Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing
- Stowers Institute for Medical Research, United States
- University of Kansas Medical Center, United States
Figures
Figure 1 with 2 supplements
Reporter-ChIP-nexus captures paused Pol II.
(A) Reporter-ChIP-nexus is performed by cloning Drosophila pseudoobscura promoters or synthetic promoters into a simple GFP reporter and transfecting into D. melanogaster Kc167 cells. The whole cell …
Figure 1—figure supplement 1
A simple GFP reporter that allows the fast insertion of any promoter sequence.
To perform reporter-ChIP-nexus, we constructed a simple GFP reporter plasmid with upstream regulatory sequences. An EcoRV cutting site was used to linearize the plasmid and insert a promoter of …
Figure 1—figure supplement 2
Workflow for gene-specific 5’ RNA sequencing, a method similar to RNA amplification of cDNA ends (RACE).
RNA transcribed form the reporter was reverse transcribed using a primer that has 3’ sequences complementary to the GFP sequence. The 5’ of this primer contains a BamHI restriction cutting site and …
Figure 2 with 4 supplements
Reporter-ChIP-nexus recapitulates the endogenous Pol II pausing profile.
(A) To obtain the endogenous Pol II pattern of D. pseudoobscura promoters in the genome, ChIP-nexus was performed in a D. pseudoobscura cell line. Results are shown for the pepck, comm2 and pk …
Figure 2—figure supplement 1
Paused Pol II stability measurements at eight D. pseudoobscura promoters.
Pol II ChIP-nexus was performed on the D. pseudoobscura cell line ML83-63. The stability of paused Pol II was analyzed by comparing the Pol II profile under control and triptolide (TRI) treated …
Figure 2—figure supplement 2
Reporter-ChIP-nexus recapitulates endogenous Pol II pausing.
The ChIP-nexus profile of endogenous D. pseudoobscura promoters (first row) strongly resembles the profile detected by reporter-ChIP-nexus after cloning the D. pseudoobscura promoters into the …
Figure 2—figure supplement 3
Larger promoter region insertion is required for recapitulating Pol II pausing at RpL13A on the plasmid.
Pol II ChIP-nexus and H3K4me3 ChIP-seq profiles at D. pseudoobscura RpL13A and Act5C endogenous loci and that on the plasmids with 300 bp or 2 kb promoter region insertion. Both RpL13A and Act5C …
Figure 2—figure supplement 4
Reporter-ChIP-nexus recapitulates gene-specific Pol II pausing stability.
The stability of paused Pol II was examined on the plasmids and at the endogenous loci by treatment with triptolide (TRI). After TRI treatment, paused Pol II is lost, and the degree of persistence …
Figure 3 with 1 supplement
Changes in downstream promoter sequences alter paused Pol II stability.
(A) The pepck and Act5C downstream sequences were replaced with that from the stably paused promoter pk or dve (fusion site: 8 bp after the TSS). (B) Pol II ChIP-nexus data on the plasmids after …
Figure 3—figure supplement 1
Downstream promoter sequences influences Pol II pausing at Act5C.
Pol II ChIP-nexus profile at Act5C and Act5C-pk-down fusion promoters under control condition and after 5 min Triptolide (TRI) treatment.
Figure 4 with 1 supplement
A TATA insertion may reduce Pol II pausing.
(A) Analysis of paused Pol II half-lives as a function of various core promoter element combinations. Median paused Pol II half-life of promoters with different combinations of core promoter …
Figure 4—figure supplement 1
Correlation between paused Pol II half-life and core promoter elements using a mutually exclusive model.
To better understand the relationship between core promoter elements and the half-live of paused Pol II genome-wide, we calculated the median half-life for each element and its possible combination …
Figure 5 with 1 supplement
The initiator sequence plays an important role in Pol II pausing.
(A) Experimental series on the dve promoter testing the role of the Inr in conjunction with a TATA-containing region. Either only the upstream region was replaced with that of the Act5C promoter …
Figure 5—figure supplement 1
Effect of TATA insertion at comm2 and dve.
Pol II ChIP-nexus profile at comm2, Act5C-up-comm2, TATA-comm2, dve, Act5C-up-dve and TATA-dve promoters under control condition and after Triptolide (TRI) treatment (40 min for comm2 and 1 hr for dv…
Figure 6
Initiator sequences differ between TATA and stably paused promoters.
(A) The sequence of stably paused promoters (132 randomly selected from the 490 promoters that did not have a TATA box and had a paused Pol II half-life longer than 60 min) are shown on the left as …
Figure 7 with 3 supplements
The G at Inr + 2 position is critical for stable Pol II pausing.
(A) Analysis of paused Pol II half-lives as a function of core promoter element combinations after separating the Inr sequences into those that contain a G at position +2 (Inr-G) versus those that …
Figure 7—figure supplement 1
Correlation between paused Pol II half-life and Inr variants using a non-mutually exclusive model.
(A) Median paused Pol II half-life (left) and promoter numbers (right) at promoters with different combinations of core promoter elements using a non-mutually exclusive model. (B) Paused Pol II …
Figure 7—figure supplement 2
Mutating the G at Inr + 2 position reduces Pol II pausing at SCP and pk.
Pol II ChIP-nexus profile at SCP, SCP-Inr-GtoT, pk and pk-Inr-GtoT fusion promoters under control condition and Triptolide (TRI) treatment (30 min for SCP, 1 hr for pk).
Figure 7—figure supplement 3
Transcript level changes after altering the promoter sequence.
