Comparison of the DNA methylation of bovine blastocysts and bovine sperm
ABSTRACT: DNA from expanded bovine blastocysts and bovine sperm were extracted then subjected to methylation-sensitive enzymatic digestion and LM-PCR enrichment before being hybridized onto a microarray. Two-condition experiment, bovine blastocysts (pools of 10) vs bovine sperm. Four biological replicates of each tissue were hybridized to four two-color arrays in a dye-balanced design.
Project description:Transcriptome (total RNA) profiling of bovine in vitro cultured expanded blastocysts (EB) comparing control non-treated expanded blastocysts with SAM-treated expanded blastocysts. S-Adenosyl methionine (SAM) is the global methyl donor providing methyl group for variety of biomolecules such as DNA , histone, RNA, lipids and etc. Two-condition experiment, bovine non-treated expanded blastocysts (pools of 10) vs bovine SAM-treated expanded blastocysts. Four biological replicates of each tissue were hybridized to four two-color arrays in a dye-balanced design.
Project description:Epigenetics (DNA methylation) profiling of sperm from Monozygotic (MZ) twin bull brothers comparing with each other Two-condition experiment, DNA methylation analysis of sperm from Monozygotic twin bull brothers which were hybridized as dye-swap in two-color arrays in a dye-balanced design.
Project description:During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the 4-cell, 8-cell, 16-cell and blastocyst stages. These were produced by in vitro fertilization of Bos t. taurus oocytes with sperm from a Bos t. indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10^3 different genes were detected in the various developmental stages. EGA was analyzed by i) detection of embryonic transcripts which are not present in oocytes; ii) detection of transcripts from the paternal allele; and iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the 4-cell to the blastocyst stage. The largest proportion of gene activation, i.e. (i) 59%, (ii) 42%, and (iii) 58%, was found in 8-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the 4-cell stage identified categories related to translation, RNA processing and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and new insight into the timing of embryonic activation of specific genes. RNA-Seq profiles from pools of 10 ooytes/embryos from bovine Bos t. taurus GV and MII oocytes and a cross-breed of Bos t. taurus x Bos t. indicus from 4-cell, 8-cell, 16-cell and blastocysts generated using Illumina GAIIx
Project description:Blastocyst formation is a primordial event of pre-implantation development since it is required for pregnancy establishment and progression. The blastocyst plays a pivotal role because it is the stage at which the embryo is transferred and starts coordinated cross-talks with the mother. It is also the terminal step of pre-implantation developmentbefore transfer; it reflects all stresses the embryo may have faced during the process of in-vitro treatment. Achieving the formation of a morphologically healthy blastocyst following normal kinetics is a good sign but remains a poor indicator of embryo quality. Considering the limitation of the invasive methods for competence assessment, the analysis of blastocysts’ gene expression is a promising way to improve our understanding of blastocyst formation and to study the effects of different treatments on gene expression. Methods: Early, expanded and hatched blastocysts were collected for RNA extraction, amplification, and cDNA microarray hybridization. Gene candidates (IFNt, PLAC8, SSLP1, AKR1B1, HNRNPA2B1, ARGFX, NANOS, CCNB1) were selected and confirmed using Q-RT-PCR to validate the microarray data. Results: Our analysis show that hatched blastocysts are enriched in genes transcripts implicated in implantation, cell adhesion and extracellular matrix digestion. Early blastocysts expressed genes mainly involved in cell cycle control, transcription and translation. Q-RT-PCR validated most microarray results (87.5%). Some of the differentially expressed genes are interesting as potential markers of competence. Conclusions: Overall, our study provides new insights into the molecular regulation of blastocyst formation. In addition, it could help assess blastocyst staging and select better embryos based on the expression of quality markers. In the present study the Laval/Sirard_bovine_embryo_3K_V3.0 array was used to conduct a series of 18 hybridizations (for three Blastocysts stages (early, expended, hatched) with three biological replicates and dye swaps) in a loop design experiment.
Project description:Bovine IVP blastocysts (day 8) were produced with and without thyroid hormones and gene expression profiles were compared. The IVC media (SOF) was supplemented with 50ng/ml of each T3 and T4. Four independent IVF run, each gave 1 control and 1 treated sample. Each sample is a pool of 5 blastocyst. Total Rna was extracted from each sample, amplified, labeled (also with dye swap) and hybridized to the EmbryoGene custom Agilent array (GPL13226). FlexArray software was used for data analysis.
Project description:The genomic DNAs of strains 263 of L. infantum and five derived independent resistant mutants to 5-fluorouracil were used in comparative genomic hybridizations to reveal the deletion and/or amplification events occured by drug resistance mechanisms. The human protozoan parasites Leishmania are prototrophic for pyrimidines and de novo pyrimidine biosynthesis is necessary for their growth. Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed amplification and deletion events as well as point mutations in metabolic genes involved in either the uridine salvage, folate or dTMP biosynthesis pathways. In particular, a dhfr-ts containing amplicon was observed in two mutants and a deletion of part of chromosome 10 was detected in one mutant. Point mutations in uridine phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also discovered. Transfection experiments confirmed that these molecular alterations were responsible for the 5-FU resistance phenotype. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import although the identity of the transporter remains elusive. This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist in a cell to lead to resistance. Each independent resistant mutant to 5-fluorouracil was hybridizated with the wild-type L. infantum 263 to 10 microarrays, each with three biological replicates (independent cultures).
Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards. Six strains of three Leishmania species were hybridizated to 12 microarrays, each with four biological replicates (independent cultures). Supplementary file: Represents final results obtained after statistical analysis of all replicates.
Project description:An integrative functional genomics of multiple forms of data is vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified Fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene due to the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with Cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD induced glioma cell migration. siRNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on Integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C terminus LRR11 domain (FMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I-FMOD interaction in integrin activation. ChIP-PCR experiments revealed that TGF-ß1 regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of SMAD2 and CBP. FMOD silencing inhibited the TGF-ß1 mediated glioma cell migration significantly. In univariate and multivariate cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, the present study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-ß1 pathway and forms a potential basis for therapeutic intervention in GBM. We used DNA isolated from 17 diffuse astrocytoma, 16 anaplastic astrocytoma, 36 glioblastoma and 9 control brain tissue for the array. P53 status: All cases were subjected to histopathology and the diagnosis of GBM was confirmed. Paraffin sections (4 μm) from the tissues were collected on silane-coated slides, and the protein expression of p53 was assessed by immunohistochemistry (IHC). Antigen retrieval was done by heat treatment at 850W in citrate buffer or at 700W. After the initial processing steps, sections were incubated overnight with primary antibody for p53 (Mouse Monoclonal DO-7; Biogenex, USA)at 4°C with 1: 200 dilution. MIB: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 µm thick sections were collected on silane-coated slides, and IHC was performed using MIB-1 (anti Ki-67 monoclonal BGX 297, diluted to 1:30) EGFR status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 µm thick sections were collected on silane-coated slides, and IHC was performed using EGFR (monoclonal E-30, diluted to 1:50) GFAP status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 µm thick sections were collected on silane-coated slides, and IHC was performed using glial fibrillary acidic protein (GFAP, monoclonal GA-5, diluted to 1:100).
Project description:Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and the expression rates of both proteins are low under standard growth conditions. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression and perform a ChIP-seq experiment. This revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region.