The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
- Hokkaido University Graduate School of Medicine, Japan
- Tokyo Metropolitan Institute of Medical Science, Japan
Figures
Figure 1
TRIM23 is expressed during adipocyte differentiation.
(A) Real-time PCR analysis of mRNA expression of Trim23 during 3T3-L1 differentiation. Total RNA was isolated from 3T3-L1 cells on the indicated days. Trim23 mRNA was normalized to that of Gtf2b. (B)…
Figure 2 with 2 supplements
TRIM23 is required for 3T3-L1 adipocyte differentiation.
(A) Immunoblot analysis of TRIM23 knockdown before induction of adipogenesis is shown. (B) Cells were stained with Oil Red O to visualize the accumulation of lipid droplets at day 6. (C) The amounts …
Figure 2—figure supplement 1
Quantitative analysis of Trim23, Cidec, Klf15, Adipoq and Retn mRNA during 3T3-L1 differentiation.
mRNA levels of Trim23, Cidec, Klf15, Adipoq and Retn were determined by real-time PCR at days 0, 2, 4, and 6. Expression level of each gene was normalized to the level of Gtf2b.
Figure 2—figure supplement 2
TRIM23 is required for human visceral preadipocyte differentiation.
Short interfering RNAs targeting TRIM23 (siTRIM23) and a non-targeting control siRNA (siControl) were introduced into human primary visceral preadipocytes, and the preadipocytes were differentiated …
Figure 3
TRIM23 is required for induction of Pparg1, Pparg2 and Cebpa but not for induction of Cebpb and Cebpd during adipogenesis.
RNA levels of Pparg, Cebpa, Cebpb and Cebpd were determined by real-time PCR at days 0, 2, 4 and 6. Expression level of each gene was normalized to that of the Gtf2b. The data represent means ± s.d. …
Figure 4
TRIM23 knockdown does not affect the induction and occupancy of C/EBPβ and C/EBPδ at the Pparg promoter during adipogenesis.
(A) RNA levels of Trim23, Cebpb and Cebpd were determined by real-time PCR at 0, 6, 12, 24 and 48 hr. Expression level of each gene was normalized to that of Gtf2b. (B) Schematic representation of …
Figure 5 with 1 supplement
TRIM23 knockdown does not affect the epigenetic marks for activated genes and chromatin opening but decreases the occupancy of Pol II at the Pparg promoters.
(A and B) ChIP analysis of H3K4me3 (A) and H3K27ac (B) at the Pparg promoters during adipocyte differentiation. Ct values of each ChIP were normalized to that of input. (C) Formaldehyde-assisted …
Figure 5—figure supplement 1
Depletion of TRIM23 does not affect H3K9me2 and H4K20me1 marks at the Pparg promoter.
(A) Control IgG ChIP does not occupy a subset of gene promoters during adipogenesis. Ct values of each ChIP were normalized to that of input. (B and C) ChIP analysis of H3K9me2 (B) and H4K20me1 (C) …
Figure 6
TRIM23 does not affect PPARγ2 transcriptional activity.
(A) TRIM23 does not affect PPARγ-mediated transcriptional activity in HEK293T cells. The peroxisome proliferator-activated receptor response element firefly luciferase reporter vector (PPRE–Luc), …
Figure 7 with 1 supplement
TRIM23 stabilizes PPARγ2.
(A) Immunoblot analysis of C/EBPα, C/EBPβ, C/EBPδ and PPARγ proteins during 3T3-L1 cell differentiation. (B) The intensity of the immunoreactive bands of PPARγ2 obtained by immunoblot analysis with …
Figure 7—figure supplement 1
The presence of TRIM23 is sufficient to inhibit basal degradation of PPARγ.
Immunoblot analysis of PPARγ protein in the absence and presence of MG132. 3T3-L1 preadipocytes with stable knockdown of TRIM23 or the corresponding control cells were treated with MG132 (10 mM) for …
Figure 8
TRIM23 interacts with PPARγ2 and promotes ubiquitination of PPARγ2.
(A) In vivo assay for interaction between TRIM23 and PPARγ2. FLAG-TRIM23 and HA-PPARγ2 were transfected into HEK293T cells. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted …
Figure 9
TRIM23 mediates atypical polyubiquitin conjugation of PPARγ2, leading to reduced recognition of ubiquitinated PPARγ2 by the proteasomal ubiquitin receptor S5a.
(A) PPARγ2 polyubiquitination by TRIM23 leads to reduced recognition by the proteasomal ubiquitin receptor S5a. HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or …
Figure 10 with 1 supplement
PPARγ2 expression rescues the adipogenesis defect in TRIM23-knockdown 3T3-L1 cells.
(A) Immunoblot analysis of ectopic PPARγ2 expression before induction of adipogenesis is shown. (B) Changes in Trim23 mRNA during adipocyte differentiation in 3T3-L1 cells. Total RNA was isolated …
Figure 10—figure supplement 1
PPARγ2 expression rescues the adipogenesis defect that occurred in TRIM23-knockdown 3T3-L1 cells.
