Enhancer Release and Retargeting” Activates Disease Susceptibility Genes (ChIP-Seq)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE115602 | GEO | 2020/08/31
REPOSITORIES: GEO
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Project description:Purpose: We used Global run-on sequencing, a gold standard assay to measure transcriptional activity to examine the change of genome transcription in the absence of RAD21 subunit of cohesin, a critical chromatin architectural protein complex. In particularl, we tested how the loss of chromatin looping regulator cohesin would impact the enhancers and their target promoter transcription. Methods: Transfection of small interfering RNAs (siRNAs) into MCF-7 cells was performed using Lipofectamine 2000 (Life technologies) following manufacturer's instructions. For all experiments, two rounds of siRNA transfection (40nM each time) were performed to achieve higher efficiency. The siRNAs used in this study include: Qiagen Negative Control siRNA (Qiagen Cat# 1027310) and/or Sigma Mission siRNA universal control #2 (SIC002); siRAD21 (Sigma SASI_Hs02_00341219 and SASI_Hs01_00195799). The transfected MCF7 cells were subjected to GRO-seq experiments. GRO-Seq experiments were performed as previously reported (Li et al., 2013, Nature). Briefly, ~10-20 millions of MCF-7 cells were washed 3 times with cold PBS and then sequentially swelled in swelling buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5 min on ice, harvested, and lysed in lysis buffer (swelling buffer plus 0.5% NP-40 and 10% glycerol). The resultant nuclei were washed one more time with 10mL lysis buffer and finally re-suspended in 100uL of freezing buffer (50mM Tris-Cl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, re-suspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase-IN, 1% sarkosyl, 500uM ATP, GTP, and Br-UTP, 2uM CTP) and incubated for 5 min at 30°C . The resultant nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Life Technologies) following manufacturer’s instructions. NRO-RNA was fragmented to ~300-500nt by alkaline base hydrolysis on ice and followed by treatment with DNase I and Antarctic phosphatase. These fragmented Br-UTP labeled nascent RNA was then immunoprecipitated with an anti-BrdU argarose beads (Sc32323ac, Santa Cruz Biotechnology) in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween) for 3 hrs at 4°C with rotation. Subsequently, T4 PNK was used to repair the end of the immunoprecipitated BrU-NRO-RNA, at 37°C for 1hr. The RNA was extracted and precipitated using acidic phenol-chloroform. cDNA synthesis was performed as per a published method (54) with few modifications. The RNA fragments were subjected to poly-A tailing reaction by poly-A polymerase (NEB) for 30 min at 37°C. Subsequently, reverse transcription was performed using oNTI223 primer and superscript III RT kit (Life Technologies). The cDNA products were separated on a 10% polyacrylamide TBE-urea gel and only those migrated between ~100-500bp were excised and recovered by gel extraction. After that, the first-strand cDNA was circularized by CircLigase (Epicentre) and re-linearized by APE1 (NEB). Re-linearized single strand cDNA (sscDNA) was separated by a 10% polyacrylamide TBE gel as described above and the product of needed size was excised (~170-400bp) for gel extraction. Finally, sscDNA template was amplified by PCR (usually between 10-14 PCR cycles) using the Phusion High-Fidelity enzyme (NEB) according to the manufacturer’s instructions. The resulted library was subjected to deep sequencing. Results: Using an optimized data analysis workflow, we generated three biological replicates of GRO-seq in the presence or absence of cohesin, and with or without treatment by estrogen.
2020-08-31 | GSE115603 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2020-08-31 | GSE115604 | GEO
Project description:RAD21 is a subunit of cohesin. Cohesin is maninly known for its role in holding sister chromatids together during cell-division but it also plays a role in gene regulation. Genome wide study in MCF7 cells have shown that cohesin co-binds with estrogen receptor alpha at numerous sites. To identify estrogen sensitive genes that are influenced by cohesin, a microarray expression analysis was conducted in MCF7 cells lacking the cohesin subunit RAD21. Global changes in gene expression were identified in cells treated with Control/RAD21 siRNA and stimulated with estrogen/vehicle thereafter.
