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3
*
Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown, MA and
Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan
3To whom correspondence should be addressed. E-mail: okano{at}cdb.riken.go.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONDISCUSSIONLITERATURE CITED |
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KEY WORDS: • DNA methylation • Dnmt1 • Dnmt3a • Dnmt3b • ES cells • knockout mouse
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONDISCUSSIONLITERATURE CITED |
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,2
). In mammals, DNA methylation is essential for normal development and plays a crucial role in gene expression, X-inactivation, genomic imprinting and silencing of retrotransposable elements (3
–5
). Abnormal DNA methylation patterns are associated with many human disorders including cancer, immunodeficiency and mental retardation (6
,7
). DNA methylation occurs at the cytosine-5 (C-5) (4
) position of cytosine guanine dinucleotides (CpG4
), and about 70% of all CpG sites are methylated, mainly in the repressive heterochromatin region and in repetitive sequences such as retrotransposable elements (8
). Most unmethylated CpG sites are seen in CpG islands, consisting of CpG-rich sequences about 1 kilobase (kb) in length and located in many functional promoters (9
). While these general DNA methylation patterns are common features for mammalian somatic cells, tissue-specific, allele-specific or developmental-stage-specific DNA methylation patterns also are found in genes, regions and repetitive sequences (10
).
DNA methylation patterns are reprogrammed during mouse embryogenesis by genome-wide demethylation and de novo methylation (11
). The fertilized egg loses its DNA methylation in both replication-dependent and replication-independent ways during preimplantation development. These processes erase significant parts of the parental DNA methylation. After implantation, the embryo undergoes a wave of de novo methylation that establishes a new embryonic DNA methylation pattern. Genome-wide reprogramming of DNA methylation also occurs during gametogenesis and plays a critical role in establishing the parental-specific methylation marks in imprinted genes.
Mammalian cells have at least two distinct DNA methylation activities (1
). Maintenance methylation activity has a preference for hemimethylated sites over unmethylated sites and ensures clonal transmission of DNA methylation patterns to daughter cells through mitosis. On the other hand, de novo methylation activity does not require preexisting methylation and establishes a new DNA methylation pattern. De novo methylation activity is present predominantly in early embryos and in specific cell types such as embryonic stem (ES) cells.
To date, four DNA methyltransferase genes have been characterized in mammals: Dnmt1, Dnmt2, Dnmt3a and Dnmt3b. Dnmt1 encodes a polypeptide of 1619 amino acid residues, consisting of a carboxy (C)-terminal catalytic domain of about 500 amino acid residues and an amino (N)-terminal region of about 1100 amino acid residues with several functional domains (12
–14
). In vitro activity of the Dnmt1 enzyme has a preference for hemimethylated DNA over unmethylated DNA. Dnmt1 localizes at the replication foci in S-phase and interacts with proliferating cell nuclear antigen (PCNA) (15
–17
). Dnmt1 is expressed highly in proliferating cells and is ubiquitous in all somatic tissues.
Dnmt2 contains all of the essential motifs for exhibiting DNA methyltransferase activity, but no enzymatic activity has been detected (18
,19
). The ES cells in which a putative catalytic center of Dnmt2 was disrupted by gene targeting show no detectable changes in DNA methylation (19
). The biological function of this gene in mammals is not yet known.
Two homologous DNA methyltransferase genes, Dnmt3a and Dnmt3b, encode polypeptides with 908 and 859 amino acid residues, respectively (20
,21
). Like the Dnmt1 enzyme, these enzymes also contain a C-terminal catalytic domain and an N-terminal region with a proline-tryptophan-tryptophan-proline (PWWP) domain and a plant homeodomain (PHD)-like Zn-finger domain homologous to the X-linked chromatin remodeling factor ATR-X (
thalassemia/mental retardation syndrome X-linked). The Dnmt3a and Dnmt3b enzymes share 80% amino acid similarity at their catalytic domain and about 60% amino acid similarity at the PHD-like Zn-finger domain. In spite of architectural similarity, very little homology was found between the Dnmt1 and Dnmt3 enzymes. Dnmt3a and Dnmt3b localize at the distinct nuclear compartment including the heterochromatin region but not at the replication foci (22
). Dnmt3a and Dnmt3b are highly expressed in ES cells and early embryos where active de novo methylation occurs. In late embryos as well as adults, Dnmt3a is expressed broadly in various tissues, whereas Dnmt3b is expressed at a very low level in most tissues except for certain cell types (23
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONDISCUSSIONLITERATURE CITED |
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Dnmt1.
