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| Status |
Public on Jun 20, 2024 |
| Title |
Basis of gene-specific transcription regulation by the Inegrator Complex (RNA-Seq) |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Integrator (INT) is a multi-subunit modular RNA processing complex, which exhibits both RNA endonuclease and protein phosphatase activity. It is responsible for transcription termination at the 3’ ends of a diverse array of RNA polymerase II (RNAP2) transcribed non-coding RNAs, as well as transcription regulation at a large number of protein coding genes. There, it terminates RNAP2 at promoter proximal pausing sites, cleaves the nascent transcript and competes with elongation factors.This INT-mediated tapering of gene expression attenuates stimulus responsive genes and is essential for cell differentiation. Although INT affects transcription at varying classes of RNAs in diverse biological contexts, how it achieves specificity in a gene- and context-dependent manner has remained elusive. Using a combination of proteomics, interaction studies and structural characterization, we identified a diverse set of transcription factors (TFs) that associate directly with defined surfaces on INT. Stress conditions lead to changes in the types of TFs bound by INT, and quantitative binding studies suggest that TF affinities can be modulated by altering the phosphorylation states of specific residues in their INT-binding motifs. Integrated multi-omics data show that INT and its TF interactors regulate significantly overlapping sets of genes and indicate that these TFs recruit INT to specific genomic loci. Consistently, we find that starvation induced formation of primary cilia, which is a cellular stress response reliant on INT-mediated transcription regulation, depends on intact TF-INT binding. Taken together, our data suggest that TFs lend INT specificity to elicit targeted gene regulation as a transcriptional response in defined biological contexts.
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| Overall design |
To assess the functional relationship between INT and its TF factor interactors, we depleted one subunit of INT (INTS13) as well as each TF individually with siPOOLs in HEK293T cells and subjected these samples to total RNA-sequencing. In order to obtain efficient knockdown of each protein, we subjected the cells to double transfections with the first one occurring 24 hours after seeding and the second one occuring 24 hours later. The cells were then left to proliferate for 48 hours, after which RNA was extracted, Dnase treated. In order to determine the role of INTS13 and the TFs in glucose starvation response, we repeated the depletions of INTS13, HDGF and ZMYND8 under starved conditions, where growth medium was changes to glucose-free medium 24 hours after the second transfection. After DNase treatment, ribosomal RNAs were depleted and sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA kit. Libraries were sequenced on an Illumina NovaSeq 6000 using 100 bp single-end reads at a depth of 40M reads per library.
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| Contributor(s) |
Sabath K, Nabih A, Arnold C, Moussa R, Domjan D, Zaugg J, Jonas S |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Jun 01, 2023 |
| Last update date |
Jun 20, 2024 |
| Contact name |
Stefanie Jonas |
| E-mail(s) |
stefanie.jonas@mol.biol.ethz.ch
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| Organization name |
ETH Zurich
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| Department |
Biology
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| Street address |
Otto-Stern-Weg 5
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| City |
Zürich |
| State/province |
Zürich |
| ZIP/Postal code |
8093 |
| Country |
Switzerland |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (35)
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| GSM7439790 |
HEK293T cells, control siPOOL repllicate 1a |
| GSM7439791 |
HEK293T cells, control siPOOL repllicate 1b |
| GSM7439792 |
HEK293T cells, control siPOOL repllicate 2 |
| GSM7439793 |
HEK293T cells, TSPYL2 siPOOL, replicate 1 |
| GSM7439794 |
HEK293T cells, TSPYL2 siPOOL, replicate 2 |
| GSM7439795 |
HEK293T cells, ZEB1 siPOOL, replicate 1 |
| GSM7439796 |
HEK293T cells, ZEB1 siPOOL, replicate 2 |
| GSM7439797 |
HEK293T cells, ZNF608 siPOOL, replicate 1 |
| GSM7439798 |
HEK293T cells, ZNF608 siPOOL, replicate 2 |
| GSM7439799 |
HEK293T cells, ZNF609 siPOOL, replicate 1 |
| GSM7439800 |
HEK293T cells, ZNF609 siPOOL, replicate 2 |
| GSM7439801 |
HEK293T cells, ZNF687 siPOOL, replicate 1 |
| GSM7439802 |
HEK293T cells, ZNF687 siPOOL, replicate 2 |
| GSM7439803 |
HEK293T cells, HDGF siPOOL, replicate 3 |
| GSM7439804 |
HEK293T cells, HDGF siPOOL, replicate 4 |
| GSM7439805 |
HEK293T cells, HDGF siPOOL, 24h glucose starvation, replicate 3 |
| GSM7439806 |
HEK293T cells, HDGF siPOOL, 24h glucose starvation, replicate 4 |
| GSM7439807 |
HEK293T cells, INTS13 siPOOL, replicate 3 |
| GSM7439808 |
HEK293T cells, INTS13 siPOOL, replicate 4 |
| GSM7439809 |
HEK293T cells, INTS13 siPOOL, 24h glucose starvation, replicate 3 |
| GSM7439810 |
HEK293T cells, INTS13 siPOOL, 24h glucose starvation, replicate 4 |
| GSM7439811 |
HEK293T cells, control siPOOL repllicate 3a |
| GSM7439812 |
HEK293T cells, control siPOOL repllicate 3b |
| GSM7439813 |
HEK293T cells, control siPOOL repllicate 4a |
| GSM7439814 |
HEK293T cells, control siPOOL repllicate 4b |
| GSM7439815 |
HEK293T cells, control siPOOL, 24h glucose starvation, repllicate 3a |
| GSM7439816 |
HEK293T cells, control siPOOL, 24h glucose starvation, repllicate 3b |
| GSM7439817 |
HEK293T cells, control siPOOL, 24h glucose starvation, repllicate 4a |
| GSM7439818 |
HEK293T cells, control siPOOL, 24h glucose starvation, repllicate 4b |
| GSM7439819 |
HEK293T cells, control siPOOL (for ZMYND8 depletion) repllicate 3 |
| GSM7439820 |
HEK293T cells, control siPOOL (for ZMYND8 depletion) repllicate 4 |
| GSM7439821 |
HEK293T cells, ZMYND8 siPOOL repllicate 3 |
| GSM7439822 |
HEK293T cells, ZMYND8 siPOOL repllicate 4 |
| GSM7439825 |
HEK293T cells, ZNF655 siPOOL repllicate 3 |
| GSM7439826 |
HEK293T cells, ZNF655 siPOOL repllicate 4 |
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| This SubSeries is part of SuperSeries: |
| GSE233958 |
Basis of gene-specific transcription regulation by the Inegrator Complex |
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| Relations |
| BioProject |
PRJNA978643 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE233957_geneCounts_beforeDuplicatesRemoval_clean_raw.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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