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Chromatin immunoprecipitation sequencing of heterochromatin-associated proteins in Tetrahymena

ABSTRACT: In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the novel heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1 (HP1)-like protein Pdd1p. Through the identification and mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore suggest that heterochromatin bodies are assembled by the Pdd1p-RNA interaction. Jub1p and Pdd1p dephosphorylation are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body of itself plays an essential role in DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type different mutant cells at 12 hpm, shared chromatin was immunoprecipitated and precipitated DNA was analyzed by high-throughput sequencing.

ORGANISM(S): Tetrahymena thermophila

SUBMITTER: Kazufumi Mochizuki

PROVIDER: E-GEOD-70081 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Chromatin Immunoprecipitation sequencing of heterochromation associated proteins in Tetrahymena

Project description:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. We previously reported that dephosphorylation of the HP1-like protein Pdd1p is required for the formation of heterochromatin bodies during the process of programmed DNA elimination in the ciliated protozoan Tetrahymena. Here, we show that the heterochromatin body component Jub4p is required for Pdd1p phosphorylation, heterochromatin body formation and DNA elimination. Moreover, our analyses of unphosphorylatable Pdd1p mutants demonstrate that Pdd1p phosphorylation is required for heterochromatin body formation and DNA elimination, while it is dispensable for local heterochromatin assembly. Therefore, both phosphorylation and the following dephosphorylation of Pdd1p are necessary to facilitate the formation of heterochromatin bodies. We suggest that Jub4p-mediated phosphorylation of Pdd1p creates a chromatin environment that is a prerequisite for subsequent heterochromatin body assembly and DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type and various mutant cells at 12 hpm (hours post-mixing), sheared chromatin was immunoprecipitated andprecipitated DNA was analyzed by high-throughput sequencing

2016-07-15 | E-GEOD-83249 | biostudies-arrayexpress

Phosphorylation of an HP1-like protein regulates RNA-bridged heterochromatin body assembly for DNA elimination

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2015-12-10 | E-GEOD-70083 | biostudies-arrayexpress

Jub4p-mediated phosphorylation of Pdd1p is a prerequisite for heterochromatin body assembly and DNA elimination

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2016-07-15 | E-GEOD-83252 | biostudies-arrayexpress

DNA elimination analysis of Tetrahymena cells defective in heterochromatin body formation

Project description:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the novel heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1 (HP1)-like protein Pdd1p. Through the identification and mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore suggest that heterochromatin bodies are assembled by the Pdd1p-RNA interaction. Jub1p and Pdd1p dephosphorylation are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body of itself plays an essential role in DNA elimination. Micronuclei (MICs) and new macronuclei (MACs) of exconjugants were isolated from different mutants at 36 hpm, and the genomic DNA was analyzed by high-throughput sequencing.

2015-12-10 | E-GEOD-70082 | biostudies-arrayexpress

DNA elimination analysis of scnRNA production-defective Tetrahymena

Project description:Analysis of genome-wide IES elimination of Late-scnRNA accumulation-defective cells inducates that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. new MACs of exconjugants were isolated from different mutants at 36 hpm, and the genomic DNA was analyzed by high-throughput sequencing

2015-06-15 | E-GEOD-68457 | biostudies-arrayexpress

Two paralogous PHD finger proteins participate in Paramecium tetraurelia's natural genome editing

Project description:The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sexual reproduction, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique Internal Eliminated Sequences (IESs) located throughout the genome require precise excision to guarantee a functional new MAC genome. Here, we characterize a pair of paralogous PHD finger proteins involved in DNA elimination. DevPF1, the early-expressed paralog, is present in only some of the gametic and post-zygotic nuclei during meiosis. Both DevPF1 and DevPF2 localize in the new developing MACs, where IESs excision occurs. In DevPF2 knockdown (KD), long IESs are preferentially retained and late-expressed small RNAs decrease; no length preference for retained IESs was observed in DevPF1-KD and development-specific small RNAs were abolished. The expression of at least two genes from the new MAC seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functionalities, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.

2024-08-30 | PXD046704 | Pride

RNA polymerase in pre-B-ALL cell lines

Project description:[Gro-seq] Precursor B acute leukemia cells measured using global nuclear run-on sequencing [ChIP-Seq] The genome-wide occupancy of ser2 and ser5 phosphorylated RNA pol2 and H3K4me3 was measured in precursor B acute leukemia cells measured using chip-seq. [Gro-seq] Nascent RNA expression profiles were generated at cells in various basal culture conditions. [ChIP-Seq] Performed from REH and Nalm6 cells cultured under basal culture conditions. Mnase digestion was used for DNA fragmentation. Antibodies against Ser2 and Ser5 phosphorylated RNA polymerase and H3K4me3 compared to input. ****************************** This study includes reanalysis of Samples in Series GSE39878 (GSM980645, GSM980644), GSE60454 (GSM1480326), and GSE41009 (GSM1006728, GSM100672). The processed data files for the reanalyses are linked to GSE67540 as supplementary files (see the GSE67540_README.txt file for additional information).

