Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.

Epigenetic modifications are thought to be important for gene expression changes during development and aging. However, besides the Sir2 histone deacetylase in somatic tissues and H3K4 trimethylation in germlines, there is scant evidence implicating epigenetic regulations in aging. The insulin/IGF-1 signaling (IIS) pathway is a major life span regulatory pathway. Here, we show that progressive increases in gene expression and loss of H3K27me3 on IIS components are due, at least in part, to increased activity of the H3K27 demethylase UTX-1 during aging. RNAi of the utx-1 gene extended the mean life span of C. elegans by ~30%, dependent on DAF-16 activity and not additive in daf-2 mutants. The loss of utx-1 increased H3K27me3 on the Igf1r/daf-2 gene and decreased IIS activity, leading to a more "naive" epigenetic state. Like stem cell reprogramming, our results suggest that reestablishment of epigenetic marks lost during aging might help "reset" the developmental age of animal cells.

BACKGROUND Wnt‐1 and β‐catenin expression levels play an important role in several malignancies. The authors determined the level of production of Wnt‐1 and β‐catenin in normal and malignant human prostate carcinoma cell lines. Surgical pathology specimens from primary prostatic adenocarcinoma, lymph node metastases, and skeletal metastases were used to establish a correlation between the level of Wnt‐1/β‐catenin expression, Gleason score, serum prostate‐specific antigen (PSA) status, and androgen receptor (AR) status. METHODS Immunohistochemical analysis was used to investigate the expression of Wnt‐1 and β‐catenin in human prostate carcinoma cell lines and in paraffin embedded sections of archival samples from 67 patients with prostate carcinoma. Comparison was made with the expression of tumoral AR and lymph node and skeletal metastases. These results were used to establish a correlation with the clinicopathologic status of patients with prostate carcinoma. RESULTS Levels of both Wnt‐1 and β‐catenin were low in normal prostate cells and were expressed highly in human prostate carcinoma cell lines. Wnt‐1 and cytoplasmic/nuclear β‐catenin expression was observed in 52% and 34%, respectively, of primary prostate carcinoma specimens. High levels of expression of Wnt‐1 and β‐catenin were seen in 77% of lymph node metastases and in 85% of skeletal metastases. These increased levels of expression were related directly to the Gleason score and to serum PSA levels in these patients. Maximum levels of Wnt‐1 and β‐catenin production were observed in skeletal metastases, whereas normal prostatic tissue failed to exhibit any detectable nuclear staining for β‐catenin. CONCLUSIONS High levels of Wnt‐1 and β‐catenin expression were associated with advanced, metastatic, hormone‐refractory prostate carcinoma, in which they can serve as markers of disease progression. Cancer 2004. © 2004 American Cancer Society.

We tested the hypothesis that cell invasiveness and tumorigenesis are driven by hypomethylation of genes involved in tumor progression. Highly invasive human prostate cancer cells PC-3 were treated with either the methyl donor S-adenosylmethionine (SAM) or methyl DNA-binding domain protein 2 antisense oligonucleotide (MBD2-AS). Both treatments resulted in a dose-and time-dependent inhibition of key genes, such as urokinase-type plasminogen activator (uPA), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor expression to decrease tumor cell invasion in vitro. No change in the levels of expression of genes already known to be methylated in late-stage prostate cancer cells, such as glutathione S-transferase P1 and androgen receptor, was seen. Inoculation of PC-3 cells pretreated with SAM and MBD2-AS into the flank of male BALB/c nu/nu mice resulted in the development of tumors of significantly smaller volume compared with animals inoculated with PC-3 cells treated with vehicle alone or MBD2 scrambled oligonucleotide. Immunohistochemical analysis of tumors showed the ability of SAM and MBD2-AS to significantly decrease tumoral uPA and MMP-2 expression along with levels of angiogenesis and survival pathway signaling molecules. Bisulfite sequencing analysis of tumoral genomic DNA showed that inhibition of both uPA and MMP-2 expression was due to methylation of their 5 ¶ regulatory region. These studies support the hypothesis that DNA hypomethylation controls the activation of multiple tumor-promoting genes and provide valuable insight into developing novel therapeutic strategies against this common disease, which target the demethylation machinery.

1 0 2 3 a r t i c l e s RESULTS Pax3 binds to major satellite repeatsTo examine transcription factors operating at heterochromatic regions, we first investigated transcription factors that had been shown to associate with heterochromatic core components 15 . Pax3 emerged as a particularly notable candidate because it has been reported to interact with both heterochromatin protein-1 (HP1) and the transcriptional regulator Kap1(Trim28) 16 . In addition, subrepeat Heterochromatin is important for genome integrity and stabilization of gene-expression programs. We have identified the transcription factors Pax3 and Pax9 as redundant regulators of mouse heterochromatin, as they repress RNA output from major satellite repeats by associating with DNA within pericentric heterochromatin. Simultaneous depletion of Pax3 and Pax9 resulted in dramatic derepression of major satellite transcripts, persistent impairment of heterochromatic marks and defects in chromosome segregation. Genome-wide analyses of methylated histone H3 at Lys9 showed enrichment at intergenic major satellite repeats only when these sequences retained intact binding sites for Pax and other transcription factors. Additionally, bioinformatic interrogation of all histone methyltransferase Suv39h-dependent heterochromatic repeat regions in the mouse genome revealed a high concordance with the presence of transcription factor binding sites. These data define a general model in which reiterated arrangement of transcription factor binding sites within repeat sequences is an intrinsic mechanism of the formation of heterochromatin.npg