Dynamics of transcriptome and chromatin accessibility revealed sequential regulation of potential transcription factors during the brown adipose tissue whitening in rabbits

Abstract

Brown adipose tissue (BAT) represents a valuable target for treating obesity in humans. BAT losses of thermogenic capacity and gains a “white adipose tissue-like (WAT-like)” phenotype (BAT whitening) under thermoneutral environments, which could lead to potential low therapy responsiveness in BAT-based obesity treatments. However, the epigenetic mechanisms of BAT whitening remain largely unknown. In this study, BATs were collected from rabbits at day0 (D0), D15, D85, and 2 years (Y2). RNA-sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed to investigate transcriptome and chromatin accessibility of BATs at the four whitening stages, respectively. Our data showed that many genes and chromatin accessible regions (refer to as “peaks”) were identified as significantly changed during BAT whitening in rabbits. The BAT-selective genes downregulated while WAT-selective genes upregulated from D0 to Y2, and the de novo lipogenesis-related genes reached the highest expression levels at D85. Both the highly expressed genes and accessible regions in Y2 were significantly enriched in immune response-related signal pathways. Analysis of different relationships between peaks and their nearby genes found an increased proportion of the synchronous changes between chromatin accessibility and gene expression during BAT whitening. The synergistic changes between the chromatin accessibility of promoter and the gene expression were found in the key adipose genes. The upregulated genes which contained increased peaks were significantly enriched in the PI3K-Akt signaling pathway, steroid biosynthesis, TGF-beta signaling pathway, osteoclast differentiation, and dilated cardiomyopathy. Moreover, the footprinting analysis suggested that sequential regulation of potential transcription factors (TFs) mediated the loss of thermogenic phenotype and the gain of a WAT-like phenotype of BAT. In conclusion, our study provided the transcriptional and epigenetic frameworks for understanding BAT whitening in rabbits for the first time and might facilitate potential insights into BAT-based obesity treatments.

Integrated analysis of microRNAs, circular RNAs, long non-coding RNAs, and mRNAs revealed competing endogenous RNA networks involved in brown adipose tissue whitening in rabbits

“…Based on the lncRNA expression patterns, we found that a small number of mRNAs at D0 and D15 and a large number of mRNAs at D85 and Y2 regulated by lncRNAs involved ceRNA networks, which might suggest that lncRNAs mainly regulate BAT whitening in the later stage of BAT whitening in rabbits. A previous study showed that de novo lipid (DNL) synthesis was a key biological process during BAT whitening in mice [ 12 ], and our previous study showed that DNL-related genes reached the highest expression levels at D85 during BAT whitening [ 29 ]. In this study, the most significantly enriched GO-BP terms by the D85-selective lncRNAs involved ceRNA networks was positive regulation of cell growth.…”

“…BATs were collected from the interscapular regions of 12 male rabbits at four growth stages of day0 (D0), D15, D85, and 2 years (Y2) (three individuals per stage). These samples were isolated from the same rabbits subjected to our previous RNA-seq study, which revealed the downregulation of UCP1 , COX1 , and PPARGC1A and the upregulation of ACSS2 , ACLY , SNCG , LEP , and CCDC80 [ 29 ] (Figure S 1 A). On the other hand, a well reproducibility among replicates in corresponding group was also found in the previous RNA-seq data [ 29 ] (Figure S 1 B).…”

“…These samples were isolated from the same rabbits subjected to our previous RNA-seq study, which revealed the downregulation of UCP1 , COX1 , and PPARGC1A and the upregulation of ACSS2 , ACLY , SNCG , LEP , and CCDC80 [ 29 ] (Figure S 1 A). On the other hand, a well reproducibility among replicates in corresponding group was also found in the previous RNA-seq data [ 29 ] (Figure S 1 B). For miRNA-seq and circRNA-seq, the adipose tissues were immediately isolated from rabbits under sterile conditions, snap-frozen in liquid nitrogen, and stored at -80 °C until RNA extraction.…”

“…The miRNAs with the thresholds of p-value < 0.05 and [log2(Fold-change) > 1 (log2FC >1) and < -1] were considered DEmiRNAs. To more accurately predict the miRNA target, we retrieved the mRNA expression data of these samples from our previous study [ 29 ]. The 3' UTR sequences of mRNAs were used for target prediction of miRNAs using miRanda software (v3.3a) [ 36 ] with default parameters, and the mRNAs that had an opposite expression with miRNAs (Spearman correlation coefficient < -0.6 with p-value < 0.05 across the 12 samples) was considered the potential target genes of miRNAs.…”

“…The interscapular regions are the major BAT depots of rabbits [ 27 ]. Our previous studies showed that the cell size of classical BAT increased during BAT whitening in rabbits and revealed this process was accompanied by the downregulation of BAT-selective genes (e.g., UCP1, PPARGC1A, and CYTB) and the upregulation of WAT-selective genes (e.g., LEP, SNCG, and CCDC80) and lipogenesis-related genes (e.g., FABP4 and GSN) using RT-qPCR and RNA-seq [ 29 ]. To investigate the ceRNA networks during BAT whitening, we performed miRNA-seq to identify the miRNA dynamics of BATs during BAT whitening in rabbits.…”