Alternative mRNA splicing of corepressors generates variants that play opposing roles in adipocyte differentiation - PubMed (original) (raw)
Alternative mRNA splicing of corepressors generates variants that play opposing roles in adipocyte differentiation
Michael L Goodson et al. J Biol Chem. 2011.
Abstract
The SMRT and NCoR corepressors partner with, and help mediate repression by, a wide variety of nuclear receptors and non-receptor transcription factors. Both SMRT and NCoR are expressed by alternative mRNA splicing, resulting in the production of a series of interrelated corepressor variants that differ in their tissue distribution and in their biochemical properties. We report here that different corepressor splice variants can exert opposing transcriptional and biological effects during adipocyte differentiation. Most notably, the NCoRω splice variant inhibits, whereas the NCoRδ splice variant promotes, adipogenesis. Furthermore, the ratio of NCoRω to NCoRδ decreases during adipogenic differentiation. We propose that this alteration in corepressor splicing helps convert the cellular transcriptional program from one that maintains the pre-adipocyte in an undifferentiated state to a new transcriptional context that promotes differentiation and helps establish the proper physiology of the mature adipocyte.
Figures
FIGURE 1.
Alternative corepressor mRNA splicing. A, schematic of alternative mRNA splicing at the NCoR and SMRT loci (GenBankTM Accession numbers NM_011308.2 and NM_011424.2). Open reading frames are indicated by black lozenges; 5′ and 3′ untranslated regions are indicated by gray lines. Receptor interaction domains (RIDs) are shown as ovals. Vertical white lines indicate exon/exon boundaries (numbered in base pairs relative to the start of transcription). Exons removed (
) or added (Δ) relative to the reference sequence by alternative splicing are indicated below each schematic. Note that our standardization on the RefSeq database in this report results in a change in the SMRT exon numbering system compared with our prior publications. B, schematic of individual corepressor proteins of interest. Exon sequences included in each corepressor protein are shown as thick lozenges; exon sequences deleted by alternative mRNA splicing are depicted as thin horizontal lines. RIDs are shown as ovals. Nomenclature has been described previously (69).
FIGURE 2.
Relative expression of different corepressor splice variants in different murine tissues. Messenger RNA was isolated from the organs and tissues indicated and was subjected to RT-PCR using primers spanning the relevant splice sites (see Fig. 1_A_ and
supplemental Table S1_A_
); the PCR products were resolved by gel electrophoresis and quantified to determine the percentage of each alternatively spliced mRNA produced at each splice site (total = 100%). The means ± S.E. (n ≥ 3) are presented. Skel Mscl, skeletal muscle; WAT, white adipose tissue; BAT, brown adipose tissue.
FIGURE 3.
Relative expression of different corepressor splice variants during 3T3-L1 cell adipogenesis. Messenger RNA was isolated from 3T3-L1 cells at different times after inducing adipocyte differentiation with: A, Dex/insulin/IBMX followed by insulin, or B, Dex/insulin/IBMX followed by rosiglitazone. The two protocols differ beginning differentiation Day 3, so differentiation Days 0 and 2 in both panels represent the same cell populations. The mRNA was subjected to RT-PCR using primers spanning the relevant splice sites and was analyzed as in Fig. 2 to determine the percentage of each alternatively spliced mRNA produced at each splice site. The means ± S.E. (n ≥ 3) are presented.
FIGURE 4.
Different corepressor splice variants display different specificities for different nuclear receptors. Proteins representing GST-only, or GST fused to the receptor interaction domains of the corepressor variants indicated below the panels, were expressed in Sf9 cells, immobilized on glutathione-agarose beads, and incubated with the radiolabeled nuclear receptors indicated within each panel. Incubations were either in the absence (solid bars) or presence (open bars) of ligand agonist (1 μ
m
T3 for TRα1, 1 μ
m
TN0901317 for LXRa, 1 μ
m
all-trans retinoic acid for RARa, 2.5 m
m
rosiglitazone for PPARγ, and 100 μ
m
chenodeoxycholic acid for Farnesoid X Receptor-α). The immobilized GST constructs were then washed, and the nuclear receptors remaining bound to each construct were eluted and quantified (input = 100%). The means ± S.E. (n ≥ 3) are shown. p values are provided in
supplemental Table S2
.