2016-07-05 | E-GEOD-67540 | biostudies-arrayexpress

NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program

Project description:Cell fate specification relies on the action of critical transcription factors that become available at distinct stages of embryonic development. One such factor is NeuroD1, which is essential for eliciting the neuronal development program and possesses the ability to reprogram other cell types into neurons. Given this capacity, it is important to understand its targets and the mechanism underlying neuronal specification. Here, we show that NeuroD1 directly binds regulatory elements of neuronal genes that are developmentally silenced by epigenetic mechanisms. This targeting is sufficient to initiate events that confer transcriptional competence, including reprogramming of transcription factor landscape, conversion of heterochromatin to euchromatin and increased chromatin accessibility, indicating potential pioneer factor ability of NeuroD1. The transcriptional induction of neuronal fate genes is maintained via epigenetic memory despite a transient NeuroD1 induction during neurogenesis. Our study not only reveals the NeuroD1-dependent gene regulatory program driving neurogenesis but also increases our understanding of how cell-fate specification during development involves a concerted action of transcription factors and epigenetic mechanisms. 1. Ectopic NeuroD1 was induced for 48 hours (+Dox) in ES cells for checking initiation of neuronal transcriptional program in comparison to uninduced condition (-Dox) 2. ChIP-seq was performed after 24 hours of NeuroD1 induction in ES cells.

2015-11-02 | E-GEOD-65072 | biostudies-arrayexpress

Long-range chromosome interactions mediated by cohesin shape circadian gene expression [ChIP-Seq]

Project description:Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes. As genome-wide transcription is organized under the high-order chromosome structure, it is unclear how circadian gene expression is influenced by chromosome structure. In this study, we focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. We analyzed the interactome of a Bmal1-bound enhancer upstream of a clock gene, Nr1d1, by 4C-seq and observed that cohesin binding sites are enriched in the interactome. Integrating circadian transcriptome data and cistrome data, we found that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites facilitate the interaction between circadian enhancer and promoter. A coarse-grained model integrating the long-range effect of cohesin and CTCF markedly improved our mechanistic understanding of circadian gene expression. This model is subsequently supported by our RNA-seq data from cohesin knockout cells. Cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. Taken together, our study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure. Bmal1 ChIP-Seq in WT mouse embryonic fibroblast cells

2016-04-04 | E-GEOD-77162 | biostudies-arrayexpress

Genome-wide analysis of Estrogen Receptor-mediated response to acute Estradiol treatment in liver (ChIP-seq)

Project description:Estrogen-based treatments have numerous extra-reproductive effects. On the one hand, they have protective metabolic actions against abdominal fat accumulation, type 2 diabetes, liver steatosis,â¦ But, on the other hand, the oral route of administration, presumably due to a hepatic impact on liver-derived coagulation factors, appears to increase risks of venous thrombosis and pulmonary embolism. The characterization of liver genes expression regulations by these hormones thereby appears of utmost interest, but the estrogen-sensitive transcriptome of liver and ER cistrome was so far explored under chronic hormonal treatment. To explore the early steps of these mechanisms, we determined here the short-term changes in liver transcriptional responses to acute E2 administration, and aimed to characterize the mechanisms allowing these programs to be engaged. Collectively, through the comparison of mice expressing or not ER, our data demonstrate that acute administration of E2 provokes genes expression variations which fit with a crucial role of ER in the prevention of liver steatosis in mice fed with a high fat diet by E2. They also evidenced higher proportion of ER binding sites (BSs) located at the direct vicinity (distance <3kb) of regulated genes than what is determined in human cancer cell models. Besides this specific aspect of ER cistrome in vivo, we unexpectedly found that 40 % of the BSs of the pioneer factor Foxa2 were dependent upon the expression of ER that indirectly influences the distribution of the nucleosomes harbouring a dimethylated H3K4 around these Foxa2 sites. Whole-genome analysis of estrogen receptor (ER) and FOXA2 binding events associated with the cartography of two histone marks (H3K4me2 and H3K27ac) in livers from wild-type or ERKO mice.

2016-05-13 | E-GEOD-70346 | biostudies-arrayexpress

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