183 EFFECT OF WELL IN WELL CULTURE OF BOVINE EMBRYOS ON GENE EXPRESSION PROFILE

2009 ◽  
Vol 21 (1) ◽  
pp. 190 ◽  
Author(s):  
N. Ghanem ◽  
M. Hoelker ◽  
C. Phatsara ◽  
K. Schellander ◽  
D. Tesfaye
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Relative Abundance ◽  
Large Scale ◽  
Gene Expression Pattern ◽  
Cdna Array ◽  
Transcript Abundance ◽  
Oxidoreductase Activity ◽  
Group Culture

To culture embryos in small groups, the well in well culture system (miniwells harboring 1 single embryo within the well) has been developed previously. In this work, we aimed to examine the effects of the microenvironment provided by well in well culture and embryo density on the relative abundance of transcripts in the resulting embryos. Cumulus–oocyte complexes (COCs) were aspirated from small follicles (2 to 8 mm), and COCs were cultured in 400 μL of modified TCM (TCM-199, Sigma, Taufkirchen, Germany) supplemented with 12% heat-inactivated estrous cow serum and 10 μg mL–1 of FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO2 in air. Fertilization was performed in Fert-TALP supplemented with 1 μg mL–1 of heparin. Zygotes were allocated randomly in 2 groups, namely: well in well culture (16 miniwells of 0.7 diameter and deepness each containing 1 embryo per well) and group of 16 (group culture of 16 embryos per well). Six pools each containing 20 Day 7 blastocysts derived from the first 2 groups were used to investigate large-scale gene expression analysis using BlueChip cDNA-Array. Three pools each containing 5 blastocysts were used for Array data validation by real-time PCR using primers specific to 5 selected genes (ATP5, PLAC8, KRT8, S100A10, and ZP3). During validation in vivo-derived bovine blastocysts were included to be used as standard. Significance Analysis of Microarray identified 75 transcripts differentially expressed between the 2 groups. Blastocysts derived from well in well culture were found to be enriched with genes regulating different molecular functions including structural constituent of ribosome (RPS29), protein binding (Cul1), calcium ion binding (S100A10, NPTX2), nitric oxide synthase regulator activity (HSPCA), and RNA polymerase II transcription factor activity (UHRF1). However, blastocysts derived from group of 16 culture were found to be enriched with genes involved in oxidoreductase activity (ALOX15, AKR1B), cytochrome-c oxidase activity (COX7A2), hydrogen ion transporting ATP synthase activity (ATP5O), transcription (PTTG1), and cell redox homeostasis (TXN). According to their biological process, genes enriched in blastocysts derived from well in well culture belong to small molecule transport and signal transduction, whereas most downregulated genes have a metabolic function. Comparison of the transcript abundance of the 5 selected genes in the 3 embryo groups showed that the expression of ATP5, PLAC8, and KRT8 in embryos from well in well culture resembles to the relative abundance in blastocyst derived from in vivo culture. However, with respect to the expression of S100A10 and ZP3 genes, blastocysts derived from group culture showed similarity with embryos derived from in vivo. In conclusion, microenvironment affects the gene expression pattern of the resulting embryos.

Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells

2005 ◽  
Vol 54 (5) ◽  
pp. 497-504 ◽  
Author(s):  
Joseph Richardson ◽  
Justin Corey Craighead ◽  
Sam Linsen Cao ◽  
Martin Handfield
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Aggressive Periodontitis ◽  
Actinobacillus Actinomycetemcomitans ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

Actinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.

scholarly journals Independent Recruitment of Mediator and SAGA by the Activator Met4

2006 ◽  
Vol 26 (8) ◽  
pp. 3149-3163 ◽  
Author(s):  
Christophe Leroy ◽  
Laëtitia Cormier ◽  
Laurent Kuras
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Rna Polymerase Ii ◽  
Gene Network ◽  
Pol Ii ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Target Promoters ◽  
Sulfur Containing