(A) Immunoblot analysis of PPARγ and C/EBPα protein during 3T3-L1 differentiation. (B) RNA levels of Cidec, Klf15, Cebpb, Adipoq, Retn and Cebpd, were determined by real-time PCR at days 0, 2, 4, …
Tables
Table 1
List of SYBR Green primers for real-time PCR
| Gene | Forward primer | Reverse primer |
|---|---|---|
| PCR primers for cloning of cDNA | ||
| Trim23 | AGGATGGCTACCCTGGTTGTAAAC | AAATCAAGCAACATCCAATACTCC |
| Pparg2 | GTTATGGGTGAAACTCTGGGA | CTGCTAATACAAGTCCTTGTA |
| Oligonucleotides for shRNA | ||
| shTRIM23a | GATCCCCGAAGAAATGGCTCTAAGTGTTCAAGAGACACTTAGAGCCATTTCTTCTTTTTA | AGCTTAAAAAGAAGAAATGGCTCTAAGTGTCTCTTGAACACTTAGAGCCATTTCTTCGGG |
| shTRIM23b | GATCCCCGGTAGATGTTAAATCGCATTTCAAGAGAATGCGATTTAACATCTACCTTTTTA | AGCTTAAAAAGGTAGATGTTAAATCGCATTCTCTTGAAATGCGATTTAACATCTACCGGG |
| qRT-PCR primers for gene expression | ||
| Adipoq | GCACTGGCAAGTTCTACTGCAA | GTAGGTGAAGAGAACGGCCTTGT |
| Cebpa | TGCGCAAGAGCCGAGATAA | CGGTCATTGTCACTGGTCAACT |
| Cebpb | CAAGCTGAGCGACGAGTACA | AGCTGCTCCACCTTCTTCTG |
| Cebpd | ATCGACTTCAGCGCCTACAT | GCTTTGTGGTTGCTGTTGAA |
| Cidec | AGCTAGCCCTTTCCCAGAAG | TCAGGCAGCCAATAAAGTCC |
| Fabp4 | CATCAGCGTAAATGGGGATT | GTCGTCTGCGGTGATTTCAT |
| Klf15 | CCCAATGCCGCCAAACCTAT | GAGGTGGCTGCTCTTGGTGTACATC |
| Pparg1 +2 | TGCAGGAGCAGAGCAAAGAG | CGGCTTCTACGGATCGAAAC |
| Pparg1 | TGAAAGAAGCGGTGAACCACTG | TGGCATCTCTGTGTCAACCATG |
| Pparg2 | TGGCATCTCTGTGTCAACCATG | GCATGGTGCCTTCGCTGA |
| Retn | TTTTCTTCCTTGTCCCTGAACTG | GATCTTCTTGTCGATGGCTTCAT |
| Gtf2b | GTTCTGCTCCAACTTTTGCCT | TGTGTAGCTGCCATCTGCACTT |
| Trim23 | TTGGAATGGCTCACACAGAAC | ACATGGGCATCAACAACAC |
| qPCR primers for ChIP | ||
| Pparg1 −1.0 kb | CTGTCTATCATGTGGGCTTCAG | ACCTTACACATAGGGTGGAGA |
| Pparg1 −0.4 kb | ACAAACTTCTCCATGACAGACA | CGCCTTGCTCCTCACAG |
| Pparg1 +0.3 kb | CTGCGTAACTGACAGCCTAAC | ACTTGGTCACTCTCCGTCCT |
| Pparg2 −1.0 kb | GATACACTGCCCTGTGTAAGG | GAGCAGCCCTTGTCACATAA |
| Pparg2 −0.2 kb | GAACAGTGAATGTGTGGGTCA | CTGACTGAGAGCCAGTTGTGA |
| Pparg2 +0.5 kb | GTGAGCATTTCAGAACACTTGG | GCCTGAAGAAGAACAGAAATTCTAC |
| Negative control | TGGTAGCCTCAGGAGCTTGC | ATCCAAGATGGGACCAAGCTG |
| Myog | GGGTCTCTTCCTCTTACCCGAT | ACCTTGCTGGCCATGGAC |
| Iqck | GAAACAAAGCCTTCCCATCC | TCCTTTCTTGCTGTGGCTTC |
| Cav2 | CTCAGAAAAGGCAGGGAAAG | CCCAGTCATGACAACACCAA |
| Fabp4 -10,000 bp | CCATGAGGAAATTCGCTACAC | CCTTCCACCCTTATCTCACAC |
| Fabp4 −5500 bp | GAGAGCAAATGGAGTTCCCAGA | TTGGGCTGTGACACTTCCAC |
| Fabp4 −200 bp | CATTGCCAGGGAGAACCAA | TCCTTCATGACCAGACCCTGT |
| Fabp4 +500 bp | CAGGTGAACCCGCAAGAAAG | GCTTGGCAAAGAAGGCCAC |
