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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 10, 2014 |
| Title |
BRD4 assists elongation of both coding and enhancer RNAs guided by histone acetylation |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Non-coding RNA profiling by high throughput sequencing
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| Summary |
In serum-starved and re-fed mouse fibroblast, nascent RNA-seq analysis showed that the BET inhibitor JQ1 antagonized a process regulating PIC formation and a downstream process involved in progressive elongation. To specifically address the role of BRD4 and its interactions with acetylated histones and P-TEFb, YFP-tagged BRD4 proteins (wild type and mutant BRD4) were stably expressed in cells, endogenous BRD4 of which was knocked down by shRNA (shBRD4). Nascent RNA-seq analysis showed that BRD4 facilitated transcript elongation in a manner dependent on acetylated histone interaction but not P-TEFb. Furthermore, the role of BRD4 in enhancer activity was evaluated by mapping the intergenic distributions of Pol II, CDK9, H3K27 acetylation (Ac) and H4 Ac as well as BRD4 by ChIP-seq analysis. The results suggest that the role of intergenic BRD4 on nearby gene expression is mediated through enhanced synthesis of eRNA.
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| Overall design |
Mouse fibroblasts were synchronized by serum-starvation (48 hours) and then re-fed (30 min) so that the transcriptional initiation of serum-response genes could be easily and uniformly captured by nascent RNA sequencing. To understand the basis of the reduced transcription initiation of protein-coding genes by JQ1, the role of BRD4 in enhancer activity was evaluated by mapping the genomic distributions of Pol II, CDK9, H3K27 acetylation (Ac) and H4 Ac as well as BRD4 by ChIP-seq analysis. To specifically address the role of BRD4 and its interaction with acetylated histone via bromodomain (BD) or with P-TEFb, YFP-tagged BRD4 proteins (wild type and mutant BRD4) were stably expressed in cells, endogenous BRD4 of which was knocked down by shRNA (shBRD4). The “mBD” mutants carry a point mutation in each BD so that they do not bind acetylated histones. A short form of BRD4 termed BRD4short lacks the P-TEFb interacting C-terminal region. Nascent RNA-seq analysis was performed to monitor the progression of transcripts.
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| Contributor(s) |
Kanno T, Kanno Y, LeRoy GT, Campos EI, Brooks SR, Sun H, Vahedi G, Heightman TD, Garcia BA, Ozato K, Reinberg DF, O'Shea JJ, Siebenlist U |
| Citation(s) |
25383670 |
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| Submission date |
Jun 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuka Kanno |
| E-mail(s) |
kannoy@mail.nih.gov
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| Phone |
301-402-3008
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| Organization name |
NIH
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| Department |
NIAMS
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| Lab |
LCBS-MIIB
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| Street address |
10 Center Drive, Rm13C120
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-1930 |
| Country |
USA |
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| Platforms (1) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
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| Samples (41)
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| Relations |
| BioProject |
PRJNA253302 |
| SRA |
SRP043498 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE58731_RAW.tar |
300.1 Mb |
(http)(custom) |
TAR (of BEDGRAPH, BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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