My research interests are in the area of epigenetics, specifically DNA methylation, and its role in cancer and development.  Most of our work centers on the enzymes that carry out the DNA methylation reaction in mammalian cells, the DNA methyltransferases (or DNMTs).  We want to understand how cells determine which sequences will be methylated and which will not, and how this regulation goes awry in cancer cells as well as how it is involved in cell differentiation.  Specific research interests fall into four general areas:  (1) The DNMTs are expressed in recombinant form and their properties studied using in vitro and in vivo enzyme assays.  (2) Their protein-interaction partners in vivo are identified using biochemical methods and yeast two-hybrid assays and the functional consequences of the interactions are studied in cells and in reconstituted in vitro systems.  (3) We also use high-throughput gene expression techniques, coupled with pharmacologic manipulation of DNA methylation levels, to uncover genes aberrantly silenced by DNA methylation in cancer.  (4) Lastly, we are interested in understanding the epigenetic make-up of pluripotent cells and how DNA methylation is involved in regulating their differentiation.

To learn more about each of these areas, please continue reading.

 
1) DNA Methylation in Mammalian Cells.

With the completion of the human genome project we now have a complete list of all genes needed to produce a human being.  Years of intensive research, however, have revealed that the situation is far more complex than a simple catalog of genes.  Of equal importance is a second system that cells use to determine when and where a particular gene will be expressed during development.  This system, which encodes spatiotemporal information, is overlaid upon the DNA in the form of epigenetic marks, which are heritable during cell division but do not alter the DNA sequence.  The only known epigenetic modification of DNA in mammalian cells is the methylation of C-5 of cytosines that occur as part of a CpG dinucleotide (Bird 2002) (Fig. 1).  By contrast, the other major group of epigenetic modifications — post-translational modification of the core histone N-terminal tails — shows a high level of diversity and complexity (Fischle et al. 2003).  Recent important studies from our lab and others have shown that most of the DNA and histone epigenetic modification machineries are coupled in their regulation through the interaction of their protein components.  Groundbreaking new research has also revealed that RNA plays an important role in regulating gene expression and chromatin structure, and likely influences DNA methylation patterns as well (Novina et al. 2004).

The mammalian DNA methylation machinery is made up of two components, the DNA methyltransferases (DNMTs) that establish and maintain DNA methylation patterns genome-wide, and the methyl-CpG binding proteins (MBDs), which are involved in ‘reading’ the methylation mark (Fig. 1).  Properly established and maintained DNA methylation patterns are essential for mammalian development and normal functioning of the adult organism.  DNA methylation is a potent mechanism for silencing gene expression and maintaining genome stability in the face of a vast amount of repetitive DNA (Fig. 1).  Embryonic stem cells deficient for DNA methyltransferase are viable but die when induced to differentiate (Panning et al. 1996).  Loss of normal DNA methylation patterns in somatic cells results in loss of cell growth control.  In normal cells, DNA methylation is found predominantly in the repetitive fraction of the genome, including satellite DNA and parasitic elements (LINES, SINES, endogenous retroviruses) (Yoder et al. 1997).  CpG islands, particularly those associated with gene promoters, are generally unmethylated, although an increasing number of exceptions are being identified (Bird 1986; Song et al. 2005).  Little is known about how DNA methylation is targeted to specific regions, however it most likely involves interactions between the DNMTs and chromatin-associated proteins (Figs. 2 & 4) (Robertson 2002).

There are five known DNA methyltransferase (DNMT) family members, DNMT1, 2, 3A, 3B, and 3L (Fig. 2) in mammalian cells that establish genome-wide DNA methylation patterns during embryonic development and maintain them in somatic cells (Bestor 2000; Robertson 2002).  DNMT1 is the most abundant and catalytically active enzyme in most cell types, which associates with S-phase replication foci via interaction with PCNA (Leonhardt et al. 1992; Chuang et al. 1997; Yokochi et al. 2002).  Its primary role is believed to be that of a maintenance methyltransferase (Bestor et al. 1996; Bestor 2000), copying DNA methylation patterns following DNA replication.  Murine knockouts of Dnmt1 are embryonic lethal at day E8.5.  The function of DNMT2 remains unclear since it possesses very low enzymatic activity in vitro and knockout of the gene in mice produces no discernable phenotype (Okano et al. 1998; Yoder et al. 1998; Hermann et al. 2003).  DNMT3A and DNMT3B are regarded as de novo methyltransferases since they are highly expressed at the stage of murine embryonic development (embryo implantation) when waves of de novo methylation are occurring in the genome (Okano et al. 1999).  DNMT3A and DNMT3B methylate hemimethylated and unmethylated DNA equally well and our work demonstrated that DNMT3A actually has a 3-4-fold preference for unmethylated DNA (Okano et al. 1998; Yokochi et al. 2002).  Murine Dnmt3a knockout mice are born live but die before reaching four weeks of age.  Dnmt3b knockout mice are embryonic lethal by ~day E14.5.  Dnmt3a knockout mice exhibit subtle DNA methylation defects in maternally imprinted regions (Hata et al. 2002), while Dnmt3b knockout mice show marked demethylation of pericentromeric satellite repeats (Okano et al. 1999).  Interestingly, knockout of Dnmt3L, which is not a functional enzyme due to lack of critical catalytic site motifs, results in maternal DNA methylation imprint failure and male sterility in mice (Hata et al. 2002).