ABSTRACT Mediator is a key RNA polymerase II (Pol II) cofactor in the regulation of eukaryotic gene expression. It is believed to function as a coactivator linking gene-specific activators to the basal Pol II initiation machinery. In support of this model, we provide evidence that Mediator serves in vivo as a coactivator for the yeast activator Met4, which controls the gene network responsible for the biosynthesis of sulfur-containing amino acids and S-adenosylmethionine. In addition, we show that SAGA (Spt-Ada-Gcn5-acetyltransferase) is also recruited to Met4 target promoters, where it participates in the recruitment of Pol II by a mechanism involving histone acetylation. Interestingly, we find that SAGA is not required for Mediator recruitment by Met4 and vice versa. Our results provide a novel example of functional interplay between Mediator and coactivators involved in histone modification.

152 SPECIES-SPECIFIC DIFFERENCES IN THE METHYLATION REPROGRAMMING DURING EARLY PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 184
Author(s):  
S. Canovas ◽  
E. Ivanova ◽  
S. Garcia-Martinez ◽  
R. Romar ◽  
N. Fonseca-Balvis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Profiles ◽  
Differential Expression Analysis ◽  
Gene Expression Pattern ◽  
Reproductive Technologies ◽  
Global Methylation ◽  
Species Specific

Studies in mouse and human have shown extensive DNA methylation reprogramming in pre-implantation development followed by remethylation from implantation. However, the extent to which such reprogramming is conserved in mammals and the timing of demethylation and remethylation are unknown. As part of a major objective to characterise methylation dynamics in the bovine and porcine species from the oocyte to the blastocyst stage, we aimed here to compare the distribution of methylation at single-base resolution in both species at Day 7.5 of development. The DNA methylation profiles were obtained from individual blastocysts at Day 7.5 [pig: 3 in vivo, 3 in vitro; cow: 3 in vivo, 3 in vitro, 3 inner cell mass (ICM) and 3 trophoectoderm (TE) dissected from in vitro blastocysts] using the post-bisulphite adaptor tagging method and Illumina sequencing. For oocytes, data (GEO: GSE63330) from Schroeder et al. 2015 were analysed. Raw sequences were mapped, methylation calls made using Bismark and data analysis and visualisation was done within the SeqMonk platform. Gene expression profiles from individual blastocysts (3 pig, 3 cow) were obtained by RNA-seq. Annotated mRNA features were quantitated in SeqMonk and these were fed into DESeq2 for differential expression analysis (P < 0.05) as previously reported (Love et al. 2014 Genome Biol. 15, 550). Global methylation levels in whole blastocysts differed substantially between porcine and bovine embryos (in vivo: 12.33 ± 3.6 v. 28.33 ± 3.5%; in vitro: 15.02 ± 3.3 v. 24.41 ± 4.1%). In addition, the distribution of methylation differed: the pattern of cytosine methylated seemed random in the porcine genome, but was highly structured in the bovine genome, with methylation predominantly over gene bodies, resembling the profile previously reported in oocytes (Schroeder et al. 2015 PLoS Genet. 11, e1005442). Regarding correlation analysis, gene expression versus methylation were plotted. It suggested that gene body methylation reflected gene expression pattern in oocytes as well as in bovine blastocysts. Pair-wise comparison of isolated ICM and TE was filtered to require 5% change, and replicate set statistics were applied. This revealed very similar total and regional methylation levels in the 2 compartments, indicating that remethylation does not initiate preferentially in one compartment in bovine pre-implantation embryos. This confirms, from a viewpoint of the genome-wide DNA methylation, what has been observed in mouse for specific genes: the trophoblast-specific DNA methylation occurs after the segregation of the TE and ICM (Nakanishi et al. 2012 Epigenetics 7, 173–183). Our study is the first to provide whole genome methylation profiles from single blastocysts of economically important livestock species. Our data demonstrate that methylation reprogramming in early pre-implantation development is species specific. Knowledge of these specific patterns may have high importance when decisions are taken regarding the use of assisted reproductive technologies, cloning, or generation of transgenic animals. This work was funded by AGL2015–66341-R (MINECO-FEDER), PRX14/00348 (MECD), 19595/EE/14 (F. Séneca).

