| Brown adipose tissue(BAT)is a main thermogenic source for maintaining body temperature in newborn livestock and a target for treating obesity in humans.The development and function of BAT are regulated by transcription factors(TFs)and noncoding RNAs.BAT gradually loses its thermogenic phenotype and transforms into white adipose tissue-like(WAT-like)tissue with age.In livestock,BAT whitening can be used to screen for thermogenesis-related genetic factors.In human medicine,understanding the molecular mechanism of BAT whitening is beneficial for treating obesity.Rabbits are an important economically livestock and an ideal model animal for studying BAT.Currently,chromatin accessibility that provides spatial conditions for TF binding in BAT has not been reported in rabbits,and it is also unknown whether non-coding RNA affects the BAT whitening of rabbits.In this study,the interscapular BATs of Tianfu black rabbits aged 0days(D0),15 days(D15),85 days(D85),and 2 years(Y2)were used as experimental materials to detect the chromatin accessibility and whole-transcriptome of BATs using techniques such as assay for transposase-accessible chromatin with high throughput sequencing(ATAC-seq)and strand-specific RNA-seq(ssRNA-seq).And then,we constructed a differentiation model of rabbit brown adipocytes to validate the function of the selected miRNA.This study is aim to explore the molecular mechanisms of the BAT whitening in rabbits and provides a theoretical basis for analyzing the cold resistance mechanism of newborn rabbits.The main results of this study are as follows:(1)Histological examination revealed that the cell sizes of rabbit brown adipocytes increased from D0 to D15 and the lipid droplets gradually accumulated from D0 to Y2.The proportion of multilocular adipocytes decreased,while that of unilocular adipocytes increased from D0 to Y2.The expression of uncoupling protein 1(UCP1)and mitochondrial-related genes gradually decreased from D0 to Y2.These results indicated that the thermogenic capacity and lipid deposition of rabbit BATs decreased and increased with age,respectively.(2)ATAC-seq identified 214,204 chromatin accessibile regions in the BATs,of which25464 regions showed different chromatin accessibility among four different stages.Clustering and enrichment analysis found that the regions with reduced chromatin accessibility after D0 may promote the whitening of rabbit BATs by regulating adjacent genes associated with vascular homeostasis and energy metabolism.The functional regions of the thermogenic regulation decreasing chromatin accessibility from D0 to D15 and the functional regions of the lipid metabolism and immune response increasing chromatin accessibility at D85 and Y2 may be the epigenetic factors that lead to the gradual transformation of rabbit BATs into white adipose tissue-like tissues.Footprint analysis indicated that the sequential regulation of some TFs involved in the thermogenesis,some TFs affecting adipocytes differentiation and expansion,some TFs that were WAT-specific expression,and some TFs inhibiting BAT thermogenesis may affect the whitening of rabbit BAT at different stages.(3)SsRNA-seq identified 3780 differentially expressed m RNAs among the four stages.The dynamic changes of transcriptome affect the thermogenesis,proliferation,lipid metabolism and immune function of BAT.The proportion of synchronous changes between chromatin accessibility and adjacent gene expression increased from D0 to Y2,indicating the cis-regulation of chromatin accessibility gradually increased with the process of BAT whitening.Combined analysis of TF footprint and gene expression showed that the extensive reduction of chromatin accessibility of TF target genes was an important epigenetic factor for the loss of rabbit BAT thermogenic capacity,and 17 TFs were considered to be the key TFs to promote rabbit BAT thermogenesis.Thirteen key SNPs involved in lipid metabolism were identified at the putative binding sites of thermogenic TFs.(4)MiRNA-seq found that 298 known miRNAs were differentially expressed among the four stages.Target genes of differentially expressed miRNA from D0 to D15 significantly enriched in lysosomal assembly,lipid metabolism,and thyroid hormone signaling pathways.The target genes of differentially expressed miRNAs from D15 to D85 are mainly enriched in biological processes and signal pathways related to neural development.The target genes of differentially expressed miRNAs of D85 to Y2 are mainly enriched in biological processes and signaling pathways related to immune response.(5)Analysis of ssRNA-seq data found that 631 lnc RNAs were differentially expressed among the four stages.By constructing lnc RNA-related competitive endogenousRNA(ce RNA)network,24 lnc RNA/miRNA/m RNA regulatory axes related to lipid metabolism and thyroxine metabolism(such as MSTRG.4180.1/miR-195-5p/MED27)were screened from D0 to D15,59 lnc RNA/miRNA/m RNA regulatory axes related to neural development were screened from D15 to D85,and 100 lnc RNA/miRNA/m RNA regulatory axes related to immune response were screened from D85 to Y2.Integrated analysis of circ RNA-seq and ssRNA-seq detected 297 differentially expressed circ RNAs among the four stages.The back-splicing sequences of circ RNAs might interact with miRNAs that have thermogenic regulatory functions.By constructing a circ RNA-related ce RNA network,13 lnc RNA/miRNA/m RNA regulatory axes related to lysosomal and thyroxine metabolism were screened from D0 to D15(such as 20＿9053986＿9054398＿P/miR-195-5p/MED27),and 48 circ RNA/miRNA/m RNA regulatory axes related to lipid metabolism were screened from D85 to Y2.The selected lnc RNA,circ RNA,and miRNA on the regulatory axis are candidate non-coding RNAs affecting the thermogenic function of rabbit BAT.(6)The function of miR-195-5p that screened from the ce RNA networks was verified on the differentiation model of rabbit brown adipocytes in vitro.The results showed that overexpression of miR-195-5p promoted the deposition of brown fat and the expression of FABP4,while inhibited the expression of UCP1.The interference of miR-195-5p inhibited the deposition of brown fat and the expression of FABP4,while promoted the expression of UCP1.These results suggested that miR-195-5p may be a gene inhibiting brown adipose tissue thermogenesis.This study revealed the dynamic changes of chromatin accessibility and whole the transcriptome during the whitening process of rabbit BATs and identified key chromatin accessibile regions,TFs,SNPs,and non-coding RNAs regulating BAT thermogenic capacity.Our results enriched the epigenetic data of rabbit adipose tissue and provided a reference for understanding rabbit BAT thermogenesis and human BAT whitening. |
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