Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Phosphorylation of the Neurospora circadian clock protein FREQUENCY by several kinases promotes its degradation and is important for the function of the circadian feedback loop. Here, we show that FRQ is less stable in a ppp-1 (catalytic subunit of PP1) mutant, resulting in its advanced phase and short period. In contrast, FRQ stability is not altered in a rgb-1 (a regulatory subunit of PP2A) mutant, but levels of frq protein and mRNA are low, resulting in a low-amplitude and long-period oscillation of the clock. Furthermore, PP1 and PP2A expressed in Neurospora can dephosphorylate the endogenous FRQ in vitro, suggesting that these two phosphatases may differentially regulate FRQ and, consequently, the behavior of the circadian clock.     

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.     

The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa

Transposable elements are common in genomes and must be controlled. Many organisms use DNA methylation to silence such selfish DNA, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. We identified a JmjC domain protein in Neurospora, DNA METHYLATION MODULATOR-1 (DMM-1), that prevents aberrant spreading of DNA and histone H3K9 methylation from inactivated transposons into nearby genes. Mutation of a conserved residue within the JmjC Fe(II)-binding site abolished dmm-1 function, as did mutations in conserved cysteine-rich domains. Mutants defective only in dmm-1 mutants grow poorly, but growth is restored by reduction or elimination of DNA methylation using the drug 5-azacytosine or by mutation of the DNA methyltransferase gene dim-2. DMM-1 relies on an associated protein, DMM-2, which bears a DNA-binding motif, for localization and proper function. HP1 is required to recruit the DMM complex to the edges of methylated regions.     

Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation

Histone post-translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. While significant progress has been made characterizing individual effector domains, the role of paired domains and how they function in a combinatorial fashion within chromatin are poorly defined. Here we show that the linked tandem Tudor and plant homeodomain (PHD) of UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1) operates as a functional unit in cells, providing a defined combinatorial readout of a heterochromatin signature within a single histone H3 tail that is essential for UHRF1-directed epigenetic inheritance of DNA methylation. These findings provide critical support for the “histone code” hypothesis, demonstrating that multivalent histone engagement plays a key role in driving a fundamental downstream biological event in chromatin.     

DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia

Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregnancy outcomes, but their underlying causes are mostly unknown. Although multiple studies have investigated gene expression changes in these disorders, few studies have examined epigenetic changes. Analysis of the DNA methylation pattern associated with such pregnancies provides an alternative approach to identifying cellular changes involved in these disorders. We analyzed methylation of 1505 CpG sites associated with 807 genes in 26 placentas from early-onset preeclampsia (EOPET), late-onset preeclampsia, IUGR and control subjects using an Illumina GoldenGate Methylation panel. Thirty-four loci were hypomethylated (false discovery rate <10% and methylation difference >10%) in the early-onset preeclamptic placentas while no and only five differentially methylated loci were found in late-onset preeclamptic and IUGR placentas, respectively. Hypomethylation of 4 loci in EOPET was further confirmed by bisulfite pyrosequencing of 26 independent placental samples. The promoter of TIMP3 was confirmed to be significantly hypomethylated in EOPET placentas (P=0.00001). Our results suggest that gene-specific hypomethylation may be a common phenomenon in EOPET placentas, and that TIMP3 could serve as a potential prenatal diagnostic marker for EOPET.     

DNA methylation is required for the control of stem cell differentiation in the small intestine

The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3–5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.     

HOXD13 methylation status is a prognostic indicator in breast cancer

Homeobox protein Hox-D13 is encoded by HOXD13 gene which is frequently methylated in cancer and has been recognized as a tumor suppressor in pancreatic cancer. In this study, we examined HOXD13 mRNA expression in 40 pairs of breast cancers and corresponding normal breast tissues. Bisulfite sequencing of HOXD13 promoter was performed in 6 pairs of breast tumors and corresponding normal breast tissues to examine the potential HOXD13 CpG methylated sites. HOXD13 DNA methylation frequency analysis was performed using MethyLight in 196 pairs of breast cancers and corresponding normal breast samples. DNA methylation status and clinico-pathological features were investigated. Kaplan-Meier survival analysis and Cox proportional hazards models were utilized to assess the effect of methylation status on overall survival. We found that 60% (24/40) of breast cancers showed low HOXD13 mRNA expression when compared with corresponding normal breast tissue. The predicted CpG island was located in the -1325 bp to +675 bp region. Next, the -332 bp site in HOXD13 gene promoter was further examined and in 57.7% (113/196) samples methylation was detected at this site. HOXD13 methylation was correlated with larger tumor size (P = 0.004), but not with other clinico-pathological parameters. In addition, patients with methylated -HOXD13 promoter had worse overall survival (OS) (P = 0.005). Based on our results we conclude that HOXD13 methylation is a common event in primary breast cancer and is associated with poor survival of breast cancer patients. HOXD13 methylation could therefore potentially be used as a prognostic factor for breast cancer.     

PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis

The histone methyl transferase PR-Set7 mediates histone H4 Lys 20 methylation, a mark of constitutive and facultative heterochromatin. We isolated a null mutation in Drosophila PR-Set7 that suppresses position effect variegation, indicating that PR-Set7 indeed functions in silencing general gene expression. In PR-Set7 larval leg and eye discs, the number of cells is lower than normal, and the DNA content in these cells is significantly increased. These data show that PR-Set7-dependent methylation is essential for the process of mitosis. The methylation mark is highly stable and is maintained even in the absence of PR-Set7 protein.     

