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| Status |
Public on Sep 10, 2024 |
| Title |
Single-molecule chromatin configurations link transcription factor binding to expression in human cells [RNA-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF’s capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.
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| Overall design |
K562 cells were cultured in 12-well plates and treated with 10 ng/mL interferon beta (PeproTech, #300-02BC) for 24 hours, after which 1 x 106 cells were harvested by centrifugation at 300 x g for 5 minutes. Cells were lysed and homogenized using QIAshredder columns (Qiagen #79654) and RNA was extracted with the RNeasy Mini Kit (Qiagen #74104). The RNA Integrity Number (RIN) for all samples was 10 as assessed by an RNA Nano Kit (Agilent #5067-1511) on an Agilent Bioanalyzer, provided by the Stanford Protein and Nucleic ACID (PAN) Biotechnology Facility. 500 ng of purified RNA for each sample was enriched for polyadenylated mRNA with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and then used as input for the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770S). Sample preparation was performed as per manufacturer’s specifications, with nine PCR cycles used for final library amplifications. Library size distributions were confirmed using the High Sensitivity DNA Kit (Agilent #5067-4626) on an Agilent Bioanalyzer. Sample concentrations were measured using the Qubit dsDNA HS Assay Kit on a Qubit 4 Fluorometer, and all samples were pooled at equimolar ratios and sequenced on a NextSeq 550 with 2 x 37 cycles. Sequencing reads were demultiplexed with bcl2fastq. Hisat2-build was used to build a reference transcriptome with a FASTA of the GRCh38 human reference genome and the accompanying GTF genome annotation file, and hisat2 was used to align the paired reads to the reference. Output SAM files were converted to BAM files using samtools and differential expression analysis was performed in R with the Bioconductor DESeq2 package72 using a set of custom R scripts (IFN_RNA_processing.R) that were largely based on the workflow and commands described in the following tutorial: http://bioconductor.org/help/course-materials/2016/CSAMA/lab-3-rnaseq/rnaseq_gene_CSAMA2016.pdf.
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| Contributor(s) |
Doughty BR |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| BioProject |
PRJNA1071686 |
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| Submission date |
Sep 06, 2024 |
| Last update date |
Sep 10, 2024 |
| Contact name |
Benjamin Rosenstock Doughty |
| E-mail(s) |
benjamin.doughty@gmail.com, bgrd@stanford.edu
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| Organization name |
Stanford University
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| Department |
Genetics
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| Street address |
279 Campus Drive W
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platforms (1) |
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| Samples (4)
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE276515_DEseq_results.csv.gz |
996.3 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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