|
| Status |
Public on Sep 15, 2017 |
| Title |
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis and Decay in response to hypoxia in HUVEC cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output.
|
| |
|
| Overall design |
Exponentially growing non-synchronized HUVEC were exposed to normoxia or hypoxia (21% or 1% oxygen respectively) for 8 hours and pulse-labelled with 4‐thiouridine during the last two hours of treatment. RNA was extracted from samples in each condition (total RNA) and an aliquot was subjected to affinity chromatography to purify the 4‐thiouridine-labelled (newly transcribed RNA, Newly Tr) and non-labelled (Pre-existent) RNA fractions. All three RNA fractions (total, newly transcribed and pre-existent) from each sample were analyzed by high-throughput sequencing. Submission includes 12 samples corresponding to 3 independent biological replicates.
|
| |
|
| Contributor(s) |
Tiana M, del Peso L |
| Citation(s) |
29059365 |
| |
| Submission date |
Nov 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Luis del Peso |
| E-mail(s) |
luis.peso@uam.es
|
| Phone |
+34 91 585 4440
|
| Organization name |
Universidad Autonoma de Madrid
|
| Department |
Biochemistry
|
| Lab |
IIB-252
|
| Street address |
Arzobispo Morcillo
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platforms (1) |
| GPL10999 |
Illumina Genome Analyzer IIx (Homo sapiens) |
|
| Samples (18)
|
|
| This SubSeries is part of SuperSeries: |
| GSE89841 |
Transcriptional repression in hypoxia is mediated by the Sin3A histone deacetylase complex |
|
| Relations |
| BioProject |
PRJNA353498 |
| SRA |
SRP093315 |
Less...
More...