DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression

Budding yeast cells suffering a single unrepaired DNA double-strand break (DSB) trigger the ATR (Mec1)-dependent DNA damage checkpoint and arrest prior to anaphase for 12–15 h, following which they adapt and resume cell division. When the DNA lesion can be repaired, the checkpoint is extinguished and cells “recover” and resume mitosis. In this autophagic punctum, we report that hyperactivation of autophagy—specifically via the cytoplasm-to-vacuole targeting (Cvt) pathway—prevents both adaptation to, and recovery from, DNA damage, resulting in the permanent arrest of cells in G₂/M. We show that Saccharomyces cerevisiae deleted for genes encoding the Golgi-associated retrograde protein transport (GARP) complex are both adaptation- and recovery-defective. GARP mutants such as vps51Δ exhibit mislocalization of the key mitotic regulator, securin (Pds1), and its degradation by the vacuolar protease Prb1. In addition, separase (Esp1), is excluded from the nucleus, accounting for pre-anaphase arrest. Pds1 is degraded via the Cvt pathway. Many of the same defects seen by deleting GARP genes can be mimicked by hyperactivation of the Cvt pathway by overexpressing an unphosphorylatable form of ATG13 or by adding the TORC1 inhibitor rapamycin. These results suggest that nuclear events such as DNA damage can have profound effects on cytoplasmic processes and further expand the burgeoning connections between DNA damage and autophagy.     

Sh3glb1/Bif-1 and mitophagy

Evasion of apoptosis, which enables cells to survive and proliferate under metabolic stress, is one of the hallmarks of cancer. We have recently reported that SH3GLB1/Bif-1 functions as a haploinsufficient tumor suppressor to prevent the acquisition of apoptosis resistance and malignant transformation during Myc-driven lymphomagenesis. SH3GLB1 is a membrane curvature-inducing protein that interacts with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the upregulation of anti-apoptotic proteins, including MCL1 and BCL2L1. Notably, we found that Sh3glb1 haploinsufficiency increases mitochondrial mass in overproliferated prelymphomatous Eμ-Myc cells. Moreover, loss of Sh3glb1 suppresses autophagy-dependent mitochondrial clearance (mitophagy) in PARK2/Parkin-expressing mouse embryonic fibroblasts (MEFs) treated with the mitochondrial uncoupler CCCP. Interestingly, PARK2-expressing Sh3glb1-deficient cells accumulate ER-associated immature autophagosome-like structures after treatment with CCCP. Taken together, we propose a model of mitophagy in which SH3GLB1 together with the class III phosphatidylinositol 3-kinase complex II (PIK3C3CII) (PIK3R4-PIK3C3-BECN1-UVRAG) regulates the trafficking of ATG9A-containing Golgi-derived membranes (A9⁺GDMs) to damaged mitochondria for autophagosome formation to counteract oncogene-driven tumorigenesis.     

Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia

The objectives of this study were to identify tissue-specific differentially methylated regions (T-DMR’s) in the folate transport genes in placental tissue compared with leukocytes, and from placental tissues obtained from normal infants or with neural tube defects (NTDs). Using pyrosequencing, we developed methylation assays for the CpG islands (CGIs) and the CGI shore regions of the folate receptor α (FOLR1), proton-coupled folate transporter (PCFT) and reduced folate carrier 1 (RFC1) genes. The T-DMRs differed in location for each gene and the difference in methylation ranged between 2 and 54%. A higher T-DMR methylated fraction was associated with a lower mRNA level of the FOLR1 and RFC1 genes. Methylation fractions differed according to RFC1 80G > A genotype in the NTD cases and in leukocytes from subjects with high total plasma homocysteine (tHcy). There were no differences in methylated fraction of folate transporter genes between NTD cases and controls. We suggest that T-DMRs participate in the regulation of expression of the FOLR1 and RFC1 genes, that the RFC1 80G > A polymorphism exerts a gene-nutrition interaction on DNA methylation in the RFC1 gene, and that this interaction appears to be most prominent in NTD-affected births and in subjects with high tHcy concentrations.     

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.     

KIBRA gene methylation is associated with unfavorable biological prognostic parameters in chronic lymphocytic leukemia

Ras-association domain family (RASSF) members are a family of genes containing an RA domain in either the C-terminus (RASSF1-RASSF6) or in the N-terminus (RASSF7-RASSF10). Members of this gene family are core members of the Salvador/Warts/Hippo (SWH) tumor suppressor network and have been shown to be involved in human tumorigenesis. Among the RASSF genes, RASSF1A is one of the most frequently methylated genes in a wide range of epithelial cancers, and we previously demonstrated that RASSF6 and RASSF10 genes are frequently epigenetically inactivated in acute leukemias, particularly in those of the B cell type. We here determined the methylation profiles of all members of the RASSF gene family as well as two recently identified (KIBRA, CRB3) upstream members of the SWH pathway in the leukemic B cells obtained from a well-characterized cohort of 95 patients with chronic lymphocytic leukemia (CLL). Among the RASSF genes, RASSF10 (50%) was the most frequently methylated gene, followed by RASSF6 (16%). The remaining RASSF genes were either unmethylated or showed a frequency of methylation < 10%. The upstream SWH member KIBRA was also frequently methylated in CLL (35%) in contrast to CRB3. Interestingly, the analysis of clinical-pathological parameters showed that KIBRA methylation was associated with unfavorable biological prognostic parameters, including unmutated IGHV genes (p = 0.007) and high CD38 expression (p < 0.05).     

Birthweight is associated with DNA promoter methylation of the glucocorticoid receptor in human placenta

Birthweight has been associated with a number of health outcomes throughout life. Crucial to proper infant growth and development is the placenta, and alterations to placental gene function may reflect differences in the intrauterine environment which functionally contribute to infant growth and may ultimately affect the child’s health. To examine if epigenetic alteration to the glucocorticoid receptor (GR) gene was linked to infant growth, we analyzed 480 human placentas for differential methylation of the GR gene exon 1F and examined how this variation in methylation extent was associated with fetal growth. Multivariable linear regression revealed a significant association (p &lt; 0.0001) between differential methylation of the GR gene and large for gestational age (LGA) status. Our work is one of the first to link infant growth as a measure of the intrauterine environment and epigenetic alterations to the GR and suggests that DNA methylation may be a critical determinant of placental function.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.     

Evidence that the methylation state of the monoamine oxidase A (MAOA) gene predicts brain activity of MAO A enzyme in healthy men

Human brain function is mediated by biochemical processes, many of which can be visualized and quantified by positron emission tomography (PET). PET brain imaging of monoamine oxidase A (MAO A)—an enzyme metabolizing neurotransmitters—revealed that MAO A levels vary widely between healthy men and this variability was not explained by the common MAOA genotype (VNTR genotype), suggesting that environmental factors, through epigenetic modifications, may mediate it. Here, we analyzed MAOA methylation in white blood cells (by bisulphite conversion of genomic DNA and subsequent sequencing of cloned DNA products) and measured brain MAO A levels (using PET and [¹¹C]clorgyline, a radiotracer with specificity for MAO A) in 34 healthy non-smoking male volunteers. We found significant interindividual differences in methylation status and methylation patterns of the core MAOA promoter. The VNTR genotype did not influence the methylation status of the gene or brain MAO A activity. In contrast, we found a robust association of the regional and CpG site-specific methylation of the core MAOA promoter with brain MAO A levels. These results suggest that the methylation status of the MAOA promoter (detected in white blood cells) can reliably predict the brain endophenotype. Therefore, the status of MAOA methylation observed in healthy males merits consideration as a variable contributing to interindividual differences in behavior.