One of the major goals of my laboratory is to identify protein-protein interactions involving the DNA methyltransferases using biochemical methods and yeast two-hybrid screening assays.  Once we identify interactions, their functional consequences are monitored using a variety of different enzymatic assays, siRNA knock-down technology, chromatin immunoprecipitation, and immunofluorescence microscopy.  In this way, we hope to decipher how each of the DNMTs works to properly methylate the genome.

DNA Methyltransferase 3B (DNMT3B) and its role in cancer, chromatin structure, and genome stability.
DNMT3B, first cloned in 1998, has come to be regarded as one of the de novo methyltransferases (Okano et al. 1998).  DNMT3B has a number of fascinating properties which is the reason we have focused significant effort on studying its regulation.  DNMT3B contains a plant homeodomain (PHD, or ATRX-like region) within its N-terminal domain (Fig. 2), a motif common to other chromatin-associated proteins (Aasland et al. 1995), and it localizes to DAPI-dense heterochromatin regions and co-localizes and interacts with HP1 proteins (Bachman et al. 2001; Lehnertz et al. 2003; Geiman et al. 2004).  DNMT3B appears to have some target sequences in common with DNMT3A but also unique ones, and its site specificity may depend on the particular splice variant expressed (there are six known, termed DNMT3B1-6, Fig. 2) (Chen et al. 2003).  DNMT3B is required for de novo and maintenance methylation (Okano et al. 1999; Chen et al. 2003).  Antisense depletion of DNMT3B in certain cancer cell lines (but not normal cells) results in apoptosis and demethylation of aberrantly methylated tumor suppressor genes, indicating that it is essential for the survival of at least some cancers (Beaulieu et al. 2002).  Antisense knock down of DNMT3B in immortalized cells inhibited their capacity to grow in soft agar, while DNMT3B-negative transformed cells were significantly less tumorigenic than their counterparts expressing DNMT3B, and demonstrated reactivation of aberrantly silenced growth regulatory genes (Soejima et al. 2003).  Remarkably, while some of the reactivated genes became demethylated following DNMT3B depletion in this study, suggesting that hypermethylation was the event responsible for silencing, other genes were reactivated with no change in their methylation status, suggesting that DNMT3B may regulate genes via its HDAC-dependent transcriptional repression functions (Bachman et al. 2001; Soejima et al. 2003).  Curiously, although point mutations in DNMT3B result in satellite repeat hypomethylation and cause Immunodeficiency, Centromere instability, Facial anomalies (ICF) syndrome in humans, antisense depletion or somatic cell knock out in culture does not (Beaulieu et al. 2002; Rhee et al. 2002).  ICF syndrome is a rare recessive disorder caused by mutations in the DNMT3B gene and is the only known human disease associated with mutations in a DNA methyltransferase (Hansen et al. 1999; Okano et al. 1999; Xu et al. 1999).  ICF individuals demonstrate hypomethylation of juxtacentromeric heterochromatin on chromosomes 1,9 and 16, numerous structural aberrations in these chromosomes (breaks, translocations, and multi-radials for example), and immunoglobulin deficiency (Ehrlich 2003).  DNMT3B is over-expressed in many tumors (Robertson et al. 1999; Kanai et al. 2001; Saito et al. 2001).  It can clearly contribute to aberrant hypermethylation based on the studies just mentioned, paradoxically however, over-expression of splice variants believed to be catalytically inactive results in satellite DNA hypomethylation, a frequent and early event in hepatocarcinogenesis associated with heterochromatin instability (Saito et al. 2002).

Click this link for relevant publications from our lab.

 
2) DNA Methylation and Cancer.

