NF-κB-repressing factor phosphorylation regulates transcription elongation via its interactions with 5'→3'
Sascha Rother1, Myriam Bartels1, Aike Torben Schweda1
1*Institute of Pharmacology, Hannover Medical School, Hannover, Germany; and Department of Plastic and Reconstructive Surgery, Hand Surgery and Burn Center, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University Hospital, Aachen, Germany.
Abstract:
NF-κB-repressing factor (NKRF) inhibits transcription elongation by binding to specific sequences in target promoters. Stimuli such as IL-1 have been shown to overcome this inhibitory action and enable the resumption of transcription elongation machinery by an unknown mechanism. Using mass spectrometry and in vitro phosphorylation analyses, we demonstrate that NKRF is phosphorylated within 3 different domains in unstimulated HeLa cells. Phosphoamino acid mapping and mutation analysis of NKRF further suggest that only Ser phosphorylation within aa 421-429 is regulated by IL-1 stimulation. In copurification studies, aa 421-429 is required for interactions between NKRF, 5'→3' exoribonuclease 2 (XRN2) and the negative elongation factor (NELF)-E in HeLa cells. Chromatin immunoprecipitation experiments further show that IL-1 stimulation leads to decrease in NKRF aa 421-429 phosphorylation and dissociation of NELF-E and XRN2 by concomitant resumption of transcription elongation of a synthetic reporter or the endogenous NKRF target gene, IL-8. Together, NKRF phosphorylation modulates promoter-proximal transcription elongation of NF-κB/NKRF-regulated genes via direct interactions with elongation complex in response to specific stimuli.
Insights
NF-κB-repressing factor (NKRF) phosphorylation regulates gene transcription. IL-1 stimulation reduces NKRF phosphorylation, releasing transcription elongation factors and resuming gene expression.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cell Signaling
Background:
- NF-κB-repressing factor (NKRF) inhibits transcription elongation at target gene promoters.
- The mechanism by which stimuli like IL-1 overcome NKRF-mediated repression is unknown.
Purpose of the Study:
- To elucidate the mechanism of IL-1-mediated relief of NKRF transcriptional repression.
- To identify the specific regulatory role of NKRF phosphorylation in gene expression.
Main Methods:
- Mass spectrometry and in vitro phosphorylation assays to analyze NKRF.
- Phosphoamino acid mapping and mutation analysis of NKRF.
- Copurification and chromatin immunoprecipitation experiments.
Main Results:
- NKRF is phosphorylated in multiple domains in HeLa cells.
- IL-1 stimulation specifically regulates Ser phosphorylation within amino acids 421-429 of NKRF.
- This region is critical for NKRF interaction with 5'→3' exoribonuclease 2 (XRN2) and negative elongation factor (NELF)-E.
- IL-1 stimulation decreases NKRF phosphorylation at aa 421-429, leading to NELF-E and XRN2 dissociation and transcription resumption of IL-8.
Conclusions:
- NKRF phosphorylation status modulates promoter-proximal transcription elongation.
- Regulation occurs through direct interactions with the transcription elongation complex in response to stimuli.
- This provides a mechanism for controlling NF-κB/NKRF-regulated gene expression.
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