To understand the roles of DNA methyltransferase genes in the establishment and maintenance of DNA methylation patterns and in mouse development, our studies have been focused on the genetic analyses of DNA methyltransferase genes with mouse ES cells and with knockout mice. First, knockout mice of Dnmt1 showed embryonic lethality at 10.5 d post coitum (dpc) and significant loss of global DNA methylation, demonstrating that DNA methylation is essential for normal mammalian development (24
). This first mutant allele was revealed to be hypomorphic, i.e., with a partial loss of function for Dnmt1, since about 5% of the Dnmt1 enzyme still was expressed. To understand the role of Dnmt1, we generated a functionally null allele of Dnmt1 by homologous recombination in which a genomic fragment encoding the catalytic center was deleted (25
). Consistent with the nature of the mutation, the homozygous mouse embryos with the Dnmt1-null allele displayed more severe phenotypic changes than did the corresponding embryos with the hypomorphic allele. Development of the mutant embryos was arrested between presomite stage and 8 somite stage around 8.5 dpc. Characterization of the ES cells homozygous for Dnmt1 revealed the following: 1) nearly all DNA methylation was lost in all sequences examined in the mutant ES cells, indicating that Dnmt1 plays a major role in maintaining global DNA methylation patterns; 2) although the mutant ES cells with extensive hypomethylated genome grew normally under undifferentiated status, their growth stopped right after differentiation occurred, suggesting that DNA methylation is essential for somatic cell growth but not for undifferentiated stem cells; and 3) the ES cells with inactivated Dnmt1 still contained low but stable amounts of DNA methylation and in vitro DNA methyltransferase activity. Furthermore, the mutant ES cells retained de novo methylation activity toward exogenous proviral DNA sequences integrated in their genome. These results provided the first genetic evidence for the existence of DNA methyltransferases other than Dnmt1. Motivated by this evidence, we pursued and cloned the novel DNA methyltransferase genes, Dnmt3a and Dnmt3b (20
).
Dnmt3a and Dnmt3b.
To examine whether Dnmt3a and Dnmt3b were required for de novo methylation, we generated the ES cells single homozygous for either Dnmt3a (Dnmt3a-/-) or Dnmt3b (Dnmt3b-/-), and double homozygous for both Dnmt3a and Dnmt3b ([Dnmt3a-/-, Dnmt3b-/-]) (23
). Whereas the Dnmt3a-/- or Dnmt3b-/- single homozygous ES cells did not show significant changes, the [Dnmt3a-/-, Dnmt3b-/-] ES cells completely lacked de novo methylation activity towards proviral DNA sequences integrated in their genome, even though active Dnmt1 enzyme was present. These results demonstrate that Dnmt3a and Dnmt3b are essential for de novo methylation in ES cells and that these two gene products are redundant in this function.
To determine whether Dnmt3a and Dnmt3b also function in de novo methylation in early embryogenesis, we generated Dnmt3a and Dnmt3b knockout mice (23
). The [Dnmt3a-/-, Dnmt3b-/-] double homozygous embryo revealed a phenotype that was strikingly similar to that of the Dnmt1-/- embryo and showed developmental arrest at the presomite stage and a distorted neural tube at around 8.5 dpc. The DNA methylation level of the [Dnmt3a-/-, Dnmt3b-/-] double homozygous embryo was significantly lower than that of the wild-type embryo. However, the level was much higher than that of the Dnmt1-/- embryo and was at the same low level as that of the blastocyst, which did not yet undergo genome-wide de novo methylation, suggesting that de novo methylation in early embryogenesis did not occur in the [Dnmt3a-/-, Dnm3b-/-] embryo. Together with the results from the ES cells, we concluded that Dnmt3a and Dnmt3b function in de novo methylation in early embryogenesis. Consistent with these results, the overexpression of Dnmt3 DNA methyltransferases in transgenic flies or mammalian cells caused de novo methylation in Drosophila genomic DNA (26
) and in stable episomal DNA (27
).
Dnmt3a-/- mice developed to term and appeared to be normal at birth. However, most of the homozygous mice were stunted and died at about 4 wk of age. Dnmt3b-/- mice suffered embryonic lethality with multiple developmental defects and growth impairment between 9.5 and 16.5 dpc. These results demonstrate that Dnmt3a-dependent and Dnmt3b-dependent DNA methyltransferase machineries not only are essential for early embryogenesis but also are required for later development and even for the normal physiological functions as adults.
Further characterization of DNA methylation of endogenous sequences in the [Dnmt3a-/-, Dnmt3b-/-] ES cells suggests that Dnmt3a and Dnmt3b have a function other than de novo methylation. The ES cells that inactivated both Dnmt3a and Dnmt3b show moderate loss of global DNA methylation in repetitive sequences. DNA methylation of some genes, such as insulin-like growth factor 2 (Igf2) and inactive X-specific transcript (Xist), is extensively reduced, whereas that of other genes, such as insulin-like growth factor 2 receptor (Igf2r) and H19, apparently is not affected in these cells (23
). Also, DNA methylation of the mouse centromeric minor satellite repeat was decreased specifically in the Dnmt3b-/- single homozygous ES cells (23
), whereas that of the human pericentromeric repeat was decreased in the patient for the immunodeficiency centromeric instability and facial anomalies (ICF) syndrome, in which the human DNMT3B gene is mutated (23
,28
,29
). These results suggest that Dnmt3a and Dnmt3b have a function to "maintain" DNA methylation in certain sequences, most likely by a mechanism other than methylation toward hemimethylated DNA.