It was recognized nearly twenty years ago that DNA methylation patterns in tumor cells are altered relative to those of normal cells (Goelz et al. 1985; Feinberg et al. 2004).  Tumor cells exhibit global hypomethylation of the genome accompanied by region-specific hypermethylation events (Baylin et al. 2001).  Most of the hypomethylation occurs in repetitive DNA that is normally heavily methylated (Yoder et al. 1997).  This results in increased transcription from transposable elements and an elevated mutation rate due to mitotic recombination (Chen et al. 1998; Eden et al. 2003).  Regions that are frequent targets of hypermethylation events are CpG islands.  Abnormal methylation of CpG islands can efficiently repress transcription of the associated gene in a manner akin to deletion and act as one of the ‘hits’ in the Knudsen model (Baylin et al. 2001; Jones et al. 2002) (Fig. 3).  There are now numerous lines of evidence indicating that aberrant DNA methylation patterns have a direct role in carcinogenesis.  An elegant study by Costello et al., utilizing a restriction landmark genomic scanning (RLGS) approach to analyze the methylation status of 1,184 CpG islands from 98 tumor samples, revealed that CpG island de novo methylation in tumor cells is widespread and differed between individual tumors and tumor types (Costello et al. 2000).  An average of 608 (and potentially up to 4,500) CpG islands were aberrantly hypermethylated in tumors in a non-random manner indicating that certain CpG islands may be more susceptible to de novo methylation than others (Costello et al. 2000).  Thus, ample evidence exists to support the notion that DNA hypermethylation acts as a primary inactivating event contributing directly to tumorigenesis.  A recent study demonstrated that the growth rate of a cancer cell can indeed depend on the silencing of a specific tumor suppressor gene ( p16^INK4a) due to DNA methylation (Bachman et al. 2003).

DNA Methylation as a Biomarker for Cancer.
Early detection results in an improved clinical outcome for most cancers, therefore significant effort is being devoted to developing early detection strategies (Kelloff et al. 2004).  DNA methylation changes in cancer, particularly CpG island hypermethylation, have been extensively studied and are very frequent and early events (Kopelovich et al. 2003).  Genes targeted for aberrant DNA methylation-mediated silencing in many tumor types include  APC, HIC1, p16^INK4a, MGMT, TIMP3, E-cadherin, and  hMLH1 (Kopelovich et al. 2003; Paz et al. 2003).  Hypermethylation of a number of these genes, such as MGMT, a gene involved in DNA repair, also occurs frequently in colon adenomas (a precursor to colon cancer) and is thought to predispose these tumors to KRAS mutations (Esteller et al. 2000; Rashid et al. 2001).  There is also significant evidence that DNA methylation changes occur even earlier in the progression of colon cancer, the pre-adenoma stage.  Aberrant crypt foci in colorectal mucosa are believed to be the earliest known precursor lesions to colorectal cancer.  Promoter hypermethylation of several genes, including  p16^INK4a and MGMT, has also been detected in aberrant crypt foci (Chan et al. 2002).  Therefore, available evidence strongly suggests that DNA methylation changes are early events in tumorigenesis that directly contribute to tumor development.  Lastly DNA methylation abnormalities have been detected in patients with ulcerative colitis, a chronic inflammatory condition of the large intestine that predisposes to cancer.  Another essential component of a good early detection biomarker is a robust assay to detect methylation changes.  Assays such as methylation specific PCR (MSP) are sufficiently robust and sensitive to detect promoter hypermethylation in bodily fluids (serum, plasma, urine) and fecal material (Laird 2003; Muller et al. 2004).  The large body of evidence demonstrating that abnormalities in DNA methylation are one of the earliest changes in cancer progression (much of this work comes from studying colon cancer), combined with the availability of robust, sensitive, and non-invasive detection methods, makes DNA methylation an excellent early detection biomarker for colon cancer.  One of the main interests of the lab is to identify methylated genes that will serve as useful early detection biomarkers for a variety of tumor types.