2016-01-30 | GSE59908 | GEO
Project description:Enhancer Release and Retargeting” Activates Disease Susceptibility Genes
| PRJNA475556 | ENA
Project description:Enhancer Release and Retargeting” Activates Disease Susceptibility Genes (ChIP-Seq)
| PRJNA475557 | ENA
Project description:Enhancer Release and Retargeting” Activates Disease Susceptibility Genes (GRO-Seq)
| PRJNA475558 | ENA
Project description:The Cohesin complex has recently been described to regulate gene expression. We wanted to determine the gene expression profile specific in mouse ES cells after depletion of the Cohesin subunit Rad21. We used microarrays to detail the global programme of gene expression underlying depletion of Rad21 and identified distinct early development related genes up-regulated and many pluripotency related genes downregulated. Rad21 was depleted in R1/E ES cells for 48h using esiRNAs against Rad21. An esiRNA against non-targeting Luciferase was used as a negative control
2011-05-19 | E-GEOD-23923 | biostudies-arrayexpress
Project description:H3K27Ac ChIP-seq in wild type and cohesin-deficient thymocytes Rad21 was deleted in CD4+ CD8+ double positive (DP) thymocytes by crossing a Rad21 floxed allele with a Cd4-driven Cre transgene. DP positive thymocytes were FACS-sorted from control and Rad21-/- littermates, which were then used to perform chromatin immunoprecipitation for histone H3 acetylated on lysine 27 (H3K27Ac).
2015-02-12 | E-GEOD-61428 | biostudies-arrayexpress
Project description:MTD project_description Inflammation and decreased stem cell function characterize organism aging, yet the relationship between these factors remains incompletely understood. This study shows that aged hematopoietic stem and progenitor cells exhibit increased ground-stage NF-κB activity, which enhances their responsiveness to undergo differentiation and loss of self-renewal in response to inflammation. The study identifies Rad21/cohesin as a critical mediator of NF-κB signals, by increasing chromatin accessibility of inter-/intra-genic and enhancer regions. Rad21/NF-κB are required for normal differentiation, but limit self-renewal of hematopoietic stem cells (HSCs) during aging and inflammation in an NF-κB dependent manner. HSCs from aged mice fail to downregulate Rad21/cohesin and inflammation/differentiation inducing signals in the resolution phase after acute inflammation. and The inhibition of cohesin/NF-κB is sufficient to revert the hypersensitivity of aged HSPCs to inflammation-induced differentiation. During aging, myeloid-biased HSCs with disrupted and naturally occurring reduced expression of Rad21/cohesin are increasingly selected over lymphoid-biased HSCs. Together, Rad21/cohesin mediated NF-κB signaling limits HSPC function during aging and selects for cohesin deficient HSCs with myeloid skewed differentiation.
2018-11-28 | PXD011537 | Pride
Project description:Rad21 is a subunit of cohesin. The main function of cohesin is to hold replicated chromosomes together until cells divide, but it also plays a role in gene expression. To find out which genes might be regulated by cohesin, a study was conducted to look for global changes in gene expression in zebrafish embryos lacking cohesin component Rad21. The zebrafish Rad21 mutant used for expression analysis was rad21nz171, an allele isolated in a forward genetic screen for regulators of runx1. Experiment Overall Design: RNA from rad21nz171 mutant and wild type zebrafish embryos collected at 24 hours post-fertilization (h.p.f.) and 48 h.p.f. was hybridized to Affymetrix microarrays (Gene Chip zebrafish genome arrays cat. no. 900488). Four pools of 50 embryos for each genotype and time point were used as the RNA source, and RNA from each pool was hybridized independently such that the experiment had four biological replicates.
2009-11-10 | E-GEOD-18795 | biostudies-arrayexpress