scholarly journals Genome-Wide Relationships between TAF1 and Histone Acetyltransferases in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (7) ◽  
pp. 2791-2802 ◽  
Author(s):  
Melissa Durant ◽  
B. Franklin Pugh
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Histone Acetylation ◽  
Rna Polymerase Ii ◽  
Histone Acetyltransferases ◽  
Histone H4 ◽  
Promoter Regions ◽  
Genome Wide ◽  
Acetylated Histone H4

ABSTRACT Histone acetylation regulates gene expression, yet the functional contributions of the numerous histone acetyltransferases (HATs) to gene expression and their relationships with each other remain largely unexplored. The central role of the putative HAT-containing TAF1 subunit of TFIID in gene expression raises the fundamental question as to what extent, if any, TAF1 contributes to acetylation in vivo and to what extent it is redundant with other HATs. Our findings herein do not support the basic tenet that TAF1 is a major HAT in Saccharomyces cerevisiae, nor do we find that TAF1 is functionally redundant with other HATs, including Gcn5, Elp3, Hat1, Hpa2, Sas3, and Esa1, which is in contrast to previous conclusions regarding Gcn5. Our findings do reveal that of these HATs, only Gcn5 and Esa1 contribute substantially to gene expression genome wide. Interestingly, histone acetylation at promoter regions throughout the genome does not require TAF1 or RNA polymerase II, indicating that most acetylation is likely to precede transcription and not depend upon it. TAF1 function has been linked to Bdf1, which binds TFIID and acetylated histone H4 tails, but no linkage between TAF1 and the H4 HAT Esa1 has been established. Here, we present evidence for such a linkage through Bdf1.

scholarly journals c-Jun Homodimers Can Function as a Context-Specific Coactivator

2007 ◽  
Vol 27 (8) ◽  
pp. 2919-2933 ◽  
Author(s):  
Benoit Grondin ◽  
Martin Lefrancois ◽  
Mathieu Tremblay ◽  
Marianne Saint-Denis ◽  
André Haman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Expression Profiles ◽  
Basic Domain ◽  
Interaction Mode

ABSTRACT Transcription factors can function as DNA-binding-specific activators or as coactivators. c-Jun drives gene expression via binding to AP-1 sequences or as a cofactor for PU.1 in macrophages. c-Jun heterodimers bind AP-1 sequences with higher affinity than homodimers, but how c-Jun works as a coactivator is unknown. Here, we provide in vitro and in vivo evidence that c-Jun homodimers are recruited to the interleukin-1β (IL-1β) promoter in the absence of direct DNA binding via protein-protein interactions with DNA-anchored PU.1 and CCAAT/enhancer-binding protein β (C/EBPβ). Unexpectedly, the interaction interface with PU.1 and C/EBPβ involves four of the residues within the basic domain of c-Jun that contact DNA, indicating that the capacities of c-Jun to function as a coactivator or as a DNA-bound transcription factor are mutually exclusive. Our observations indicate that the IL-1β locus is occupied by PU.1 and C/EBPβ and poised for expression and that c-Jun enhances transcription by facilitating a rate-limiting step, the assembly of the RNA polymerase II preinitiation complex, with minimal effect on the local chromatin status. We propose that the basic domain of other transcription factors may also be redirected from a DNA interaction mode to a protein-protein interaction mode and that this switch represents a novel mechanism regulating gene expression profiles.

scholarly journals Retrotransposon expression as a defining event of genome reprograming in fertilized and cloned bovine embryos

