Micro-C of Drosophila embryos to investigate chromatin conformation during dorsal-ventral patterning
ABSTRACT: Micro-C was carried out for control embryos and embryos produced from gd7 mutant mothers. The embryos from mutant mothers produce only a single type along the dorsal-ventral axis (dorsal ectoderm). We used these embryos to compare chromatin conformation across tissues.
Project description:Hi-C was carried out for control embryos and embryos produced from gd7, Tollrm9/rm10, and Toll10B mutant mothers. The embryos from mutant mothers produce only a single type along the dorsal-ventral axis. We used these embryos to compare chromatin conformation across tissues.
Project description:Purpose: The goal of this study was to compare gene expression in whole embryos to identify transcriptomic changes that result from maternal exposure to predation risk. Methods: Whole embryo mRNA profiles of 3 day post-fertilizationstickleback embrosof mothers exposed to simulated predation risk and control embryos were generated by RNA-sequencing of pooled embryos using Illumina Hiseq2000. The sequence reads that passed quality filters were aligned to the stickleback reference genome and analyzed at the gene level (EdgeR) and at the transcript level (Cufflinks/Cuffdiff). Subsets of embryos were also measured for embryo length and eye diameter, and data were analyzed with a general linear model (SPSS). Results: We mapped ~22 million sequence reads per sample to the stickleback reference genome (BROADS1, Ensembl database version 71.1, Feb 2006) and identified 17440 transcripts with the Tophat workflow. Differential expression analysis using both EdgeR and Cufflinks/Cuffdiff identified 455 transcripts were differentially expressed in embryos of mothers exposed to simulated predation risk as compared to control embryos, with an FDR <0.05 (Cuffdiff) or <0.10 (EdgeR). Gene ontology and pathway analysis (DAVID, IPA) of the differentially expressed gene list revealed enrichment of genes involved in growth, metabolism, neurogenesis, and epigenetics. Embryos of mothers exposed to predation risk had elevated expression of growth and metabolism genes and were also larger than control embryos, suggesting at least some of the genes differentially expressed in this study are involved in the transfer of maternal experience to offspring. Conclusions: Our results suggest that early stickleback embryos respond to maternal exposure to predation risk via changes in gene expression, and a general acceleration of the developmental program. Further study is needed to elucidate the myriad molecular interactions between genes that are differentially-regulated as a result of maternal exposure to predation risk and to understand their relationships to previously-observed maternal effects in this system. Whole embryo mRNA profiles of 3dpf stickleback embryos of mothers exposed to simulated predation risk [E] and control mothers [C] were generated by barcoded, multiplexed high-throughput RNA-sequencing on Illumina Hiseq-2000.
Project description:We carried out ChIP-seq for H3K27ac and H3K27me3 in embryos from Tollrm9/rm10 mutant mothers. The embryos from mutant mothers produce only a single tissue type (neuroectoderm) along the dorsal-ventral axis. We used these embryos to compare chromatin state across tissues.
Project description:Precise nucleosome-positioning patterns at promoters are thought to be crucial for faithful transcriptional regulation. However, the mechanisms by which these patterns are established and dynamically maintained and subsequently contribute to transcriptional control are poorly understood. The Swi/Snf (Baf) chromatin remodeling complex is a master developmental regulator and tumor suppressor that is capable of mobilizing nucleosomes in biochemical assays. Yet, its role in establishing the nucleosome landscape in vivo is unclear. Here we have inactivated Snf5 and Brg1, core subunits of the mammalian Swi/Snf complex, to evaluate their effects on chromatin structure and transcription levels genome-wide. We find that inactivation of either subunit leads to disruptions of specific nucleosome patterning combined with a loss of overall nucleosome occupancy at a large number of promoters, regardless of their association with CpG islands. These rearrangements are accompanied by gene expression changes that promote cell proliferation. Collectively, these findings define a direct relationship between chromatin-remodeling complexes, chromatin structure, and transcriptional regulation. Nucleosome profiling in three different cell types under two digestion conditions
Project description:We find a high concordance between the binding of the Drosophila transcription factor Dorsal and the co-activator CBP during early embryogenesis. This relationship was furter examined by comparing CBP distribution in Drosophila embryos derived from wt and mutant flies lacking intranuclear Dorsal (gd7). Our data suggests a specific involvemet of CBP in initiating early dorsoventral patterning, but not in anterioposterior. CBP ChIP seq of 2-4 hours old Drosophila embryos derived from w1118 (wild-type) or gd7 homozygous mutant mothers
Project description:Left-right axis determination is a fundamental and conserved developmental process, with consequences for human pathology. At the molecular level, this process requires at least two centers of nodal-type TGFbeta signals: the first center, containing nodal/Xn; Microarrays were used to determine differences in gene expression in posterior mesoderm of Xenopus embryos. We compared controls and Derriere depleted embryos. Experiment Overall Design: Microarrays were used to determine differences in gene expression in posterior mesoderm of Xenopus embryos. We compared controls and embryos depleted of Derriere protein using MO oligonucleotides.
