Project description:Methylthioadenosine phosphorylase (MTAP), a key enzyme in the adenine and methionine salvage pathways, catalyzes the hydrolysis of methylthioadenosine (MTA), a compound suggested to affect pivotal cellular processes in part through the regulation of protein methylation. MTAP is expressed in a wide range of cell types and tissues and its deletion is common to cancer cells and in liver injury. The aim of this study was to investigate the proteome and methyl proteome alterations triggered by MTAP deficiency in liver cells to define novel regulatory mechanisms that may explain the pathogenic processes of liver diseases. iTRAQ analysis resulted in the identification of 216 differential proteins (p<0.05) that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis lead to the identification of 74 differentially methylated proteins between SK-Hep1 and SK-Hep1+ cells, including 116 new methylation sites. Restoring normal MTA levels in SK-Hep1+ cells parallels the specific methylation of 56 proteins, including KRT8, TGF and CTF8A, which provides a novel regulatory mechanism of their activity with potential implications in carcinogenesis. Inhibition of RNA binding proteins methylation is especially relevant upon accumulation of MTA. As an example, methylation of quaking protein in R242 and R256 in SK-Hep1+ cells may play a pivotal role in the regulation of its activity as indicated by the up-regulation of its target protein p27 kip1. The phenotype associated with a MTAP deficiency was further verified in the liver of MTAP+/- mice. Our data support that MTAP deficiency leads to MTA accumulation and deregulation of central cellular pathways, increasing proliferation and decreasing the susceptibility to chemotherapeutic drugs, which involves differential protein methylation.
Project description:We show that Polθ is recruited to mitotic Double-strand breaks (DSBs) to slow down cell cycle progression and allow DNA repair. Because Polθ is one of the only repair protein to forms repair foci during mitosis, we investigated its regulation during mitosis. We performed immunoprecipitation (IP) of Polθ and assessed phosphorylation by immunoblot analysis (using pan phospho antibodies). We observed a phosphorylation signal corresponding to the size of Polθ when IP was performed from mitotic cell extracts. This phosphorylation signal was abolished when cells where treated with two different PLK1 inhibitors (PLK1i), indicating that PLK1 is responsible for Polθ phosphorylation in mitosis. In order to elucidate the regulation of mitotic Polθ activity, we performed mass spectrometry (MS)-based phosphorylation analysis of Polθ in mitosis with or without the PLK1 inhibitor Volasertib. We found 5 phosphorylated residues. To assess the functional consequences of Polθ phosphorylation by PLK1, we mutated some of the identified residues and found that the phospho dead mutant of Polθ fails be recruited to DSBs in mitosis. This indicates that PLK1-mediated regulation of mitotic Polθ repair is essential for its proper functioning
Project description:Post-partum uterine inflammation (endometritis) is associated with lower fertility at both the time of infection and after the inflammation has resolved. It was hypothesized that aberrant DNA methylation may be involved in the sub-fertility associated with post-partum uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with sub-clinical endometritis. Endometrial tissues were obtained at 29 days post-partum (n=12) and Agilent two-colour microarrays were used to characterize transcription and DNA methylation profiles. Analyses revealed 1,856 probes to be differentially expressed in animals with subclinical endometritis (SUI, n=6) compared with control cows (NUI, n=6, P<0.05, Storey Multiple testing correction). No significant associations among DNA methylation and gene expression were detected. Further analysis of gene expression data using GeneGo Metacore and Gene Set Enrichment Analysis identified several pathways and processes enriched in the comparison. Several pathways that are involved in the innate immune response were enriched in SUI cows. Consistent with the presence of microorganisms in the uterus, there was enrichment for the Toll-like receptor (TLR) signaling pathway, including increased expression of the transcription factor NFKB1, the pro-inflammatory cytokines IL1A and IL1B, downstream chemokines, cytokines, and acute phase and antimicrobial proteins in the endometrium of SUI cows. Furthermore, the chemokine signaling pathway was enriched in SUI cows, with increased expression of genes that attract cells of the innate immune system. Increased expression of IL-8 and CXCL6, chemotactic factors for recruitment of neutrophils along with the immune cell surface marker PTPRC in SUI cows is consistent with the greater number of polymorphonuclear cells present in the uterus of these cows. Several antimicrobial peptides (LAP, TAP, DEFB1, DEFB10, DEFB103B, DEFB7) and acute phase proteins, including SAA3, LBP, and the complement gene CFB, had greater expression in SUI cows. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local pro-inflammatory environment in the uterus. If this period of inflammation is prolonged, it could result in tissue damage or failure to complete involution of the uterus, which may create a sub-optimal environment for future pregnancy. Agilent two-colour microarrays were used to characterize DNA methylation profiles in cows with subclinical endometritis (SUI, n=6) compared to control cows (NUI, n=6). Endometrial tissues (caruncular, intercaruncular) were obtained at 29 days post-partum.
