TET-family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem (ES) cells. ES cells depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1, and display hyperactive Nodal signalling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions that favor derivation of trophoblast stem (TS) cells, Tet1-depleted ES cells activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in mid-gestation embryo chimeras. Consistent with these findings, Tet1-depleted ES cells form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm and ectopic appearance of trophoblastic giant cells. Thus Tet1 functions to regulate the lineage differentiation potential of ES cells. Here, we performed whole-genome transcriptome profiling of ES cells stably depleted of Tet1 by shRNA knockdown (Tet1-kd) cultured in either standard ES cell or in TS cell culture conditions. Gene expression changes in Tet1-kd ES cells were fairly modest compared to control (GFP-kd) cells, although gene ontology (GO) analysis of differentially expressed genes yielded many terms related to embryonic development and cell cycle regulation. In TS cell culture conditions, a core set of genes defining trophectodermal cell differentiation, including Cdx2, Eomes and Tead4, was enriched in Tet1-kd compared to GFP-kd cells.
Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.
Specimen partView Samples
The difference in X chromosome copy number creates a potential difference in X chromosomal gene expression between males and females. In many animals, dosage compensation mechanisms equalize X chromosome expression between sexes. Yet, X chromosome is also enriched for sex-biased genes due to differences in the evolutionary history of the X and autosomes. The manner in which dosage compensation and sex-biased gene expression exist on the X chromosome remains an open question. Most studies compare gene expression between two sexes, which combines expression differences due to X chromosome number (dose) and sex. Here, we uncoupled the effects of sex and X dose in C. elegans and determined how each process affects expression of the X chromosome compared to autosomes. We found that in the soma, sex-biased expression on the X chromosome is almost entirely due to sex because the dosage compensation complex (DCC) effectively compensates for the X dose difference between sexes. In the germline where the DCC is not present, X chromosome copy number contributes to hermaphrodite-biased gene expression. These results suggest that X dose contributes to sex-biased gene expression based on the level of dosage compensation in different tissues and developmental stages. Overall design: RNA-Seq profiles of C. elegans XO hermaphrodite and XX male L3 larvae and adults
Untangling the Contributions of Sex-Specific Gene Regulation and X-Chromosome Dosage to Sex-Biased Gene Expression in Caenorhabditis elegans.
Specimen part, Cell line, SubjectView Samples
Mrhl is a non coding RNA identified from mouse chromosome 8. It is a 2.4kb poly adenylated, nuclear restricted RNA expressed in multiple tissues. The 2.4 kb RNA also undergoes a nuclear processing event mediated through Drosha that generates an 80nt intermediate RNA. This study was aimed at understanding the functiion of mrhl by silencing the mrhl RNA in the mouse spermatogonial cells using a pool of siRNAs targeted against the mrhl and analyse the global gene expression change using Affymetrix mouse expression array. The mRNAs that showed significant change in expression in mrhl siRNA treated cells against control were studied further for their biological significance with respect to mrhl silencing.
mrhl RNA, a long noncoding RNA, negatively regulates Wnt signaling through its protein partner Ddx5/p68 in mouse spermatogonial cells.
Specimen part, Cell lineView Samples
We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen.
Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes.
No sample metadata fieldsView Samples
ASCL1 is known to act as transcriptional activator of notch signaling pathway. We have found that ASCL1 is over expressed in secondary glioblastoma.
System analysis identifies distinct and common functional networks governed by transcription factor ASCL1, in glioma and small cell lung cancer.
Cell lineView Samples
Women are born with millions of primordial follicles which gradually decrease with increasing age and this irreversible supply of follicles completely exhausts at menopause. The fertility capacity of women diminishes in parallel with aging. The mechanisms for reproductive aging are not fully understood. In our recent work we observed a decline in BRCA1 mediated DNA repair in aging rat primordial follicles. To further understand the age-related molecular changes, we performed microarray gene expression analysis using total RNA extracted from immature (1820 days) and aged (400450 days) rat primordial follicles. The results of current microarray study revealed that there were 1011 (>1.5 fold, p<0.05) genes differentially expressed between two groups in which 422 genes were up-regulated and 589 genes were down-regulated in aged rat primordial follicles compared to immature. The gene ontology and pathway analysis of differentially expressed genes revealed a critical biological function such as cell cycle, oocyte meiosis, chromosomal stability, transcriptional activity, DNA replication and DNA repair were affected by age and this considerable difference in gene expression profiles may have adverse influence on oocyte quality. Our data provide information on the processes that may contribute to aging and age-related decline in fertility.
Age-related changes in gene expression patterns of immature and aged rat primordial follicles.
Specimen partView Samples
Spermatogonial stem cells (SSCs) provide foundation for spermatogenesis by undergoing continuous self-renewal division. Previous studies have reported conflicting results on the role of the pituitary gland activity in SSC self-renewal. In this study, we analyzed the role of hormonal regulation of SSCs using Lhcgr (luteinizing hormone/choriogonadotropin receptor) knockout mice. Analysis of gene expression profiles showed that testes of Lhcgr-deficient mice exhibit significantly enhanced Wnt5a expression in Sertoli cells.
The Luteinizing Hormone-Testosterone Pathway Regulates Mouse Spermatogonial Stem Cell Self-Renewal by Suppressing WNT5A Expression in Sertoli Cells.
Sex, Specimen partView Samples
Traditional rice varieties found in India have many desirable characteristics. Amongst them, their differential responses to abiotic and biotic stresses are of great agricultural importance. Drought or osmotic stress is one of the major abiotic stresses afflicting crop plants in India. Indigenous varieties like Dagad deshi have been found to be drought resistant and, thereby, are being studied in great detail by plant breeders and biotechnologists alike. In this study, we have analyzed the transciptomes of two contrasting cultivars, i.e. Dagad deshi (tolerant) and IR20 (susceptible), under control and stress conditions to elucidate the differences in their responses to drought stress using Affymetrix microarray platform.
Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance.
Specimen part, TimeView Samples