DNA Methylation as a Chemoprevention Target.
Unlike tumor suppressor genes that have been inactivated by genetic mutations, genes silenced by DNA methylation are intact and can be reactivated by small molecule inhibitors of the DNMTs (Kopelovich et al. 2003).  Therapies targeting DNA methylation and the DNA methyltransferases have therefore attracted significant interest as a means for cancer prevention.  Inhibitors of DNA methylation, such as 5-aza-2'-deoxycytidine (5-azadC), zebularine (Cheng et al. 2004), antisense oligonucleotides to DNMT1 (MacLeod et al. 1995), and procainamide (Lin et al. 2001), are capable of reversing aberrant promoter hypermethylation, resulting in gene reactivation and restoration of cell growth control, apoptosis, and DNA repair capacity (Murakami et al. 1995; Bender et al. 1998; Herman et al. 1998; Zhu et al. 2001).  5-azadC administered to Apc^Min/+ mice, a model for human colon cancer, resulted in dramatically reduced rates of intestinal tumor formation (Laird et al. 1995).  In other studies, 5-azadC treatment diminished the formation of aberrant crypt foci in the colons of selenium-deficient rats that had been treated with carcinogens (Davis et al. 2002).  Administration of 5-azadC to mice also prevented lung tumor development in animals treated with a carcinogen found in tobacco smoke (Belinsky et al. 2003).  Thus there is ample evidence that targeting epigenetic changes in ‘normal’ tissues with small molecule DNMT inhibitors results in dramatic reductions in certain cancers in model systems.  Although existing demethylating agents such as 5-azadC may not be ideal for use in a prevention strategy (requiring long-term administration) due to safety concerns, other agents such as procainamide, zebularine, and valproic acid (Detich et al. 2003) may be safer for long-term use.  In addition, there is now significant interest in the pharmaceutical industry in developing new and more specific inhibitors of epigenetic modifications; therefore new drugs better suited for a chemoprevention strategy will likely be available within the next few years.  DNA methylation changes are very early events directly contributing to many cancers, they can be readily detected with non-invasive assays, and DNMT inhibitors dramatically reduce the incidence of colon cancer in high-risk model systems.  These aspects make promoter hypermethylation an excellent early detection biomarker and target for cancer chemoprevention.

Another one of the major goals of my laboratory is to use high-throughput methods (such as gene-chip expression microarrays) to identify genes that are silenced with high frequency at a very early stage of cancer by aberrant DNA methylation.  With these genes in hand, we will attempt to validate them as early detection cancer biomarkers by monitoring their DNA methylation status in individuals at risk for a particular type of cancer (due to family history or exposure to some carcinogen such as tobacco smoke for example).  We hope that one or more of these biomarkers will be useful for detecting cancer earlier and less invasively than currently available methods, thereby enhancing the success of treatment.

Click this link for relevant publications from our lab.

 
3) Connections between DNA Methylation and Chromatin Remodeling.

Modifications to the chromatin and the core histone ‘tails’ impact gene expression and the ability of proteins that act on DNA to access their target sequences.  Recent studies indicate that these epigenetic processes, as well as chromatin remodeling, cooperate to control chromatin structure and ultimately gene expression and DNA methylation itself.  Histone H3 and H4 tail modifications include acetylation, phosphorylation, and methylation (Jenuwein et al. 2001).  An intricate interplay exists between different modifications on various sites of the histone tails of H3 and H4, some of which act antagonistically to regulate the conversion from an active chromatin state to an inactive one (termed the histone code (Lachner et al. 2003)).  In particular, methylation of lysine residues on the histone H3 tail appear to be intimately connected to the DNA methylation status of the particular chromatin region in which it occurs (Tamaru et al. 2001; Geiman et al. 2002; Jackson et al. 2002) (Fig. 4).  Treatment of human cancer cells with 5-aza-CdR results in rapid loss of repressive histone H3 lysine 9 methylation and DNA methylation indicating that there is feedback between the two types of methylation (Fahrner et al. 2002; Nguyen et al. 2002).

Chromatin remodeling proteins of the SNF2 family, which utilize ATP to alter the structure of chromatin through disruption of histone/DNA contacts (Havas et al. 2001), also influence DNA methylation patterns.  The mammalian Lsh (lymphoid specific helicase) gene, an SNF2 helicase family member (Jarvis et al. 1996) exerts profound effects on DNA methylation patterns.  Lsh knockout mice exhibit drastic losses of DNA methylation, both in repetitive elements and single copy genes (Dennis et al. 2001).  ATRX, another SNF2 family member, is mutated in ATRX (alpha thalassemia and mental retardation, X-linked) syndrome and these patients exhibit both hyper- and hypomethylation changes in repetitive elements (Gibbons et al. 2000).  We have found that both DNMT1 and DNMT3B interact with the hSNF2H chromatin remodeling enzyme (Fig. 4) (Geiman et al. 2004; Geiman et al. 2004; Robertson et al. 2004).  SNF2-like proteins may be required for accessibility of DNA methyltransferases to DNA in chromatin.  in vivo the DNMTs must contend with a multitude of other DNA binding proteins and nucleosomal chromatin structure.  Therefore chromatin-associated factors probably dictate the targeting or access of the DNMTs to particular DNA sequences.  This is most likely mediated by protein-protein interactions.  For example, DNMT1 and DNMT3A are known to interact with HDAC1 and HDAC2 (Robertson et al. 2000; Rountree et al. 2000; Fuks et al. 2001; Ling et al. 2004), although the functional consequence of this interaction is unclear.  We and others have identified a number of interesting interactions between DNMT3B and components of the chromatin modification machinery, more than for any other DNMT (summarized in Figs. 2 & 4).  Thus, although many indirect connections have been established between chromatin modifications and DNA methylation, the exact mechanistic relationship, i.e. what epigenetic modification targets another and how, remains largely unknown.

