DNA methylation and the formation of heterochromatin in Neurospora crassa.

Studies of the control and function of DNA methylation in Neurospora crassa have led to a greater understanding of heterochromatin formation. DNA methylation in Neurospora is dependent on trimethylation of histone H3 lysine 9 (H3K9me3) by the histone methyltransferase, DIM-5. The linkage between these two methyl marks is facilitated by heterochromatin protein 1 (HP1), which serves as an adapter protein. HP1 binds to the H3K9me3 and recruits the DNA methyltransferase, DIM-2. Although HP1 links H3K9me3 to DNA methylation, it also serves to recruit the DNA methylation modifier complex to the edges of heterochromatin regions, where it serves to limit the spreading of the heterochromatin by countering H3K9me3.

DNA methylation, chromatin inheritance, and cancer.

Cancer is a process driven by the accumulation of abnormalities in gene function. While many of these changes are genetic, epigenetically mediated changes in gene expression are being increasingly appreciated. This latter process emphasizes the need to understand two key components of heritable, but reversible, modulation of gene promoter function that are closely tied to one another - formation of chromatin which modulates transcription and establishing patterns of DNA methylation. The link lies first in the recruitment to methylated cytosines of a family of methyl-CpG binding domain proteins (MBDs), which are direct transcriptional repressors and can complex with transcriptional corepressors including histone deacetylases (HDACs). Additionally, the proteins that catalyze DNA methylation, DNA methyltransferases (DNMTs), also directly repress transcription and associate with HDACs. Regulation of these above chromatin-DNA methylation interactions as a function of DNA replication timing is emerging as a key event in the inheritance of transcriptionally repressed domains of the genome. Importantly, synergy between HDAC activity and DNA methylation is operative for a key epigenetic abnormality in cancer cells, transcriptional silencing of tumor suppressor genes. This change has now been recognized for genes that are essential for normal regulation of virtually every major cell function including cell growth, differentiation, apoptosis, DNA repair, and cell-cell, cell-substratum interaction. Understanding the molecular determinants of both normal and abnormal patterns of chromatin formation and DNA methylation thus holds great promise for our understanding of cancer and for means to better diagnose, prevent, and treat this disease.

Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin.

We demonstrate that the recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, like DNMT1, repress transcription in a methylation-independent manner. Dnmt3a and Dnmt3b repress transcription primarily through a plant homeodomain-like motif that is shared with the ATRX protein but is not present in DNMT1. Unlike DNMT1, which localizes to replication foci during S-phase in murine embryonic fibroblasts, Dnmt3a co-localizes with heterochromatin protein 1 alpha (HP1 alpha) and methyl-CpG binding proteins throughout the cell cycle to late-replicating pericentromeric heterochromatin. In contrast to Dnmt3a, Dnmt3b remained diffuse in the nucleus of embryonic fibroblasts at all cell cycle stages. However, Dnmt3a and Dnmt3b co-localize to these pericentromeric heterochromatin regions in murine embryonic stem cells. This finding is important to the fact that mutations in DNMT3B are found in the developmental syndrome, ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies), which involves extensive loss of methylation from pericentromeric regions. The localization of Dnmt3a and Dnmt3b was unaffected in Dnmt1 null embryonic stem cells, which lose the majority of methylation at pericentromeric major satellite repeats, suggesting that these enzymes are not dependent upon preexisting methylation for their targeting. DNMT1 is then positioned to reestablish transcriptionally repressive chromatin as cells replicate, while Dnmt3a and Dnmt3b may help to establish such chromatin in late S-phase and maintain this repressive heterochromatin throughout the cell cycle in a developmentally and/or cell type manner.

Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer.

Gene function in cancer can be disrupted either through genetic alterations, which directly mutate or delete genes, or epigenetic alterations, which alter the heritable state of gene expression. The latter events are mediated by formation of transcriptionally repressive chromatin states around gene transcription start sites and an associated gain of methylation in normally unmethylated CpG islands in these regions. The genes affected include over half of the tumor suppressor genes that cause familial cancers when mutated in the germline; the selective advantage for genetic and epigenetic dysfunction in these genes is very similar. The aberrant methylation can begin very early in tumor progression and mediate most of the important pathway abnormalities in cancer including loss of cell cycle control, altered function of transcription factors, altered receptor function, disruption of normal cell-cell and cell-substratum interaction, inactivation of signal transduction pathways, loss of apoptotic signals and genetic instability. The active role of the aberrant methylation in transcriptional silencing of genes is becoming increasingly understood and involves a synergy between the methylation and histone deacetylase (HDAC) activity. This synergy can be mediated directly by HDAC interaction with DNA methylating enzymes and by recruitment through complexes involving methyl-cytosine binding proteins. In the translational arena, the promoter hypermethylation changes hold great promise as DNA tumor markers and their potentially reversible state creates a target for cancer therapeutic strategies involving gene reactivation.

DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci.

DNA methylation can contribute to transcriptional silencing through several transcriptionally repressive complexes, which include methyl-CpG binding domain proteins (MBDs) and histone deacetylases (HDACs). We show here that the chief enzyme that maintains mammalian DNA methylation, DNMT1, can also establish a repressive transcription complex. The non-catalytic amino terminus of DNMT1 binds to HDAC2 and a new protein, DMAP1 (for DNMT1 associated protein), and can mediate transcriptional repression. DMAP1 has intrinsic transcription repressive activity, and binds to the transcriptional co-repressor TSG101. DMAP1 is targeted to replication foci through interaction with the far N terminus of DNMT1 throughout S phase, whereas HDAC2 joins DNMT1 and DMAP1 only during late S phase, providing a platform for how histones may become deacetylated in heterochromatin following replication. Thus, DNMT1 not only maintains DNA methylation, but also may directly target, in a heritable manner, transcriptionally repressive chromatin to the genome during DNA replication.

DNA methylation inhibits elongation but not initiation of transcription in Neurospora crassa.

In plants, animals, and fungi, DNA methylation is frequently associated with gene silencing, yet little is known about the role of the methylation in silencing. In Neurospora crassa, repeated sequences are silenced by repeat-induced point mutation (RIP) and genes that have suffered numerous GC --> AT mutations by RIP are typically methylated at remaining cytosines. We investigated possible effects on transcription from methylation associated with RIP by taking advantage of 5-azacytidine, which prevents most methylation in Neurospora and a dim-2 mutation that abolishes all detectable methylation. Northern analyses revealed that methylation prevents the accumulation of transcripts from genes mutated by RIP. Measurements of transcription rates in vivo showed that methylation inhibits transcription severely but does not influence mRNA stability. Results of nuclear run-on experiments demonstrated that transcription initiation was not significantly inhibited by the dense methylation in the promoter sequences. In contrast, methylation blocked transcription elongation in vivo.

Epigenetic control of a transposon-inactivated gene in Neurospora is dependent on DNA methylation.

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5' noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am- or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSam alpha and beta, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.

Ectopic integration of transforming DNA is rare among neurospora transformants selected for gene replacement.

In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such "cotransformants" are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycinR), allowed to conidiate to generate homokaryons and then selected for either Am- (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycinS (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa.

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.