HMGB2 orchestrates mitotic clonal expansion by binding to the promoter of C/EBPβ to facilitate adipogenesis

Abstract

High-mobility group box 2 (HMGB2) is an abundant, chromatin-associated protein that plays an essential role in the regulation of transcription, cell proliferation, differentiation, and tumorigenesis. However, the underlying mechanism of HMGB2 in adipogenesis remains poorly known. Here, we provide evidence that HMGB2 deficiency in preadipocytes impedes adipogenesis, while overexpression of HMGB2 increases the potential for adipogenic differentiation. Besides, depletion of HMGB2 in vivo caused the decrease in body weight, white adipose tissue (WAT) mass, and adipocyte size. Consistently, the stromal vascular fraction (SVF) of adipose tissue derived from hmgb2^-/- mice presented impaired adipogenesis. When hmgb2^-/- mice were fed with high-fat diet (HFD), the body size, and WAT mass were increased, but at a lower rate. Mechanistically, HMGB2 mediates adipogenesis via enhancing expression of C/EBPβ by binding to its promoter at "GGGTCTCAC" specifically during mitotic clonal expansion (MCE) stage, and exogenous expression of C/EBPβ can rescue adipogenic abilities of preadipocytes in response to HMGB2 inhibition. In general, our findings provide a novel mechanism of HMGB2-C/EBPβ axis in adipogenesis and a potential therapeutic target for obesity.

Fig. 1. HMGB2 is required for adipogenesis.

a qPCR for mRNA of HMGB2, C/EBPβ, and PPARγ during 3T3-L1 cells differentiation. b Western blot analysis of protein levels of HMGB2, C/EBPβ, and PPARγ. c Schema of si-HMGB2 or EGFP-HMGB2 plasmid treatment during adipogenic induction. d Oil Red-O staining of cells treated with EGFP-HMGB2 plasmid. e Western blot analysis of EGFP-HMGB2 plasmid in 3T3-L1 cells. f, g Knockdown efficiency of three si-HMGB2 examined by qPCR and western bot. h Effect of siRNA transfection at different time points on adipogenesis, as determined by Oil Red-O staining. i Statistical data of Oil Red-O by OD measurements. j qPCR for mRNA of HMGB2 and adipogenic genes. k Western blot analysis for HMGB2, C/EBPα, and PPARγ. Data represent three biological replicates each with three technical replicates. (f, j: n = 3; data presented in graphs represent mean s.e.m. *P < 0.05; **P < 0.01, Students’ t test. d, e, h: cells were harvested and stained at day 10, n = 3).

Fig. 2. HMGB2 promotes mitotic clonal expansion in adipogenesis.

a–d Detection of cell proliferation at different stages after HMGB2 interference by using immunofluorescence assay. e, f qPCR for mRNA of MCE-specific genes and cell-cycle genes affected by si-HMGB2 in 3T3-L1 cells. (b, d–f: n = 3, **P < 0.01; *P < 0.05 against si-NC, Students’ t test; data presented in graphs represent mean ± s.e.m.).

Fig. 3. Deletion of HMGB2 suppresses adipogenesis in vivo.

a, b Representative body weight diagram of male (n = 5) and female mice (n = 6). c The weight of Body, fat tissue, and lean of hmgb2^−/− and WT mice. n = 5, male, 12 weeks old. d The phenotype of WT and hmgb2^−/− mice at 12 weeks postnatal. e Dissection images. Yellow arrow: ingWAT in WT mice; white arrow: ingWAT in hmgb2^−/− mice. f Anatomical images of eWAT (upper picture) and ingWAT (lower picture). g H&E staining of ingWAT slides. Scale bar: 100 μm. h Western blot analysis of protein level for adipogenic maker genes. Protein was extracted from ingWAT of male mice (n = 5). i qPCR analysis for genes related to adipogenesis, lipolysis, adipokines, and insulin in ingWAT. j Oil Red-O staining of SVFs derived from ingWAT (left) and OD measurements of stained cells (right). k Western blot analysis of protein levels for adipogenic marker genes in SVFs. (i: mean values ± s.e.m. **P < 0.01; *P < 0.05).

Fig. 4. HFD enhances adipogenic ability of hmgb2^−/− mice but at a lower rate.

a Representative comparison images of 4 different groups. b, c Abdominal WAT and ingWAT images of four mice groups. d H&E staining of ingWAT slides. e Statistical data of adipocyte size measurements from four groups. f qPCR of adipogenic genes of four mice groups. (four groups of mice: WT (chow diet), hmgb2^−/− (chow diet), WT HFD, and hmgb2^−/−-HFD. a–d: male; age: 20 weeks old; n = 5, f: mean values ± s.e.m. **P < 0.01).

Fig. 5. Analysis of HMGB2 binding targets at different stages of adipogenesis.

a Genomic distribution analysis of HMGB2 binding regions using ChIP-seq data at different sampling time points. b Classification analysis of HMGB2-target genes at MCE stage by iTAK software (version 1.7a). c Venn diagram for the numbers of genes bound by HMGB2 on different sampling time points. d GO enrichment for the biological process that HMGB2 participates in. e KEGG classification of HMGB2 at 24 h. f Classification of transcription factors and the numbers that bound by HMGB2. g, h Venn diagram about genes screened using RNA-seq and ChIP-seq data at 24 h.

Fig. 6. HMGB2 binds to the promoter of C/EBPβ in MCE stage to modulate adipogenesis.

a ChIP-seq analysis of the peak distributions of C/EBPβ, PPARγ, and FABP4 loci bound by HMGB2 at −48, 24, and 96 h. b ChIP-PCR assay for the interaction between HMGB2 and C/EBPβ promoter in 3T3-L1 cells. c Band intensity for b (n = 3, *P < 0.05). d Luciferase activity for truncated DNA fragments of C/EBPβ (***P < 0.001). Experiments were conducted in 293T cells. e Binding motif of HMGB2 on C/EBPβ. HOMER and KNOWN analysis by ChIP-seq on FDR-corrected, p values represent enrichment. f Luciferase activity of potential regions of C/EBPβ bound by HMGB2 before and after mutation (***P < 0.001). Experiments were carried out in 293T cells. g Oil Red-O staining assay in 3T3-L1 cells (left) and OD measurements (right). h Western blot for adipogenic genes. (g, h: 3T3-L1 cells transfected with si-NC, si-HMGB2, and si-HMGB2 with C/EBPβ overexpression plasmid, respectively).

Fig. 7. Regulatory mechanisms of HMGB2 in adipogenesis.

HMGB2 binds to the promoter region of C/EBPβ in MCE stage to regulate adipogenesis. High-fat diet enhances adipogenic ability of hmgb2^−/− mice but at a lower rate.