Effects of PGRMC1 phosphorylation on genome integrity and epigenetics
ABSTRACT: Progesterone receptor membrane component 1 (PGRMC1) is a protein that has been implicated in cancer biology and poor patient outcomes, which can be over-expressed in cancers, and exist in alternative states of phosphorylation.Here, we show that manipulation of PGRMC1 phosphorylation by mutagenesis results in altered cell metabolism. We examine the pathway by which nicotinamide-N-methyl transferase (NNMT) transfers a methyl group from S-adenosylmethionine (SAM) to produce 1-methylnicotinamide, lowering the levels of global methyl donor SAM. Hypothesizing that PGRMC1 phosphorylation status affects genome methylation, we discovered that each of several mutants elicited distinct patterns of CpG methylation.We conclude that PGRMC1 phosphorylation status, as controlled by unknown signaling processes, causes profound changes in cellular plasticity by affecting mechanisms also associated with early embryological tissue differentiation.
Project description:This study examines the therapeutic plausibility of using universal methyl group donor S-adenosylmethionine (SAM) to block breast cancer development, growth, and metastasis. cancer. Anti-tumor and anti-metastatic activity of SAM was evaluated through a series of studies in vitro using two different human breast cancer cell lines and in vivo using a MDA-MB-231 xenograft model of breast cancer. The data shown in this array is obtained from control and SAM-treated MDA-MB-231 cell lines. Overall design: RNA from three different biological repeats from control and SAM-treated MDA-MB-231 cells were subjected to Human Gene 2.0 ST array.
Project description:S-adenosylmethionine (SAM) is the methyl donor for biological methylation modifications that regulate protein and nucleic acid functions. Here we show that methylation of a phospholipid, phosphatidylethanolamine (PE), is the major consumer of SAM in budding yeast. The induction of phospholipid biosynthetic genes is accompanied by induction of the enzyme that hydrolyzes S-adenosylhomocysteine (SAH), a product and inhibitor of methyltransferases. Beyond its function for the synthesis of phosphatidylcholine (PC), the methylation of PE facilitates the turnover of SAM for the synthesis of cysteine and glutathione. Strikingly, cells that lack PE methylation accumulate SAM, which leads to hypermethylation of histones and the major phosphatase PP2A, dependency on cysteine, and sensitivity to oxidative stress. Without PE methylation, particular sites on histones then become methyl sinks to enable the turnover of SAM. These findings reveal an unforeseen metabolic function for phospholipid and histone methylation intrinsic to the life of a cell. Overall design: Two biological replicates of wild type and cho2Δ cells in YPL media, in SL media after 1 hour and in SL media after 3 hour were collected for sequencing.
Project description:Methionine adenosyltransferase (MAT), that catalyzes the synthesis of S-adenosylmethionine (SAM) from ATP and methionine, is involved in folate-mediated one-carbon metabolism (OCM) that is essential for preimplantation embryos in terms of both short-term periconceptional development and long-term phenotypic programming beyond the periconceptional period. SAM is the universal methyl donor for epigenetic methylation of DNA and histones. In the context of the long-term phenotypic programming by the modulation of OCM during periconceptional period, possible interactions between MAT2A and epigenetic status of specific genes during this period are of particular interest. MAT2A has been proposed to interact with many chromatin-related proteins and be recruited to their specific target genes to constitute gene-regulatory complexes. Thus, in the aim of addressing the possible interaction of MAT2A with specific genomic DNA regions in periconceptional period, we performed a ChIP-seq analysis using MAT2A antibody against bovine blastocysts. Overall design: In vitro produced bovine blastocysts were processed to ChIP-Seq using MAT2A antibody with corresponding Input and IgG control. The same procedures were duplicated using total of 669 and 677 blastocysts, respectively, for each replicate.
Project description:Maintenance of the intracellular levels of the methyl donor S-adenosylmethionine (SAM) is essential for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of MAT2A, which encodes the only SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3´ UTR. Increasing METTL16 occupancy on the MAT2A 3´ UTR is sufficient to induce efficient splicing. We propose that under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which in turn promotes MAT2A splicing. Interestingly, human and S. pombe METTL16 methylate the U6 spliceosomal snRNA at a sequence identical to hp1. These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Overall design: m6A-seq was performed on polyA-selected RNA from HEK293A-TOA cells from siCtrl or siMETTL16 knockdown conditions. 3 biological replicates were performed for each condition. Both the m6A-IP and the inputs are available for each sample.
Project description:The analysis investigates the impact of methyl donor S-adenosylmethionin on transcription and methylation profiles of prostate carcinoma cells. PC-3 cells (Prostatecarcinoma cells) were treated with 160 micromolar S-adenosylmethionine or vehicle
Project description:We examined the effect of nicotinamide on cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibited the phosphorylation of myosin light chain, suppressed actomyosin contraction, and led to improved cell survival after individualization. Then we analyzed the global gene expression profile after 24 hours of nicotinamide and ROCK inhibitor treatment, and found that the gene expression profile of human embryonic stem cell (hESC) treated with nicotinamide was much different from that of ROCK inhibitor treatment. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and CK1. We demonstrated that nicotinamide affected human embryonic stem cell (hESC) pluripotency and differentiation as a selective kinase inhibitor. Overall design: Total RNA obtained from human embryonic stem cell (hESC) culture in E8 medium (E8 24 hrs), E8 medium with nicotinamide 10 mM treatment (Nam 24 hrs), or E8 medium with 10 μM ROCK inhibitor Y27632 treatment (ROCKi 24 hrs) for 24 hours. HESCs were treated with BMP4 for 6 days as mesoderm differentiation control (Mesoderm).
Project description:The analysis investigates the impact of methyl donor S-adenosylmethionin on transcription and methylation profiles of prostate carcinoma cells. Overall design: PC-3 cells (Prostate carcinoma cells) were treated with 160 micromolar S-adenosylmethionine or vehicle
Project description:We aimed to examine the gene expression changes responding to a depletion of intracellular S-adenosylmethionine (SAM). The mouse plasma cell line X63/0 was treated with the SAM-synthetase inhibitor cycloleucine (cLEU), and the total RNA was isolated and analyzed by RNA-sequencing. As a result, we idntified 27 genes, including the ubiquitous SAM-synthetase MAT2A, whose expssions were up-regulated by two-fold or more. Overall design: Total RNA was extracted from the mouse plasma cell line X63/0 before/after a three-hour treatment with 30 mM cycloleucine. This experiment was triplicated, and the resulting six samples were applied to RNA-sequencing.