Elucidating the exact nature of the interactions between and DNA and histone methylation machineries is a major focus of the lab. Previously, we have used biochemical purification methods to show that DNMT1 interacts with histone deacetylases and the retinoblastoma tumor suppressor gene and that DNMT3B interacts with histone deacetylases, histone methylase (H3-K9), and ATP-dependent chromatin remodeling enzymes. We expect that by that by continuing to purify additional DNMT-containing complexes we will identify other novel interactions. The functions of these complexes will then be studied using recombinant proteins and chromatin templates assembled in vitro.

Click this link for relevant publications from our lab.

 
4) Epigenetics and the Regulation of Pluripotency and Differentiation of Stem Cells.

The non-random distribution of cellular DNA methylation patterns in mammalian cells is critical to normal cellular functioning, differentiation, transcriptional regulation, embryonic development, and genome stability (Dean et al. 2001; Jones et al. 2002; Li 2002; Robertson 2002).  Although much is known about the role of DNA methylation in cancer, far less is known about its role in differentiation.  DNA methylation patterns are unstable in ES cells in culture and are significantly altered in cloned mammals (Humpherys et al. 2001; Santos et al. 2003).  DNMT-deficient embryonic stem cells die when induced to differentiate in vitro and treatment of certain cell types with DNA methylation inhibitors causes them to differentiate (Jones 1985; Li et al. 1992).  Aberrant DNA methylation patterns, in addition to other epigenetic modifications to the genome such as histone acetylation and histone methylation, are believed to be major contributors to the low efficiency of cloning (Santos et al. 2003).  Epigenetic genome modifications are also likely to be intimately involved in maintaining embryonic stem cell pluripotency and in the orderly differentiation of these cells into many other cell types.  ES cells are genetically identical to the differentiated cells they give rise to, yet these differentiated cells have radically different transcription profiles and highly specialized functions, suggesting that epigenetic modifications must play a prominent role (Fig. 5).  Therefore a better understanding of the epigenetic modification machinery in pluripotent cells and its role in regulating differentiation will provide critical and novel information that will enhance our abilities to manipulate stem cells for therapeutic purposes and aid in the treatment and prevention of cancer.

A major goal of the laboratory is to define the activity and protein-interaction partners of the DNA methyltransferases in pluripotent cells and analyze how these change during the differentiation process (Fig. 5).

Click this link for relevant publications from our lab.

 
References:

1. Aasland R, Gibson TJ and Stewart AF (1995). "The PHD finger: implications for chromatin-mediated transcriptional regulation." Trends Biochem. Sci 20: 56-59.
2. Bachman KE, Park BH, Rhee I, Rajagopalan H, Herman JG, Baylin SB, Kinzler KW and Vogelstein B (2003). "Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene." Cancer Cell 3: 89-95.
3. Bachman KE, Rountree MR and Baylin SB (2001). "Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin." J. Biol. Chem. 276(34): 32282-32287.
4. Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K and Herman JG (2001). "Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer." Hum. Mol. Genet. 10(7): 687-692.
5. Beaulieu N, Morin S, Chute IC, Robert M-F, Nguyen H and MacLeod AR (2002). "An essential role for DNA methyltransferase DNMT3B in cancer cell survival." J. Biol. Chem. 277(31): 28176-28181.
6. Belinsky SA, Klinge DM, Stidley CA, Issa J-P, Herman JG, March TH and Baylin SB (2003). "Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer." Cancer Res. 63: 7089-7093.
7. Bender CM, Pao MM and Jones PA (1998). "Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines." Cancer Res. 58: 95-101.
8. Bestor TH (2000). "The DNA methyltransferases." Hum. Mol. Genet. 9(16): 2395-2402.
9. Bestor TH and Tycko B (1996). "Creation of genomic methylation patterns." Nature Genet. 12: 363-367.
10. Bird A (1986). "CpG-rich islands and the function of DNA methylation." Nature 321: 209-213.
11. Bird A (2002). "DNA methylation patterns and epigenetic memory." Genes. Dev. 16: 6-21.
12. Chan AO-O, Broaddus RR, Houlihan PS, Issa J-PJ, Hamilton SR and Rashid A (2002). "CpG island methylation in aberrant crypt foci of the colorectum." Am. J. Pathol. 160(5): 1823-1830.
13. Chen RZ, Pettersson U, Beard C, Jackson-Grusby L and Jaenisch R (1998). "DNA hypomethylation leads to elevated mutation rates." Nature 395: 89-93.
14. Chen T, Ueda Y, Dodge JE, Wang Z and Li E (2003). "Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b." Mol. Cell. Biol. 23(16): 5594-5605.
15. Cheng JC, Weisenberger DJ, Gonzales FA, Liang G, Xu G-L, Hu Y-G, Marquez VE and Jones PA (2004). "Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells." Mol. Cell. Biol. 24(3): 1270-1278.
16. Chuang LS-H, Ian H-I, Koh T-W, Ng H-H, Xu G and Li BFL (1997). "Human DNA-(cytosine-5) methyltransferase-PCNA complex is a target for p21^Waf1." Science 277: 1996-2000.
17. Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright FA, Fermisco JD, Peltomaki P, Lang JC, Schuller DE, Yu L, Bloomfield CD, Caligiuri MA, Yates A, Nishikawa R, Huang H-JS, Petrelli NJ, Zhang X, O'Dorisio MS, Held WA, Cavenee WK and Plass C (2000). "Aberrant CpG-island methylation has a non-random and tumor-type-specific patterns." Nature Genet. 25: 132-138.
18. Davis CD and Uthus EO (2002). "Dietary selenite and azadeoxycytidine treatment affect dimethylhydrazine-induced aberrant crypt formation in rat colon and in HT-29 cells." J. Nutr. 132: 292-297.
19. Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E and Reik W (2001). "Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos." Proc. Natl. Acad. Sci. USA 98(24): 13734-13738.
20. Dennis K, Fan T, Geiman T, Yan Q and Muegge K (2001). "LSH, a member of the SNF2 family, is required for genome-wide methylation." Genes. Dev. 15: 2940-2944.
21. Detich N, Bovenzi V and Szyf M (2003). "Valproate induces replication-independent active DNA demethylation." J. Biol. Chem. 278(30): 27586-27592.
22. Eden A, Gaudet F, Waghmare A and Jaenisch R (2003). "Chromosomal instability and tumors promoted by DNA hypomethylation." Science 300: 455.
23. Ehrlich M (2003). "The ICF syndrome, a DNA methyltransferase 3B deficiency and immunodeficiency disease." Clin. Immunol. 109: 17-28.
24. Esteller M, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA, Watkins DN, Issa JP, Sidransky D, Baylin SB and Herman JG (2000). "Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis." Cancer Res. 60(9): 2368-2371.
25. Fahrner JA, Eguchi S, Herman JG and Baylin SB (2002). "Dependence of histone modifications and gene expression on DNA hypermethylation in cancer." Cancer Res. 62: 7213-7218.
26. Feinberg AP and Tycko B (2004). "The history of cancer epigenetics." Nature Rev. Canc. 4: 1-11.
27. Fischle W, Wang Y and Allis CD (2003). "Binary switches and modification cassettes in histone biology and beyond." Nature 425: 475-479.
28. Fuks F, Burgers WA, Godin N, Kasai M and Kouzarides T (2001). "Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription." EMBO J. 20(10): 2536-2544.
29. Geiman TM and Robertson KD (2002). "Chromatin remodeling, histone modifications, and DNA methylation - how does it all fit together?" J. Cell. Biochem. 87: 117-125.
