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.
Project description:We used genome wide ChIP-seq to examine the chromatin histone changes after the knockdown of one of cohesin subunit, RAD21, in MCF7 breast cancer cells.
2020-08-31 | GSE115602 | GEO
Project description:Promoter Defects-mediated Enhancer Release and Retargeting Activates Disease Genes
Project description:Gene expression in metazoans is regulated by RNA Polymerase II (Pol II) promoter-proximal pausing and its release. Previously, we identified that Pol II-associated factor 1 (PAF1) modulates the release of paused Pol II into productive elongation. Here, we find that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of these enhancers. Enhancer activation as the result of Paf1 loss releases Pol II from paused promoters of nearby PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner.
Project description:2 kinds of SU-DHL-10 cell clones (Pax5-TSS2mut/deletion) were generated to investigate enhancer relocation, we campared 4C-seq data between the Pax5-TSS2mut/deletion clones and wild type SU-DHL-10.
Project description:Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase, either SMARCA4 or SMARCA2. Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser Syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.