To study the effect of altered promoter sequences on RNA expression, we performed RT-qPCR for all experiments. The relative Pol II pausing stability (green) and relative RNA expression levels …
Figure 8
Model of how promoter sequences may influence Pol II pausing through TFIID Since the core promoter elements that we analyzed are bound by TFIID, we hypothesize that TFIID affects Pol II pausing dependent on the position it binds to the promoter.
(A)At stably paused promoters, pausing elements and consensus Inr sequences with a G at +2 position promote TFIID-downstream DNA interactions, which prolong Pol II pausing. (B)At TATA-containing …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit polyclonal anti-Rpb3 | Julia Zeitlinger Lab | Zeitlinger Lab #163185–50 | 10 ug |
| Antibody | Rabbit polyclonal anti-H3K4me3 | Cell Signaling | #9727 | 10 ug |
| Strain, strain background (E. coli) | NEB 5-alpha Competent E. coli (High Efficiency) | New England Biolabs | #C2987H | |
| Strain, strain background (E. coli) | One Shot ccdB Survival 2 T1R Competent Cells | ThermoFisher Scientific | #A10460 | |
| Chemical | Chloroform | Sigma-Aldrich | #C2432 | |
| Commercial assay or kit | CircLigase ssDNA Ligase | Illumina (Epicentre) | #CL4115K | |
| Chemical compound | DMSO | Sigma-Aldrich | #276855 | |
| Chemical compound | Dynabeads Protein A | Life Technologies | #100-08D | |
| Chemical compound | Dynabeads Protein G | Life Technologies | #100-04D | |
| Commercial assay or kit | FastDigest BamHI | ThermoFisher Scientific | #FERFD0054 | |
| Chemical compound | FuGENE HD reagent | Promega | #E2311 | |
| Chemical compound | HyClone SFX-Insect Cell Culture Media | ThermoFisher Scientific | #SH3027802PM | |
| Commercial assay or kit | Klenow Fragment (3’ to 5’ exo-) | New England Biolabs | #M0212S | |
| Commercial assay or kit | Lambda Exonuclease | New England Biolabs | #M0262L | |
| Chemical compound | Opti-MEM Reduced Serum Medium | ThermoFisher Scientific | #31985062 | |
| Chemical compound | Phenol: Chloroform: iso-Amyl alcohol | VWR | #97064–824 | |
| Chemical compound | Protease Inhibitor Cocktail tablets EDTA-free | Roche Diagnostics Corporation | #5056489001 | |
| Commercial assay or kit | Proteinase K | Life Technologies | #25530–049 | |
| Commercial assay or kit | recJ | New England Biolabs | #M0264L | |
| Commercial assay or kit | Restriction emzyme AfeI | New England Biolabs | #R0652S | |
| Commercial assay or kit | Restriction emzyme EcoRV-HF | New England Biolabs | #R3195S | |
| Commercial assay or kit | Restriction emzyme SacI-HF | New England Biolabs | #R3156S | |
| Commercial assay or kit | RNase A | ThermoFisher Scientific | #EN0531 | |
| Commercial assay or kit | RNase H | New England Biolabs | #M0297S | |
| Commercial assay or kit | SuperScript II Reverse Transcriptase | ThermoFisher Scientific | #18064014 | |
| Commercial assay or kit | T4 DNA Polymerase | New England Biolabs | #M0203S | |
| Chemical compound | Triptolide | TOCRIS Bioscience | #3253 | |
| Chemical compound | TRIzol Reagent | ThermoFisher Scientific | #15596026 | |
| Commercial assay or kit | Direct-zol RNA MiniPrep kit | Genesee | #11–330 | |
| Commercial assay or kit | Fast SYBR Green Master mix | Life Technologies | #4385612 | |
| Commercial assay or kit | Gibson Assembly Master Mix | New England Biolabs | #E2611S | |
| Commercial assay or kit | High-Capacity RNA-to-cDNA Kit | ThermoFisher Scientific | #4387406 | |
| Commercial assay or kit | IBI High Speed Plasmid Mini Kit | MIDSCI | #IB47101 | |
| Commercial assay or kit | NEBNext dA-tailing module | New England Biolabs | #E6053L | |
| Commercial assay or kit | NEBNext end-repair module | New England Biolabs | #E6050L | |
| Commercial assay or kit | NEBNext Multiplex Oligos for Illumina | New England Biolabs | #E7335S | |
| Commercial assay or kit | Q5High-Fidelity 2X Master Mix | New England Biolabs | #M0492L | |
| Commercial assay or kit | Q5 Site-Directed Mutagenesis Kit | New England Biolabs | #E0554S | |
| Commercial assay or kit | Quick Ligation Kit | New England Biolabs | #M2200L | |
| Cell line (D. melanogaster) | Kc167 cells | DGRC | #1 | |
| Cell line (D. pseudoobscura) | ML83-63 cells | DGRC | #33 | |
| Sequence-based reagent | ChIP-nexus oligos | (He et al., 2015) | See Table S5 | |
| Sequence-based reagent | Gene-specific 5’ RNA sequencing oligos | This paper | See Table S6 | |
| Recombinant DNA reagent | Reporters used in this study | This paper | See Table S7 and S8 | |
| Software, algorithm | Analysis code | GitHub | https://github.com/zeitlingerlab/Shao_eLife_2019 |
Additional files
-
Supplementary file 1
2019 eLife Shao Manuscript Supplementary Information.
- https://doi.org/10.7554/eLife.41461.022
-
Transparent reporting form
- https://doi.org/10.7554/eLife.41461.023