Aberrant DNA Methylation of G-protein-coupled Bile Acid Receptor Gpbar1 (TGR5) is a Potential Biomarker for Hepatitis B Virus Associated Hepatocellular Carcinoma

Background: G-protein-coupled bile acid receptor Gpbar1 (TGR5) is a newly identified liver tumor suppressor in carcinogenesis. This present study was therefore to determine the potential value of serum TGR5 promoter methylation in identifying hepatocellular carcinoma (HCC) from chronic hepatitis B (CHB) patients.Methods: The circulating cell-free DNA (cfDNA) was extracted from a retrospective dataset including 160 HCC, 88 CHB and 45 healthy controls (HCs). Methylation status of TGR5 promoter was examined by methylation-specific polymerase chain reaction (MSP).Results: Hypermethylation of the TGR5 promoter occurred significantly more frequent in HCC (77/160, 48.13%) than CHB (12/88, 13.64%; p<0.01) and HCs (2/45, 4.44%; p<0.01). The methylation rate of TGR5 in HCC patients ≥60 years old was significantly higher than those <60 years old (p<0.05). Alpha fetoprotein (AFP) had sensitivity of 58.13%, 30.63% and 24.38% at cut-off points of 20, 200 and 400ng/ml respectively; while TGR5 methylation combined AFP had sensitivity of 81.25%, 68.13% and 65%. AFP had specificity of 47.73%, 92.05% and 98.86% at cut-off points of 20, 200 and 400ng/ml respectively; while TGR5 methylation combined AFP had specificity of 38.64%, 78.41% and 85.23%. AFP had Youden index of 0.06, 0.23 and 0.23 at cut-off points of 20, 200 and 400ng/ml respectively; while TGR5 methylation combined AFP had Youden index of 0.20, 0.47 and 0.50.Conclusions: Our findings strongly suggested the combination of serum TGR5 promoter methylation and AFP enhanced the diagnostic value of AFP alone in discriminating HCC from CHB patients.     

UV sensitive mutations in histone H3 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> that alter specific K79 methylation states genetically act through distinct DNA repair pathways

Chromatin serves as a regulator of various nuclear processes, with post-translational modifications of histone proteins serving as modulators to influence chromatin function. We have previously shown that histone H3 K79 methylation is important for repair of UV-induced DNA damage in Saccharomyces cerevisiae, acting through multiple repair pathways. To evaluate the potential role of distinct K79 methylation states in DNA repair, we identified four mutations in histone H3 that confer sensitivity to UV, each of which also has a distinct effect on specific K79 methylation states. Epistasis analyses indicate that each mutation exerts its phenotypic effects through distinct subsets of the various DNA damage response pathways, suggesting the existence of discrete roles for histone H3 in DNA damage checkpoint and repair pathways. Furthermore, we find that the distribution of K79 methylation states is altered by mutation of the acetylatable N terminal lysines in histone H4. The combined results suggest that K79 methylation states may be modulated in response to UV damage via a trans-histone regulatory pathway, and that distinct methylation states may provide a means of coordinating specific DNA repair and damage checkpoint pathways.     

DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.     

Site-specific methylation of placental HSD11B2 gene promoter is related to intrauterine growth restriction

Intrauterine growth restriction (IUGR) is associated with detrimental effects on neurodevelopmental progress in childhood and higher risk of degenerative diseases in adulthood. Placental 11β-hydroxysteroid dehydrogenase (HSD11B2) is a key gene involved in glucocorticoid metabolism, which in turn seems to be related to fetal growth impairment. As reduction of placental HSD11B2 gene expression has been associated with reduced human fetal growth, and methylation of HSD11B2 gene promoter has been shown to have an important role in HSD11B2 gene repression, we seek to investigate the relationship between IUGR and HSD11B2 gene promoter methylation in human placentas. We found that methylation levels of all studied CpG sites were significantly higher in IUGR newborns than those in controls. Further, methylation levels of the first and the third CpG sites were inversely associated with measures of fetal growth (birth weight and ponderal index). In addition, consistent with the above negative correlation, methylation levels of the first and the third CpG sites were inversely associated with HSD11B2 gene expression. These results together show a link between the site-specific methylation of placental HSD11B2 promoter and the development of IUGR.     

Alteration of DNA methylation levels in MRL lupus mice

Recent reports suggest that DNA methylation is involved in the cause of autoimmune disease. We investigated the alteration of DNA methylation levels in lupus strains of mice, MRL/lpr as a model, which develop an age-dependent lymphadenopathy and autoimmune disease. DNA methylation levels of thymus and axillary lymph nodes in 20-week-old MRL/lpr mice, which are in an autoimmune disease state, were lower than those of 4-week-old MRL/lpr mice with no symptoms as yet. No significant changes were observed in MRL/4- strain mice, which seemed normal at least 20 weeks, while DNA methylation levels in the spleen of both strains of mice increased significantly from the age of 4 to 20 weeks. However, no significant changes of DNA methylation levels in peripheral blood were observed with ageing in MRL strains. Moreover, we clarified that administration of 5-azacytidine had a strong effect on longer survival of MRL/lpr mice and reduced DNA methylation levels in the axillary lymph nodes and spleen. The possible relevance of DNA methylation levels to the progression of autoimmune disease is discussed.     

De novo telomere formation is suppressed by the Mec1-dependent inhibition of Cdc13 accumulation at DNA breaks

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability, since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. Here we report that Mec1, the ATR ortholog, couples the detection of DNA ends with the inhibition of telomerase. Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, a phosphorylation event that suppresses Cdc13 accumulation at DSBs. Conversely, telomere addition at accidental breaks is promoted by Pph3, the yeast protein phosphatase 4 (PP4). Pph3 is itself modulated by Rrd1, an activator of PP2A family phosphatases. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DNA breaks. These studies therefore identify a mechanism by which the ATR family of kinases enforces genome integrity, and a process that underscores the contribution of Cdc13 to the fate of DNA ends.