FIGURE 5.
Slower proliferation and lower saturation density of 3T3-L1 cells expressing ectopic NCoRω versus NCoRδ or GFP control. Stable transformants of 3T3-L1 cells expressing GFP, NCoRδ, or NCoRω were isolated and analyzed as indicated. A, ecoptically introduced GFP control, NCoRω, and NCoRδ are expressed at comparable levels. RNA from three independent 3T3-L1 transformants (differentiation Day 0) was analyzed by quantitative RT-PCR for expression of the ectopic GFP, NCoRω, or NCoRδ using the primer pairs noted in
supplemental Table S1_C_
. Means ± S.E. are shown. B, introduction of ectopic NCoRω or NCoRδ detectably alters the overall expression of the corresponding NCoR splice variants in the 3T3-L1 transformants. The relative abundance of mRNAs containing NCoR exon 37b− or 37b+ was assayed in each transformant by RT-PCR, using primers that span the alternative splice sites as in Fig. 2. C, NCoRω transformants proliferate slower and reach lower saturation density. Proliferating 3T3-L1 transformants expressing GFP, NCoRδ, or NCoRω were plated into culture dishes as described under “Experimental Procedures” and maintained at 37 °C. Viable cells were quantified at the times indicated using CellTiter 96 Aqueous One Solution and measuring absorbance at 490 nm. The means ± S.E. (n ≥ 6) are shown.
FIGURE 6.
Inhibition of 3T3-L1 cell adipogenesis by NCoRω and enhancement by NCoRδ. A, representative microscopic fields. Cultured 3T3-L1 transformants expressing GFP, NCoRω, or NCoRδ were induced using Dex/insulin/IBMX and stained for lipid accumulation on differentiation Day 8 using Oil Red O. Cell morphology was visualized by hematoxylin counterstain. Representative photomicrographs are presented from three independent experiments. B, quantification. Oil Red O staining was quantified in the experiments described in A by digital imaging of microscope fields and the use of ImageJ software, as described under “Experimental Procedures.” The means ± S.E. from multiple microscope fields (n ≥ 6 per transformant) from each of three independent experiments are shown. Oil Red O staining of the NCoRω and NCoRδ cells differed statistically from one another and from the control cells, all with a p value of <0.001.
FIGURE 7.
Enhanced adipogenesis in mouse embryo fibroblasts from NCoRω-/ω- mice. MEFs were isolated from wild-type or NCoRω-/ω- splice-specific knock-out mice, cultured, and induced to differentiate using Dex/insulin/IBMX as described under “Experimental Procedures.” Cells were stained for lipid accumulation on differentiation Day 8 using Oil Red O. Cell morphology was visualized by hematoxylin counterstain. A, representative photomicrographs are presented showing MEFs derived from three different wt/wt and three different NCoRω-/NCoRω- embryos. B, quantification. Oil Red O staining was quantified in the experiments described in A by digital imaging of microscope fields and the use of ImageJ software, as in A. The means ± S.E. from multiple microscope fields are presented. Oil Red O staining of the wt/wt and NCoRω-/ω- MEFS differed at a p value of <0.001. C, ablation of 37b+ NCoRω expression in the NCoRω-/ω- MEFs. RT-PCR was employed as in Fig. 2 to quantify the expression of different corepressor splice variants in the wt/wt and NCoRω-/ω- cells, as indicated. Both NCoR splicing at exon 37 (targeted by the knock-out) and SMRT splicing at exon 47 (a negative control) are shown.
FIGURE 8.
Different effects on adipocyte gene expression by NCoRω versus NCoRδ. Cultured 3T3-L1 transformants expressing GFP, NCoRω, or NCoRδ were induced to differentiate using Dex/insulin/IBMX, and mRNA was isolated on differentiation Days 0 and 8. The mRNA was analyzed by quantitative RT-PCR for expression of 23 genes associated with adipocyte differentiation and/or function, using the primer pairs noted in
supplemental Table S1_B_
. The means ± S.E. from three independent experiments are shown. p values are presented in
supplemental Table S3
. Although included in all plots, expression on Day 0 for many genes may have been too low to produce a visible bar on this graph.
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