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. 2024 May 24:15:1395750.
doi: 10.3389/fendo.2024.1395750. eCollection 2024.

Alternative isoform expression of key thermogenic genes in human beige adipocytes

Sarah Hazell Pickering  1 , Mohamed Abdelhalim  1 , Philippe Collas  1   2 , Nolwenn Briand  1
Affiliations

Alternative isoform expression of key thermogenic genes in human beige adipocytes

Sarah Hazell Pickering et al. Front Endocrinol (Lausanne). .

Abstract

Background: The beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes.

Methods: We performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing.

Results: We find 777 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 562 genes. Importantly, only 10% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte differentiation. Functional classification of alternative isoforms points to a gain of function for key thermogenic transcription factors such as PPARG and CITED1, and enzymes such as PEMT, or LPIN1. We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT.

Discussion: These results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

Keywords: adipose differentiation; alternative splicing; alternative transcript isoforms; beige adipocyte; human adipose stem cells (hASCs); human thermogenic adipocytes; transcriptomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Model validation. (A) Bodipy staining of lipid droplets of day 15 differentiated white and beige adipocytes from 6 unrelated human subjects. Scale bar: 10µM. (B) Lipid droplet area per field quantified from (A) (ns non-significant; two-way ANOVA with Tukey’s multiple comparison; n>= 15 fields per condition from 3 independent experiments). (C) Western blot analysis of Perilipin1, CITED1, and UCP1 expression in day 15 differentiated white (W) and beige (B) adipocytes from 6 subjects. γTubulin is shown as a loading control. (D) Perilipin1 signals normalized to γTubulin, quantified from western blots (ns, non-significant; two-way ANOVA with Sidák’s multiple comparisons test; n=3). (E) Overrepresentation analysis of genes upregulated in beige vs white adipocytes from all donors (p < 0.05; RNA-seq) using GO and Hallmark gene sets from MSigDb (adjusted p-values < 1.2 x10-6). The gene ratio is the fraction of differentially expressed genes in each gene set. (F) Heatmap of relative gene expression (z-score transformed FPKM) for differentially expressed beige marker genes (p < 0.01 in any donor, eBayes method, limma package) from the BATLAS gene set (37). (G) Median gene expression of beige marker genes from the BATLAS gene set in day 15 white and beige adipocytes from 6 human subjects (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA and t-tests with Holmberg adjustment).
Figure 2
Figure 2
Functional classification of beige and white alternative isoforms. (A) Volcano plot of changes in exon-exon junction percentage spliced-in (PSI) between white and beige adipocytes and adjusted p-values per cluster from LeafCutter. (B) Annotation of exon junctions to transcript databases. (C) Venn diagram showing the overlap between differentially spliced genes (DSGs) and differentially expressed genes (DEGs) (D) Mean TRIFID score for exon junctions with white and beige enrichment (| ΔPSI> 0.1 & p < 0.05) compared to junctions with low differential enrichment (| ΔPSI< 0.1) from significant clusters (p < 0.05; n subsampled to 280), and non-significant junctions (p > 0.05; n subsampled to 280; Kruskal-Wallis test and t-tests with Holmberg adjustment). (E) Change in normalized TRIFID scores (Δ TRIFID) between white and beige isoforms (averaged per exon junction) plotted against LeafCutter ΔPSI adjusted p-value. (F) Network representation of genes pertaining to “Mitochondrial Matrix” (GO:0005759) “Fatty acid metabolism” (GO:0006631) and “Adaptive thermogenesis” (GO:1990845) GO terms, colored by Δ TRIFID score.
Figure 3
Figure 3
Splice variants arise from differential TSS usage. (A) Proportion of differentially spliced junctions (DSJs) mapping to the first intron of a transcript, arranged by annotation database. (B) Enrichment of H3K4me3 ChIP-Seq (log ratio of input) at differential TSSs in white and beige adipocytes. TSSs belonging to non-significant DEGs and non-significant DSGs are used as a control (n=23,529). (C) Quantification of H3K4me3 enrichment around the TSS (-1 kb/+2 kb), scaled within each condition (ns, non-significant, ** p < 0.01: ***p < 0.001; two-way ANOVA and Wilcoxon test with Holmberg adjustment). (D) Top 10 differentially enriched transcription factor binding sites at beige vs white TSSs (-2 kb/+0.5 kb) identified by Unibind. (E) Proportion of promoters (TSSs -2 kb/+0.5 kb) that intersect PPARγ ChIP-Seq peaks (beige promoters n=170; white promoters n=174) and promoters from non-significant DSGs and DEGs (n=12 257). Overlapping white and beige promoter regions are shown separately (n=63). (F, H) PPARγ and MED1 ChIP profiles around white (left panel) and beige (right panel) TSSs. (G, I) Quantification of PPARγ (TSS ± 250 bp) and MED1 (TSS ± 500 bp) ChIP enrichment around white, beige, and non-significant TSSs (***p < 0.0001; two-way ANOVA and Wilcoxon test with Holmberg adjustment).
Figure 4
Figure 4
Beige specific isoform expression of PPARG. (A) Schematic representation of differential splicing pattern across the PPARG gene. (B) Integrative genomics viewer (IGV) tracks showing an overlay of S1-S6 RNA-seq reads on the forward strand, PPARγ and MED1 ChIP-seq over input ratios (33), and H3K4me3 and H3K27ac signals across PPARG1 and PPARG2 TSSs in beige vs white adipocytes. (C) ΔPSI on exon1-exon2 junction for PPARG1 (left panel) and PPARG2 (right panel) in white vs beige adipocytes derived from 6 ASC lines (p < 0.0001; LeafCutter). (D) Total PPARG expression level (FPKM) in white vs beige adipocytes (**p < 0.01, ns, non-significant; eBayes method, limma package; n=3) (E) Relative expression of PPARG1 (upper panel) and PPARG2 (lower panel) assessed by qPCR using isoform specific primers (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparison; n=3). (F) Representative western blot and (G, H) quantification of PPARγ isoforms and total protein expression normalized to γTubulin (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Sidák’s multiple comparisons test; n=3).
Figure 5
Figure 5
Beige-specific isoform expression of PEMT. (A) Schematic representation of differential splicing pattern across PEMT gene. (B) Integrative genomics viewer (IGV) tracks showing an overlay of S1-S6 RNA-seq reads on the reverse strand, PPARγ and MED1 ChIP-seq over input ratios (33), H3K4me3 and H3K27ac signals and Fantom CAGE-seq peaks across PEMT TSSs in white vs beige adipocytes. (C) ΔPSI on exon1-exon2 junction for PEMT-L (left panel) and PEMT-C (right panel) in beige vs white adipocytes derived from 6 ASC lines (p < 0.0001; LeafCutter). (D) Alphafold models for PEMT-S, PEMT-L and PEMT-C isoforms. The additional N-terminal amino acids in PEMT-L and PEMT-C are highlighted in yellow. (E) Total PEMT expression level (FPKM) in white vs beige adipocytes (***p < 0.0005, eBayes method, limma package, n = 3). (F) Relative expression of PEMT-C (left panel) and PEMT-L (right panel) assessed by qPCR using isoform specific primers (***p < 0.001, ns, non-significant, two-way ANOVA with Tukey’s multiple comparison, n = 3). (G) Representative western blot and (H) quantification of PEMT protein expression normalized to γTubulin (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Sidák’s multiple comparisons test; n=3).
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by the Research Council of Norway (grant 313508).

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