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| Status |
Public on Nov 27, 2020 |
| Title |
The reducing equivalent NADPH dictates histone acetylation via direct inactivation of HDAC3 |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
NADPH has been long well-recognized as a key cofactor for antioxidant defense and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We found that reduction of cellular NADPH levels by silencing malic enzyme (ME) or G6PD impairs global histone acetylation and transcription in both adipocytes and tumor cells. These effects can be reversed by supplementation of exogenous NADPH or inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH or inhibition of histone deacetylase 3 (HDAC3). Mechanistically,NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator Ncor2 (SMRT) or Ncor1, thereby impairs HDAC3 activation. Interestingly, it appears that NADPH and Ins(1,4,5,6)P4 bind to the same domains on HDAC3, and NADPH has relatively higher affinity towards HDAC3. Thus, while Ins(1,4,5,6)P4 acts as an ‘intermolecular glue’, NADPH may function as a HDAC3-Ncor assembly inhibitor. Collectively, our findings uncovered a previous unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.
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| Overall design |
Briefly, 3T3-L1 preadipocytes cells were transfected with control, ME1, or ME2 siRNAs as indicated and two days later induced to differentiate into adipocytes. After another 7 days, cells were washed in PBS buffer and subsequently cross-linked with 1% formaldehyde solution at room temperature (RT) for 15 min. After washing in cold PBS, cross-linking reaction was stopped by the addition of 0.125M glycine with gently shaking for 5min. Cells were then centrifuged and lysed in 300 μL SDS lysis buffer (50mM Tris-HCl pH 8.0, 10mM EDTA pH 8.0 , 1% SDS, protease inhibitors) for 15 minutes on ice. Cell lysates were sonicated to generate DNA fragments approximately 200-1000 bp and subjected to immunoprecipitation with indicated antibodies against Ac-H2B and Ac-H3. Immunoprecipitations were carried out overnight, samples were then washed with low salt buffer, high salt buffer, LiCl salt buffer and TE buffer sequentially. Histone-DNA complexes were eluted using elution buffer (1%SDS, 100 mM NaHCO3), incubated at 65ºC for overnight in the presence of 0.54 M NaCl. The next day, DNase-free RNase(10 mg/ml)was added into samples and incubated for 1 hour at 37℃.Samples were then incubated with Proteinase K for another 1 hour at 55ºC.Finally, DNA were purified using Qiaex II beads (Qiagen) and analyzed by quantitative PCR (qPCR). For ChIP-seq analysis, briefly, 3T3-L1 cells were treated with siRNA for 2 days and confluent cells were induced to differentiate into adipocytes. Cell were cross-linked, and the cell lysates were sonicated to generate DNA fragments and subjected to immunoprecipitation using Ac-H2B, Ac-H3 or HDAC3 antibodies as described above. Bound DNA fragments were eluted for ChIP-seq library construction and sequencing.
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| Contributor(s) |
Li W, Kou J, Qin J, Li L, Zhang Z, Pan Y, Xue Y, Du W |
| Citation(s) |
33462516 |
| Submission date |
Sep 19, 2019 |
| Last update date |
Feb 26, 2021 |
| Contact name |
wei li |
| E-mail(s) |
942013819@qq.com, liwei@ibms.pumc.edu.cn, wjdu123@hotmail.com
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| Organization name |
Peking Union Medical College
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| Street address |
No.5 Dongdansantiao street, Dongcheng district
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| City |
beijing |
| ZIP/Postal code |
100005 |
| Country |
China |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA566337 |
| SRA |
SRP222487 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE137694_RAW.tar |
6.1 Mb |
(http)(custom) |
TAR (of BED) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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