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 289-299 ◽  
Author(s):  
L C Bui ◽  
A V Evsikov ◽  
D R Khan ◽  
C Archilla ◽  
N Peynot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Ribosomal Proteins ◽  
Gene Expression Pattern ◽  
Cdna Array ◽  
Ltr Retrotransposons ◽  
Bovine Embryos ◽  
Genes Encoding ◽  
Low Efficiency ◽  
Cloned Bovine

Genome reprograming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprograming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprograming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprograming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprograming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprograming driven by oocyte cytoplasm.

scholarly journals Structure and Complexity of a Bacterial Transcriptome

2009 ◽  
Vol 191 (10) ◽  
pp. 3203-3211 ◽  
Author(s):  
Karla D. Passalacqua ◽  
Anjana Varadarajan ◽  
Brian D. Ondov ◽  
David T. Okou ◽  
Michael E. Zwick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Cell ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
A Genome ◽  
Nucleotide Resolution ◽  
Bacterial Transcriptome

ABSTRACT Although gene expression has been studied in bacteria for decades, many aspects of the bacterial transcriptome remain poorly understood. Transcript structure, operon linkages, and information on absolute abundance all provide valuable insights into gene function and regulation, but none has ever been determined on a genome-wide scale for any bacterium. Indeed, these aspects of the prokaryotic transcriptome have been explored on a large scale in only a few instances, and consequently little is known about the absolute composition of the mRNA population within a bacterial cell. Here we report the use of a high-throughput sequencing-based approach in assembling the first comprehensive, single-nucleotide resolution view of a bacterial transcriptome. We sampled the Bacillus anthracis transcriptome under a variety of growth conditions and showed that the data provide an accurate and high-resolution map of transcript start sites and operon structure throughout the genome. Further, the sequence data identified previously nonannotated regions with significant transcriptional activity and enhanced the accuracy of existing genome annotations. Finally, our data provide estimates of absolute transcript abundance and suggest that there is significant transcriptional heterogeneity within a clonal, synchronized bacterial population. Overall, our results offer an unprecedented view of gene expression and regulation in a bacterial cell.

scholarly journals A Transcriptome Fingerprinting Assay for Clinical Immune Monitoring

2019 ◽  
Author(s):  
Matthew C Altman ◽  
Nicole Baldwin ◽  
Elizabeth Whalen ◽  
Taha Al-Shaikhly ◽  
Scott Presnell ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Transcriptional Profiling ◽  
Transcript Abundance ◽  
Immune Monitoring ◽  
Immune Functions ◽  
Functional Interpretation ◽  
Clinical Tools

ABSTRACTBackgroundWhile our understanding of the role that the immune system plays in health and disease is growing at a rapid pace, available clinical tools to capture this complexity are lagging. We previously described the construction of a third-generation modular transcriptional repertoire derived from genome-wide transcriptional profiling of blood of 985 subjects across 16 diverse immunologic conditions, which comprises 382 distinct modules.ResultsHere we describe the use of this modular repertoire framework for the development of a targeted transcriptome fingerprinting assay (TFA). The first step consisted in down-selection of the number of modules to 32, on the basis of similarities in changes in transcript abundance and functional interpretation. Next down-selection took place at the level of each of the 32 modules, with each one of them being represented by four transcripts in the final 128 gene panel. The assay was implemented on both the Fluidigm high throughput microfluidics PCR platform and the Nanostring platform, with the list of assays target probes being provided for both. Finally, we provide evidence of the versatility of this assay to assess numerous immune functionsin vivoby demonstrating applications in the context of disease activity assessment in systemic lupus erythematosus and longitudinal immune monitoring during pregnancy.ConclusionsThis work demonstrates the utility of data-driven network analysis applied to large-scale transcriptional profiling to identify key markers of immune responses, which can be downscaled to a rapid, inexpensive, and highly versatile assay of global immune function applicable to diverse investigations of immunopathogenesis and biomarker discovery.

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