Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling in the endometrium of pregnant mammals

ABSTRACT: A major challenge in biology is to determine how evolutionarily novel characters originate, however, mechanistic explanations for the origin of novelties are almost completely unknown. The evolution of mammalian pregnancy is an excellent system in which to study the origin of novelties because extant mammals preserve major stages in the transition from egg-laying to live-birth. To determine the molecular bases of this transition, we characterized the pregnant/gravid uterine transcriptome from tetrapods, including species in the three major mammalian lineages, and used ancestral transcriptome reconstruction to trace the evolutionary history of uterine gene expression. We show that thousands of genes evolved or lost uterine expression during the origins of mammalian pregnancy, including the loss of genes responsible for the mineralization of the eggshell in the Mammalian and Therian stem-lineages, and the recruitment of genes into uterine expression that mediate maternal-fetal communication and maternal immunotolerance of the fetal allograft in the Therian and Eutherian stem-lineages. Our results indicate that one of the defining mammalian novelties evolved through a step-wise process that dramatically changed uterine gene expression, generating a suite of evolutionary innovations that support prolonged gestations. We report genome-wide gene expression generated by mRNA-Seq from the pregnant endometrium of dog, armadillo, and the platypus.

ORGANISM(S): Ornithorhynchus anatinus

SUBMITTER: Vincent Lynch

PROVIDER: E-GEOD-57714 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study were to compare signals from competent and abnormal human embryos impacted differently on the expression of endometrial receptivity genes in mouse uteri using transcriptome profiling (RNA-seq). Methods: Immature female (25 d C57BL/6) mice were given a hormone treatment of a single dose 1 mg progesterone and 10 mg/kg/day ÃÂ²-estradiol for a total of 3d to prime the uterus for embryo transfer. The uterine horns of control and study mice were injected with an equal volume (50 ÃÂ¼l) of either unconditioned embryo culture medium (ECM), serving as controls, or pooled conditioned media from competent (n = 9) or arresting embryos (n = 18). Uterine mRNA profiles of 25-day-old wild-type (WT) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000 platform. The sequence reads that passed quality filters were analyzed with the following methods: Bowtie Alignment followed by TopHat (splice juntions mapper) and Cufflinks (transcript abundance). qRTÃ¢ÂÂPCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the uteri WT and NrlÃ¢ÂÂ/Ã¢ÂÂ mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRTÃ¢ÂÂPCR. RNA-seq data had a linear relationship with qRTÃ¢ÂÂPCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and NrlÃ¢ÂÂ/Ã¢ÂÂ retina, with a fold change Ã¢ÂÂ¥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRTÃ¢ÂÂPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of maternal uterine reponse to signals from competent and abnormal human embryos, with biological replicates, generated by RNA-seq technology. Supernatant from competent and abnormal human embryos were flushed into the uterine horns of 25d old C57BL/6 female mice. Uterine mRNA profiles of 21-day old mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000 platform (HiSeq).

Project description:BS69 (aka ZMYND11) was initially discovered as a direct target of adenoviral E1A oncoprotein1. Subsequent studies implicated BS69 as a tumor suppressor and a transcriptional regulator2. But exactly how BS69 regulates gene expression has remained elusive. BS69 contains tandemly arranged PHD, BROMO and PWWP domains, which are known to function as chromatin recognition modalities. Here we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via these chromatin recognition modules. Using a proteomics approach, we further identified association of BS69 with a host of RNA splicing regulators including EFTUD2 and other U5 snRNP components of the spliceosome. Remarkably, RNA-seq analysis shows that BS69 mainly regulates intron retention (IR), which is one of the least well-understood RNA alternative splicing events in mammalian cells. Specifically, loss of BS69 results in a decrease in IR, while in contrast, knockdown of EFTUD2 leads to an increase in IR, consistent with its role as a core member of the splicesome machinery. Importantly, wildtype BS69, but not a BS69 mutant defective in its interaction with EFTUD2, rescues the IR events. We also show that knockdown of SETD2, the main enzyme that mediates H3K36 trimethylation3, similarly results in a decreased retention of the same introns regulated by BS69. Significantly, a BS69 mutant unable to bind H3.3K36me3 in vitro fails to rescue the reduced IR phenotype associated with the loss of BS69. Taken together, our findings identify an antagonistic relationship between BS69 and the core splicing machinery and demonstrate that BS69-mediated RNA splicing regulation is dependent both on its ability to bind chromatin decorated by H3K36me3 and its ability to physically interact with the core spliceosome machinery. Our study reveals a novel and unexpected role of BS69 in connecting histone H3.3K36 trimethylation to regulated RNA splicing, providing significant new insights into chromatin regulation of RNA splicing. HeLa cells were infected with lentiviruses carrying control and BS69 shRNA and deep sequencing data were generated, in triplicate, using Illumina HiSeq 2000

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Gene expression profiling in the endometrium of pregnant mammals

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