Project description:Immunocytochemical studies revealed that dG9a moves into nucleus after cycle 8 and appears to regulate gene expression by di-methylating H3K9 from cycle 8 to cycle 11. To determine which genes are regulated by dG9a during cycles 8-11, we examined mRNA levels by performing RNA-sequence analysis using early embryos (0-2 h after egg laying) of dG9a null mutant and wild type as a control mRNA profiles of about 0-2h-old embryos of wild type (CantonS) and dG9a-depleted (dG9aRG5) strain
Project description:The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing derivation of NTESCs from all oocyte donors tested using adult AMD patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine. Here we perform RNA-seq based transcriptome profiling in human Donor (fibroblast cells), in vitro fertilized embryos at 8-cell stages (IVF_8Cell), somatic cell nuclear transfer embryos at 8-cell stages (SCNT_8Cell), SCNT assisted by KDM4A 8-cell embryos (SCNT_KDM4A_8Cell). Besides, we also perform RNA-seq in Control human ES cells (CTR_hES) and SCNT assisted by KDM4A derived human ES cells (NTK) with duplicates.
Project description:For centuries, scientists have wondered whether the responses of animals and plants to environmental change can be transmitted to subsequent generations. Lately, there been much interest in the inheritance of information that is not encoded by differences in DNA sequence. Here we show that transient, mild heat stress results in a heritable change in the messenger RNA profile of early-stage C. elegans embryos. After heat stress is removed for one generation, abundances of most, but not all, mRNAs have relaxed to the unstressed state. Our approach was to culture worms at 20°C, then split the culture into 20°C control and 25°C treatment, followed by return of the 25°C culture to 20°C. Specifically, we compared mRNA abundances in 4-cell stage embryos collected from mothers kept at 20°C, with embryos whose mothers were also reared at 20°C but whose grandparents had been reared at 25°C. Two strains and two generations under a treatment and under a control condition were analyzed by microarray with three separately collected 50-embryo replicates for each combination, for a total of 24 samples.
Project description:Zebrafish (Danio rerio) model system have used widespread vertebrate investigations for genetic and cell biological analyses, and is suitable for small molecular screens such as chemical, toxicity and drug in order to use for human diseases and drug discovery . Recently, These powerful zebrafish model increasingly apply to human metabolic disease such as obesity and diabetes and toxicology. Despite a lot of advantages, proteomics research at zebrafish has received little interest in comparison with genetic and biological research using histology and in situ hybridization. Protein lysine acetylation is one of the most known post-translational modifications with dynamic and reversibly controlled by lysine acetyltransferase such as histone acetyltransferases and lysine deacetylase such as histone deacetylases and sirtuins family.Also, during the past year, global lysine acetylome studies using MS-based proteomics approach was in diverse species such as human, mouse, E. coli, Yeast and plants. Based on global acetylome data, our understanding of the roles of lysine acetylation in various cellular processes has increased. . The aim of this study was to identify Lysine acetylation in zebrafish embryos and determine the homology from Human at modified site level. Here we showed the global lysine acetylation study in Zebrafish embryos using MS-based zebrafish embryos.