Project description:Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions M-bM-^@M-^S and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization and reprogramming into induced pluripotent stem cells (iPSC) using high density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and occur particularly in intergenic and non-promoter regions of developmental genes. We demonstrate that ionizing irradiation, although associated with a very similar senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40 TAg) result in telomere extension but do not influence SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevented SA-DNAm changes. Our results indicate that replicative senescence is associated with an epigenetically controlled process which stalls cells in a particular differentiated state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence. Samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip
Project description:Notch signalling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighbouring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and together with the transcription factor moiety of Notch (NICD) it regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole genome binding profiles were generated for RBPJ, its coactivator p300, NICD and the histone H3 modifications H3K4me3, H3K4me1 and H3K27ac in myogenic cells under active or inhibitory Notch signalling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300, and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signalling mediate their activities and consequently impact on cell fate decisions. ChIP (chromatin immunoprecipitation) is followed by deep sequencing to generate genome-wide patterns of RBP-J binding in mouse C2C12 cells under various conditions. Cells were either Notch activated by exposure to immobilized ligand or by overexpression of NICDGFP, or Notch inhibited by treatment with DAPT. Notch activation and inhibition treatments were applied for 6h and 24h. In addition to RBP-J, p300 and NICDGFP were profiled by ChIP-Seq and gene expression was assessed by RNA-Seq.
Project description:The first step in metastasis is dissemination of tumor cells into the surrounding tissue, or stroma. In carcinomas, cancers of epithelial origin, tumor cells must first breach the basement membrane (BM), a network that encapsulates the tumor. As tumor progresses, other cell types that reside in the stroma, such as carcinoma-associated fibroblasts (CAFs) accumulate around the tumor. Whether CAFs can help cancer cells to breach the BM remains an open question. Here we show that CAFs promote cancer cell invasion by physically remodeling the BM. Using a novel 3D in vitro model we observed that primary CAFs isolated from colon cancer patients stimulate cancer cell invasion through a native mesenteric BM. This stimulated invasion is the result of the physical presence of CAFs rather than an increased invasive potential of cancer cells. In the presence of CAFs, cancer cells can invade the BM in a matrix metalloproteinase-independent manner. Using live imaging, we found that CAFs actively pull and stretch the BM, leading to the formation of gaps through which cancer cells can migrate. Notably, CAF-mediated gap-widening is independent of proteolysis but relies on actomyosin contractility. We propose that CAFs exert mechanical forces that alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion.
Project description:This SuperSeries is composed of the following subset Series: GSE37065: Long-term culture associated gene expression changes in MSC [Affymetrix] GSE37066: Pluripotent Stem Cells Escape From Senescence-Associated DNA Methylation Changes [Illumina] GSE38806: Gene expression profiles of induced pluripotent mesenchymal stromal cells [Affymetrix] Refer to individual Series
Project description:This study describes the DNA methylation profiling using whole-genome bisulfite sequencing of mouse ES cells, either derived and maintained in 2i serum-free NDiff medium, or in the presence of serum and LIF, or maintained and derived in the presence of serum and LIF and subsequently adapted to 2i serum-free NDiff medium, or maintained and derived in the presence of 2i and LIF and subsequently adapted to 2i serum. DNA methylation profiling using whole-genome bisulfite sequencing of 14 samples, 3 different lines (E14, XT67E1, Rex/GFP-2i) of pluripotent mouse ES cells as well during conversion from 2i to serum and vice versa.