30. Geiman TM, Sankpal UT, Robertson AK, Chen Y, Mazumdar M, Heale JT, Schmiesing JA, Kim W, Yokomori K, Zhao Y and Robertson KD (2004). "Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery." Nucleic Acids Res. 32(9): 2716-2729.
31. Geiman TM, Sankpal UT, Robertson AK, Zhao Y, Zhao Y and Robertson KD (2004). "DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system." Biochem. Biophys. Res. Commun. 318: 544-555.
32. Gibbons RJ, McDowell TL, Raman S, O'Rourke DM, Garrick D, Ayyub H and Higgs DR (2000). "Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the patterns of DNA methylation." Nature Genet. 24: 368-371.
33. Goelz SE, Vogelstein B, Hamilton SR and Feinberg AP (1985). "Hypomethylation of DNA from benign and malignant human colon neoplasms." Science 228: 187-190.
34. Hansen RS, Wijmenga C, Luo P, Stanek AM, Canfield TK, Weemaes CMR and Gartler SM (1999). "The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome." Proc. Natl. Acad. Sci. USA 96(25): 14412-14417.
35. Hata K, Okano M, Lei H and Li E (2002). "Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice." Development 129: 1983-1993.
36. Havas K, Whitehouse I and Owen-Hughes T (2001). "ATP-dependent chromatin remodeling activities." Cell. Mol. Life Sci. 58: 673-682.
37. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa J-PJ, Markowitz S, Willson JKV, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA and Baylin SB (1998). "Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal cancer." Proc. Natl. Acad. Sci. USA 98: 6870-6875.
38. Hermann A, Schmitt S and Jeltsch A (2003). "The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity." J. Biol. Chem. 278(34): 31717-31721.
39. Humpherys D, Eggan K, Akutsu H, Hochedlinger K, III WMR, Biniszkiewicz D, Yanagimachi R and Jaenisch R (2001). "Epigenetic instability in ES cells and cloned mice." Science 293: 95-97.
40. Jackson JP, Lindroth AM, Cao X and Jacobsen SE (2002). "Control of CpNpGp DNA methylation by the KRYPTONITE histone H3 methyltransferase." Nature 416: 556-560.
41. Jarvis CD, Geiman T, Vila-Storm MP, Osipovich O, Akella U, Candeias S, Nathan I, Durum SK and Muegge K (1996). "A novel putative helicase produced in early lymphocytes." Gene 169: 203-207.
42. Jenuwein T and Allis CD (2001). "Translating the histone code." Science 293: 1074-1080.
43. Jones PA (1985). "Effects of 5-azacytidine and its 2-deoxy derivative on cell differentiation and DNA methylation." Pharmacol. Ther. 28: 17-27.
44. Jones PA and Baylin SB (2002). "The fundamental role of epigenetic events in cancer." Nature Rev. Genet. 3: 415-428.
45. Kanai Y, Ushijima S, Nakanishi Y and Hirohashi S (2001). "DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancers." Int. J. Cancer 91: 205-212.
46. Kelloff GJ, Schilsky RL, Alberts SS, Day RW, Guyton KZ, Pearce HL, Peck JC, Philips R and Sigman CC (2004). "Colorectal adenomas: a prototype for the use of surrogate end points in the development of cancer prevention drugs." Clin. Cancer Res. 10: 3908-3918.
47. Kopelovich L, Crowell JA and Fay JR (2003). "The epigenome as a target for cancer chemoprevention." J. Natl. Cancer Inst. 95(23): 1747-1757.
48. Lachner M, O'Sullivan RJ and Jenuwein T (2003). "An epigenetic road map for histone lysine methylation." J. Cell. Sci. 116: 2117-2124.
49. Laird PW (2003). "The power and the promise of DNA methylation markers." Nature Rev. Canc. 3: 253-266.
50. Laird PW, Jackson-Grusby L, Fazell A, Dickinson SL, Jung WE, Li E, Weinberg RA and Jaenisch R (1995). "Suppression of intestinal neoplasia by DNA hypomethylation." Cell 81: 197-205.
51. Lehnertz B, Ueda Y, Derijck AAHA, Braunschweig U, Perez-Burgos L, Kubicek S, Chen T, Li E, Jenuwein T and Peters AHFM (2003). "Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin." Curr. Biol. 13: 1192-1200.
52. Leonhardt H, Page AW, Weier H and Bestor TH (1992). "A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei." Cell 71: 865-873.
53. Li E (2002). "Chromatin modification and epigenetic reprogramming in mammalian development." Nature Rev. Genet. 3: 662-673.
54. Li E, Bestor TH and Jaenisch R (1992). "Targeted mutation of the DNA methyltransferase gene results in embryonic lethality." Cell 69: 915-926.
55. Lin X, Asgari K, Putzi MJ, Gage WR, Yu X, Cornblatt BS, Kumar A, Piantadosi S, DeWeese TL, DeMarzo AM and Nelson WG (2001). "Reversal of GSTP1 CpG island hypermethylation and reactivation of p-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide." Cancer Res. 61: 8611-8616.
56. Ling Y, Sankpal UT, Robertson AK, McNally JG, Karpova T and Robertson KD (2004). "Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription." Nucleic Acids Res. 32(2): 598-610.
