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. 2024 May 3:15:1385811.
doi: 10.3389/fendo.2024.1385811. eCollection 2024.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis

Raja Gopal Reddy Mooli  1 , Bokai Zhu  1   2   3 , Saifur R Khan  4   5   6   7 , Veerababu Nagati  1 , Kulandaimanuvel Antony Michealraj  7 , Michael J Jurczak  1 , Sadeesh K Ramakrishnan  1   2
Affiliations

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis

Raja Gopal Reddy Mooli et al. Front Endocrinol (Lausanne). .

Abstract

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms.

Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays.

Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20.

Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

Keywords: GABPA; beige adipocyte; epigenetic modification; neonatal adipose tissue; subcutaneous adipose tissue.

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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
(A) Western blot showing beige markers in iWAT of P20 and P3 mice. (B) Relative mRNA expression of thermogenic adipocyte markers in iWAT of P20 and P3 mice. (C) Immunohistology images showing UCP1 and TOM20 in iWAT of P3 and P20 mice. Magnification 20× (n = 3–4 mice) (D) Relative mRNA levels showing M1 and M2 macrophage markers in iWAT. (E) Western blot for markers showing acetylated histone H3K27 in iWAT. (F) H3K27ac Chip-qPCR analysis for Ucp1 enhancer and Ucp1 proximal in iWAT from P20 and P3 mice. All the data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001 as analyzed by one-way ANOVA (Tukey multiple-comparisons test) and two-tailed Student’s t-test.
Figure 2
Figure 2
(A) Schematic showing H3K27ac ChIP-seq in iWAT of P20 and P3 mice. (B) Distribution plot for H3K27ac poised enhancers. (C) Heat map of H3K27ac poised enhancers in iWAT that are stronger in P20 mice than in P3 mice. (D) H3K27ac ChIP-seq data centered on the TSS (±5 kb) of thermogenic, general, and white adipocyte marker genes.
Figure 3
Figure 3
(A) Heat map showing differentially expressed gene profiles and GO pathway analysis of gene clusters upregulated and downregulated in P20 and P3 mice. (B) GO enrichment pathway analysis showing thermogenic genes and corresponding p-values of the upregulated genes iWAT of P20 mice. (C) Heat map of upregulated genes in the GO category of thermogenesis, fatty acid oxidation, oxidative phosphorylation, and mitochondrial metabolism (n = 4 per group) (D) Heat map of gene expression profiles of RNA-seq and ChIP-seq and GO pathways of up- or downregulated genes with positively or negatively associated H3K27ac changes in iWAT of P20 compared to P3 mice.
Figure 4
Figure 4
(A) Position weight matrix logos of beige-enriched motifs and ranks of each motif are represented by Z-score. (B) Relative mRNA levels of Gabpα in iWAT of P20 and P3 mice. (C) Immunohistology showing GABPα in iWAT. Magnification 20× (n = 2–3 mice). (D) Chip-qPCR analysis for Ucp1 enhancer and Ucp1 proximal for GABPα in iWAT from P20 and P3 mice. (E) Oil Red O staining of the SVF cell line transfected with siRNA against Gabpα or scramble and differentiated into beige adipocytes for 6 days (magnification 4×). (F) Western blot showing beige adipocyte markers in the inguinal SVF cell line transfected with siRNA against Gabpα or scramble and differentiated into beige adipocytes. (G) GO pathway of H3K27ac with potential GABPα motif upregulated in P20 mice. All the data are presented as mean ± SEM. **p < 0.01 as analyzed by one-way ANOVA (Tukey multiple-comparisons test) and two-tailed Student’s t-test.
Figure 5
Figure 5
(A) Distribution of H3K27ac poised enhancers in glycolysis-related genes in iWAT of P20 and P3 mice. (B) Heat map of upregulated genes in the “glycolysis” GO category in iWAT of P20 and P3 mice. (C) Relative mRNA expression for glycolysis-related genes in iWAT. (D) Western blot showing proteins for glycolysis in iWAT. (E) Ex vivo glycolysis analysis in iWAT of P20 and P3 mice. (F) Western blot showing glycolysis-related proteins in the inguinal SVF cell line transfected with siRNA against Gabpα and differentiated into beige adipocytes. (G) Schematic showing the mechanism of beige adipocyte induction in iWAT of P20 mice. The Schematic figure was generated using BioRender. All the data are presented as mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001 as analyzed by two-tailed Student’s t-test.
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