Project description:To identify the activity-induced gene expression programs in inhibitory neurons, we analyzed RNA extracted from cultured E14 mouse MGE-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. Mouse E14 MGE-derived neurons were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and WT RNA-Seq was performed on ABi SOLiD
Project description:We used genome-wide sequencing methods to study stimulus-dependent enhancer function in neurons. We identified ~12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Remarkably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 that is mono-methylated at lysine 4 (H3K4me1). The level of eRNA expression at neuronal enhancers positively correlates with the level of mRNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis. Examination of genome-wide binding of three types of modified histones, four transcription factors, and RNA polymerase II (ChIP-Seq), as well as RNA expression (RNA-Seq) before and after membrane depolarization via application of extracellular potassium.
Project description:AB SOLID sequencing of ribosome-depleted RNA from S. Cerevisiae, S. Paradoxus, S. Mikatae, and S. Bayanus These four yeast species were grown in complete media and total RNA was sequenced. Cross-Species Gene Expression using RNA-Seq Data was examined. Eight samples examined: two biological replicates of each species
Project description:This SuperSeries is composed of the following subset Series: GSE39977: Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals [ChIP-seq] GSE39978: Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals [RNA-seq] Refer to individual Series
Project description:The mammalian circadian clock involves a transcriptional feedback loop in which CLOCK and BMAL1 activate the Period and Cryptochrome genes, which then feedback and repress their own transcription. We have interrogated the transcriptional architecture of the circadian transcriptional regulatory loop on a genome scale in mouse liver and find a stereotyped, time-dependent pattern of transcription factor binding, RNA polymerase II (RNAPII) recruitment, RNA expression and chromatin states. We find that the circadian transcriptional cycle of the clock consists of three distinct phases - a poised state, a coordinated de novo transcriptional activation state, and a repressed state. Interestingly only 22% of mRNA cycling genes are driven by de novo transcription, suggesting that both transcriptional and post-transcriptional mechanisms underlie the mammalian circadian clock. We also find that circadian modulation of RNAPII recruitment and chromatin remodeling occurs on a genome-wide scale far greater than that seen previously by gene expression profiling. Examination of 9 transcriptional regulators, 2 RNAPII and 6 histone modifications every 4hr during the circadian cycle in mouse liver
Project description:In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of initiating Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the Pol IIB form and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. This study was performed in a human Raji cell line. It contains ChIP-seq data for H3K36me3 (two replicates), H3K4me1 (two replicates), H3K4me3 (two replicates), Pol II (three replicates), Ser2P (two replicates), Ser5P (two replicates), Ser7P (two replicates), Tyr1P 3D12 (two replicates) and Tyr1P 8G5 (one replicate). MNase-experiment for nucleosomes was performed in paired-end sequencing on one replicate, 4 replicates for the input genomic DNA was used and one replicate was generated for the short strand specific RNA experiment.
Project description:To identify the activity-induced gene expression programs in inhibitory and excitatory neurons, we analyzed RNA extracted from cultured E14 mouse MGE- and CTX-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. To identify the gene programs regulated in these cells by the activity-induced early-response transcription factor Npas4, we repeated the same experiment in the MGE- and CTX-cultures lacking Npas4 (Npas4-KO). Littermate mouse E14 MGE- or CTX-derived neurons (WT or KO for Npas4) were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and analyzed using Affymetrix GeneChip Mouse Expression Set 430 2.0 microarray platform.
Project description:Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockM-NM-^T19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockM-NM-^T19 mutation to a M-bM-^HM-<900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKM-NM-^T19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001. Examination of 3 transcriptional regulators in mouse liver at ZT8
Project description:Acute leukemia are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells. Chromatin immunoprecipitations of Spi-1, H3K36me3, RNApolII,mouse IgG followed by sequencing were performed on spleen-derived erythroleukemic cells of spi-1 transgenic mice. In case of Spi-1, reads obtained from the two different mice represent biological replicates and were merged for bioinformatic analysis. Input DNA from each ChIP experiments have been pooled and sequenced as one control.
Project description:White adipose tissue (WAT) harbors functionally diverse subpopulations of adipose progenitor cells that differentially impact tissue plasticity in a sex- and depot-dependent manner. To date, the molecular basis of this cellular heterogeneity has not been fully defined. Here, we describe a multilayered omics approach to dissect adipose progenitor cell heterogeneity from in three dimensions: progenitor subpopulation, sex, and anatomical localization. We applied state-of-the-art mass spectrometry methods to quantify 4870 proteins in eight different stromal cell populations from perigonadal and inguinal WAT of male and female mice and acquired transcript expression levels of 15477 genes using RNA-seq. Notably, our data highlight the molecular signatures defining sex differences in PDGFR+ preadipocyte differentiation and identify regulatory pathways that functionally distinguish adipose tissue PDGFRb+ subpopulations. The data are freely accessible as a resource at "Pread Profiler. Together, the multilayered omics analysis provides unprecedented insights into adipose stromal cell heterogeneity.