57. MacLeod AR and Szyf M (1995). "Expression of antisense to DNA methyltransferase mRNA induces DNA demethylation and inhibits tumorigenesis." J. Biol. Chem. 270(7): 8037-8043.
58. Muller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M, Muhlthaler M, Ofner D, Margreiter R and Widschwendter M (2004). "Methylation changes in faecal DNA: A marker for colorectal cancer screening?" Lancet 363: 1283-1285.
59. Murakami T, Li X, Gong J, Bhatia U, Traganos F and Darzynkiewicz Z (1995). "Induction of apoptosis by 5-azacytidine: drug concentration-dependent differences in cell cycle specificity." Cancer Res. 55: 3093-3098.
60. Nguyen CT, Weisenberger DJ, Velicescu M, Gonzales FA, Lin JCY, Liang G and Jones PA (2002). "Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-aza-2'-deoxycytidine." Cancer Res. 62: 6456-6461.
61. Novina CD and Sharp PA (2004). "The RNAi revolution." Nature 430: 161-164.
62. Okano M, Bell DW, Haber DA and Li E (1999). "DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development." Cell 99: 247-257.
63. Okano M, Xie S and Li E (1998). "Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases." Nature Genet. 19: 219-220.
64. Okano M, Xie S and Li E (1998). "Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells." Nucleic Acids Res. 26(11): 2536-2540.
65. Panning B and Jaenisch R (1996). "DNA hypomethylation can activate Xist expression and silence X linked genes." Genes. Dev. 10(16): 1991-2002.
66. Paz MF, Fraga MF, Avila S, Guo M, Pollan M, Herman JG and Esteller M (2003). "A systematic profile of DNA methylation in human cancer cell lines." Cancer Res. 63: 1114-1121.
67. Rashid A, Shen L, Morris JS, Issa J-P and Hamilton SR (2001). "CpG island methylation in colorectal adenomas." Am. J. Pathol. 159(3): 1129-1135.
68. Rhee I, Bachman KE, Park BH, Jair K-W, Yen R-WC, Schuebel KE, Cui H, Feinberg AP, Lengauer C, Kinzler KW, Baylin SB and Vogelstein B (2002). "DNMT1 and DNMT3b cooperate to silence genes in human cancer cells." Nature 416: 552-556.
69. Robertson AK, Geiman TM, Sankpal UT, Hager GL and Robertson KD (2004). "Effects of chromatin structure on the enzymatic and DNA binding functions of DNA methyltransferases DNMT1 and Dnmt3a in vitro." Biochem. Biophys. Res. Commun. 322(1): 110-118.
70. Robertson KD (2002). "DNA methylation and chromatin - unraveling the tangled web." Oncogene 21: 5361-5379.
71. Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL and Wolffe AP (2000). "DNMT1 forms a complex with Rb, E2F1, and HDAC1 and represses transcription from E2F-responsive promoters." Nature Genet. 25: 338-342.
72. Robertson KD, Uzvolgyi E, Liang G, Talmadge C, Sumegi J, Gonzales FA and Jones PA (1999). "The human DNA methyltransferases (DNMTs) 1, 3a, and 3b: Coordinate mRNA expression in normal tissues and overexpression in tumors." Nucleic Acids Res. 27(11): 2291-2298.
73. Rountree MR, Bachman KE and Baylin SB (2000). "DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci." Nature Genet. 25: 269-277.
74. Saito Y, Kanai Y, Sakamoto M, Saito H, Ishii H and Hirohashi S (2001). "Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis." Hepatology 33: 561-568.
75. Saito Y, Kanai Y, Sakamoto M, Saito H, Ishii H and Hirohashi S (2002). "Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis." Proc. Natl. Acad. Sci. USA 99(15): 10060-10065.
76. Santos F, Zakhartchenko V, Stojkovic M, Peters A, Jenuwein T, Wolf E, Reik W and Dean W (2003). "Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos." Curr. Biol. 13: 1116-1121.
77. Soejima K, Fang W and Rollins BJ (2003). "DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40 T antigen and activated Ras." Oncogene 22: 4723-4733.
78. Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H and Held WA (2005). "Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression." Proc. Natl. Acad. Sci. USA 102(9): 3336-3341.
79. Tamaru H and Selker EU (2001). "A histone H3 methyltransferase controls DNA methylation in Neurospora crassa." Nature 414: 277-283.
80. Xu G-L, Bestor TH, Bourc'his D, Hsieh C-L, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ and Viegas-Pequignot E (1999). "Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene." Nature 402: 187-191.
81. Yoder JA and Bestor TH (1998). "A candidate mammalian DNA methyltransferase related to pmt1 from fission yeast." Hum. Mol. Genet. 7: 279-284.
82. Yoder JA, Walsh CP and Bestor TH (1997). "Cytosine methylation and the ecology of intragenomic parasites." Trends Genet. 13: 335-340.
83. Yokochi T and Robertson KD (2002). "Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a." J. Biol. Chem. 277(14): 11735-11745.
84. Zhu W-G, Lakshmanan RR, Beal MD and Otterson GA (2001). "DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors." Cancer Res. 61: 1327-1333.

back to top