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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
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RSF1 orchestrates p53 transcriptional activity by coordinating p300 acetyltransferase and FACT complex
Yungyeong Heo1, Yonghyeon Kim1, Won Chung Lim2
1Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Republic of Korea; Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, 443-721, Republic of Korea.
Biochemical and Biophysical Research Communications
|November 23, 2024
Summary
Remodeling and spacing factor 1 (RSF1) is crucial for p53-dependent gene transcription after DNA damage. RSF1 facilitates p53 acetylation and the recruitment of key proteins for DNA repair and cell survival.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- p53-dependent gene transcription is vital for DNA repair and cell survival following DNA damage.
- Remodeling and spacing factor 1 (RSF1) has been previously identified as essential for p53-dependent gene transcription.
- The precise mechanism by which RSF1 influences p53 activity remains to be fully elucidated.
Purpose of the Study:
- To investigate the role of RSF1 in regulating p53 transcriptional activity in response to DNA damage.
- To determine how RSF1 depletion affects p53 acetylation and the recruitment of transcriptional machinery.
- To elucidate the molecular mechanisms by which RSF1 promotes p53-dependent gene expression.
Main Methods:
- Depletion of RSF1 using knockout models.
- Western blotting to assess p53 acetylation levels (acetylated-Lys382).
- Co-immunoprecipitation to detect interactions between RSF1 and p300 acetyltransferase.
- Chromatin immunoprecipitation (ChIP) assays to analyze the occupancy of p300, TBP, and FACT complex subunits (SSRP1, SPT16) at the CDKN1A gene.
- Analysis of histone modifications (H3K27ac, H3K4me1) at the CDKN1A gene locus.
Main Results:
- RSF1 depletion significantly reduced the acetylation level of p53 at Lys382, a key regulator of its transcriptional activity.
- RSF1 was found to co-precipitate with p300 acetyltransferase following etoposide-induced DNA damage.
- Chromatin immunoprecipitation assays revealed decreased accumulation of p300 and TBP at the CDKN1A gene enhancer region in RSF1 knockout cells.
- RSF1 depletion also led to reduced binding of FACT complex subunits (SSRP1, SPT16) to the promoter of the CDKN1A gene.
- Associated with these changes, histone marks indicative of active enhancers and promoters (H3K27ac, H3K4me1) were diminished at the CDKN1A locus.
Conclusions:
- RSF1 plays a critical role in promoting p53-dependent transcription of genes like p21 (encoded by CDKN1A) after DNA damage.
- RSF1 facilitates p53 transcriptional activity by enhancing p53 acetylation.
- RSF1 promotes the recruitment of p300 acetyltransferase to the enhancer and the FACT complex to the promoter of the CDKN1A gene, thereby facilitating transcription.
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