The de novo cytosine methyltransferase DRM2 requires intact UBA domains and a catalytically mutated paralog DRM3 during RNA-directed DNA methylation in Arabidopsis thaliana.

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS rearranged methyltransferase2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA-directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term domains rearranged methyltransferase3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA-directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA-directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA-directed DNA methylation systems consist of a combination of ancestral and convergent features.

Regulation of DNMT1 stability through SET7-mediated lysine methylation in mammalian cells.

Inheritance of epigenetic information encoded by cytosine DNA methylation patterns is crucial for mammalian cell survival, in large part through the activity of the maintenance DNA methyltransferase (DNMT1). Here, we show that SET7, a known histone methyltransferase, is involved in the regulation of protein stability of DNMT1. SET7 colocalizes and directly interacts with DNMT1 and specifically monomethylates Lys-142 of DNMT1. Methylated DNMT1 peaks during the S and G(2) phases of the cell cycle and is prone to proteasome-mediated degradation. Overexpression of SET7 leads to decreased DNMT1 levels, and siRNA-mediated knockdown of SET7 stabilizes DNMT1. These results demonstrate that signaling through SET7 represents a means of DNMT1 enzyme turnover.

Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression.

Pituitary tumor-transforming gene (PTTG) originally isolated from GH-secreting pituitary adenoma cells causes in vitro cell transformation, in vivo tumorigenesis, and induces basic fibroblast growth factor. These functions require an intact C-terminal proline-proline-serine-proline motif. PTTG1 is abundantly expressed in human pituitary tumors and plays a role in the early stages of experimental prolactinoma formation. We now determined direct effects of PTTG1 on hormonal phenotypes of functional pituitary tumor cells. Overexpression of PTTG1 C terminus (amino acids 147-202) containing intact proline-proline-serine-proline motifs in rat prolactin (PRL)- and GH-secreting GH3 cells markedly abrogates PRL mRNA expression by more than 90% (P < 0.001) and hormone levels (P < 0.001) and PRL promoter activity (P < 0.01) compared with control vector cells or to a PTTG1 C terminus mutant (P163A, S165Q, P166L, P170L, P172A, and P173L). Wild-type PTTG1 C-terminal transfectants formed smaller (P < 0.05) sc tumors in rats compared with control or mutated PTTG1 C-terminal transfectants. Estrogen (10 nm) treatment for 48 h partially restored PRL expression in stable wild-type PTTG1 C-terminal transfectants. These results indicate that targeting PTTG1-mediated signaling alters the hormonal phenotype in pituitary cells and disrupted PTTG1 action may be a potential subcellular therapeutic tool for repressing PRL hypersecretion.

Adenovirus small e1a alters global patterns of histone modification.

Adenovirus small early region 1a (e1a) protein drives cells into S phase by binding RB family proteins and the closely related histone acetyl transferases p300 and CBP. The interaction with RB proteins displaces them from DNA-bound E2F transcription factors, reversing their repression of cell cycle genes. However, it has been unclear how the e1a interaction with p300 and CBP promotes passage through the cell cycle. We show that this interaction causes a threefold reduction in total cellular histone H3 lysine 18 acetylation (H3K18ac). CBP and p300 are required for acetylation at this site because their knockdown causes specific hypoacetylation at H3K18. SV40 T antigen also induces H3K18 hypoacetylation. Because global hypoacetylation at this site is observed in prostate carcinomas with poor prognosis, this suggests that processes resulting in global H3K18 hypoacetylation may be linked to oncogenic transformation.

Epigenetic reprogramming by adenovirus e1a.

Adenovirus e1a induces quiescent human cells to replicate. We found that e1a causes global relocalization of the RB (retinoblastoma) proteins (RB, p130, and p107) and p300/CBP histone acetyltransferases on promoters, the effect of which is to restrict the acetylation of histone 3 lysine-18 (H3K18ac) to a limited set of genes, thereby stimulating cell cycling and inhibiting antiviral responses and cellular differentiation. Soon after expression, e1a binds transiently to promoters of cell cycle and growth genes, causing enrichment of p300/CBP, PCAF (p300/CBP-associated factor), and H3K18ac; depletion of RB proteins; and transcriptional activation. e1a also associates transiently with promoters of antiviral genes, causing enrichment for RB, p130, and H4K16ac; increased nucleosome density; and transcriptional repression. At later times, e1a and p107 bind mainly to promoters of development and differentiation genes, repressing transcription. The temporal order of e1a binding requires its interactions with p300/CBP and RB proteins. Our data uncover a defined epigenetic reprogramming leading to cellular transformation.