It is important to note that current gene knockout studies have not ruled out the possibilities that Dnmt1 also can contribute de novo methylation or can play a major role in different forms of de novo methylation for specific DNA structures or in cancer cells. Because of the high maintenance methylation activity by Dnmt1, examination of a possible de novo methylation activity by this enzyme in the Dnmt1-/- ES cells has been difficult. Overexpression of Dnmt1 in cancer cell lines leads to the de novo methylation of endogenous CpG islands (30
). Since a small portion of the N-terminal end of Dnmt1 was not known at that time, the Dnmt1 construct used in that study was truncated. The missing portion contained the Dnmt1-associated protein (DMAP1) interacting region; this fact needs to be considered in the interpretation of the results. Further, a human colorectal cancer cell line with inactivated human DNMT1 gene retains 80% of its genomic DNA methylation, suggesting contributions by enzymes other than DNMT1 to maintenance methylation in tumor cells (31
). It will be necessary to reevaluate cellular DNA methylation activities based on molecular machinery.
Future directions
DNA methylation patterns are established early in development and are inherited faithfully through cell lineage. Three distinct DNA methyltransferase genes have been shown to play key roles in these processes. An obvious direction for future studies is to understand molecular mechanisms that control DNA methylation patterns directly or indirectly through DNA methyltransferases. It is likely that DNA methyltransferases form protein complexes and that other components in these complexes may regulate enzymatic activity or target specificity. A number of proteins are reported to interact with Dnmt1 and Dnmt3a/Dnmt3b, including PCNA, DMAP1, retinoblastoma protein (pRb), histone deacetylases (HDACs) and RP58 (repressor protein with a predicted molecular mass of 58 kDa) (16
,32
–34
). Also, there are some genes that share putative regulatory domains with the DNA methyltransferases and are speculated to control the enzymes; these include such genes as Dnmt3L (DNA methyltransferase 3-like) and ATR-X (35
,36
). Chromatin-remodeling factors and core histone modification systems are closely connected with the regulation of the DNA methylation pattern (37
–39
). Further studies will reveal the biochemical nature of these factors and will delineate how these factors and chromatin structures cooperate to control the activities and localization of the DNA methyltransferases.
Another possible direction is to approach questions of what the cellular function of the DNA methylation pattern is and how it is interpreted. It is of great interest to find the target genes or the target sequences of these DNA methyltransferases and how they accomplish their biological functions during embryonic development as well as in adulthood. It is not difficult to imagine that DNA methyltransferases have multiple targets and thus multiple functions such as transcriptional regulation, DNA replication, chromosomal segregation and recombination. It is also conceivable that DNA methylation in some fraction of CpG sites may be crucial, whereas DNA methylation in other CpG sites may be neutral for biological functions. The knockout mice and the mutant cells with inactivated DNA methyltransferases will be useful for this purpose. Close examination of the relationships among DNA methylation pattern, gene expression, chromatin structures and phenotypic analyses of the mutant cells is needed. In particular, the behavior of molecules sensing DNA methylation such as the methyl-CpG binding domain protein (MBD) family and the CTCF (CCCTC-binding factor) in the mutant cells will be interesting. Recent progress in the field has provided a deeper understanding of human diseases, including cancer and neurological disorders. Further studies will shed light on this fundamental aspect of basic biology and may lead to the development of novel therapies, drug discoveries and engineering.
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FOOTNOTES |
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2 This work was supported by National Institutes of Health grants CA82389 and GM52106 to En Li. Masaki Okano is a special fellow of the Leukemia and Lymphoma Society.
4 Abbreviations used: ATR-X, thalassemia/mental retardation syndrome X-linked; C, cytosine; CpG, cytosine guanine dinucleotides; CTCF, CCCTC-binding factor; DMAP, Dnmt1-associated protein; Dnmt1, Dnmt2, Dnmt 3a, Dnmt3b, DNA methyltransferase genes; Dnmt3L, DNA methyltransferase 3-like; dpc, days post coitum; ES, embryonic stem; HDACs, histone deacetylases; ICF, immunodeficiency, centromeric instability and facial anomalies; Igf2, insulin-like growth factor 2; Igf2r, insulin-like growth factor 2 receptor; kb, kilobase; MBD, methyl-CpG binding domain protein; PCNA, proliferating cell nuclear antigen; PHD, plant homeodomain protein; pRb, retinoblastoma protein; PWWP, proline-tryptophan-tryptophan-proline; RP58, repressor protein with a predicted molecular mass of 58 kDa; Xist, inactive X-specific transcript.
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TOPABSTRACTINTRODUCTIONDISCUSSIONLITERATURE CITED |
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