Long noncoding RNAs regulate adipogenesis

Abstract

The prevalence of obesity has led to a surge of interest in understanding the detailed mechanisms underlying adipocyte development. Many protein-coding genes, mRNAs, and microRNAs have been implicated in adipocyte development, but the global expression patterns and functional contributions of long noncoding RNA (lncRNA) during adipogenesis have not been explored. Here we profiled the transcriptome of primary brown and white adipocytes, preadipocytes, and cultured adipocytes and identified 175 lncRNAs that are specifically regulated during adipogenesis. Many lncRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα). RNAi-mediated loss of function screens identified functional lncRNAs with varying impact on adipogenesis. Collectively, we have identified numerous lncRNAs that are functionally required for proper adipogenesis.

Fig. 1.

Global transcriptome profiling of lncRNAs and protein coding genes during adipogenesis via RNA-Seq. (A) This heatmap shows 1,734 coding genes and 175 lncRNAs significantly up-regulated (red) or down-regulated (blue) between preadipocytes and differentiated adipocytes. Independent hierarchical clustering of expression values for these lncRNAs and coding genes clusters differentiated and primary adipocytes together. (B) RNA-Seq read alignment of three lncRNAs as well as three adipocyte markers—AdipoQ, Fabp4, and Glut4—is displayed. RNA-Seq read alignment coverage in brown and white preadipocytes and cultured mature adipocytes is displayed above each gene. Previously identified PPARγ ChIP-Seq binding sites (10) are identified with an asterisk (*), and exons are depicted as blue bars.

Fig. 2.

Validation of selected lncRNAs. Primary brown preadipocytes (A) and primary white preadipocytes (B) were isolated, cultured, and differentiated as described in Experimental Procedures. Total RNA was extracted from D0 and D6 cells, and qRT-PCR was performed to examine the expression of selected lncRNAs. Twenty-six lncRNAs (n = 3) were validated as significantly differentially up-regulated during both brown and white fat differentiation (P < 0.05; means ± SEM). (C) RT-PCR was performed to examine the expression of lncRNAs across 12 adult mouse tissues. Their expression profiles, presented here as differences from the row mean, are plotted as a heatmap. Red represents a higher expression and blue a lower expression. BAT, brown adipose tissue; Epi, epididymal white fat.

Fig. 3.

LncRNAs are essential regulators for adipogenesis. (A) Primary white preadipocytes were isolated, cultured, and differentiated as described in Experimental Procedures. One day before differentiation, cells were transfected with siRNAs targeting each lncRNA. At 4 d after differentiation induction, ORO staining was performed to detect the lipid accumulation. RT-PCR was performed to examine the expression of the targeted lncRNAs (A, bar plots) (n = 3; *P < 0.05; means ± SEM) and several adipogenesis marker genes (B).

Fig. 4.

Microarray analysis of adipogenesis-regulated LncRNA knockdowns. Gene expression profiles for 1,727 genes selected as significant between undifferentiated (D0) and differentiated (D4) adipose precursors but not significantly regulated between D4 and a scrambled siRNA control. Knockdowns are rank-ordered by JSD to D0, and this ordering highlights the range of effects each LncRNA has on regulating the adipogenesis transcriptome. The dashed lines divide the knockdowns into three regions: those with little to no effect on adipogenesis (similar to D4 and scramble), those with moderate effects on the adipogenesis-regulated genes (middle block), and those ncRNAs whose absence severely affects the differentiation potential of adipocyte precursors (similar to D0). Distance from D0 correlates with lipid accumulation, as determined by Oil Red staining (plates).