNA methyltransferase DNMT3B in cancer cell survival. J Biol Chem 2002; 277:28176-81. 68. Chen RZ, Akbarian S, Tudor M, Jaenisch R. Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat Genet 2001; 27:327-31. 69. Guy J, Hendrich B, Holmes M, Martin JE, Bird A. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet 2001; 27:322-6. 70. Hendrich B, Guy J, Ramsahoye B, Wilson VA, Bird A. Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development. Genes Dev 2001; 15:710-23. 71. Campbell PM, Bovenzi V, Szyf M. Methylated DNA-binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo. Carcinogenesis 2004; 25:499-507. 72. Sansom OJ, Berger J, Bishop SM, Hendrich B, Bird A, Clarke AR. Deficiency of Mbd2 suppresses intestinal tumorigenesis. Nat Genet 2003; 34:145-7. 73. Millar CB, Guy J, Sansom OJ, Selfridge J, MacDougall E, Hendrich B, et al. Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice. Science 2002; 297:403-5. 74. Riccio A, Aaltonen LA, Godwin AK, Loukola A, Percesepe A, Salovaara R, et al. The DNA repair gene MBD4 (MED1) is mutated in human carcinomas with microsatellite instability. Nat Genet 1999; 23:266-8. 75. Yamada T, Koyama T, Ohwada S, Tago K, Sakamoto I, Yoshimura S, et al. Frameshift mutations in the MBD4/MED1 gene in primary gastric cancer with high-frequency microsatellite instability. Cancer Lett 2002; 181:115-20. 76. Evertson S, Wallin A, Arbman G, Rutten S, Emterling A, Zhang H, et al. Microsatellite instability and MBD4 mutation in unselected colorectal cancer. Anticancer Res 2003; 23:3569-74. 77. Dodge JE, Kang YK, Beppu H, Lei H, Li E. Histone H3-K9 methyltransferase ESET is essential for early development. Mol Cell Biol 2004; 24:2478-86. 78. Peters AH, O’Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, et al. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 2001; 107:323-37. 79. Dennis K, Fan T, Geiman T, Yan Q, Muegge K. Lsh, a member of the SNF2 family, is required for genome-wide methylation. Genes Dev 2001; 15:2940-4. 80. Picketts DJ, Higgs DR, Bachoo S, Blake DJ, Quarrell OW, Gibbons RJ. ATRX encodes a novel member of the SNF2 family of proteins: Mutations point to a common mechanism underlying the ATR-X syndrome. Hum Mol Genet 1996; 5:1899-907. 81. O’Carroll D, Erhardt S, Pagani M, Barton SC, Surani MA, Jenuwein T. The polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol 2001; 21:4330-6. 82. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 2003; 22:5323-35. 83. Koontz JI, Soreng AL, Nucci M, Kuo FC, Pauwels P, van Den Berghe H, et al. Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors. Proc Natl Acad Sci USA 2001; 98:6348-53.

www.landesbioscience.com

84. Wang J, Mager J, Chen Y, Schneider E, Cross JC, Nagy A, et al. Imprinted X inactivation maintained by a mouse polycomb group gene. Nat Genet 2001; 28:371-5. 85. Faust C, Lawson KA, Schork NJ, Thiel B, Magnuson T. The Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo. Development 1998; 125:4495-506. 86. Lehnertz B, Ueda Y, Derijck AA, Braunschweig U, Perez-Burgos L, Kubicek S, et al. Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol 2003; 13:1192-200. 87. Fuks F, Hurd PJ, Deplus R, Kouzarides T. The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 2003; 31:2305-12.

Cancer Biology & Therapy

837