Fig 1.
Generation of comprehensive epigenomic and transcriptomic reference maps during brown adipogenesis.
(A) Schematic of the adipogenesis procedures for BA (commitment and differentiation) and WA (differentiation only). (B) Snapshot of the genomic region surrounding the Cidea gene in the UCSC Genome Browser featuring a panel of chromatin marks, PPARγ binding, and mRNA levels during BA differentiation. The scale represents the normalized reads counts. For the H3K4me1 and H3K27me3 d7 tracks, the scale was adjusted to account for lower total ChIP-seq signals. For other loci see S3 Fig.
Fig 2.
Transcriptomic analysis of BA and WA differentiation.
(A) and (B) Differentiation stage-specifically expressed coding genes during BA and WA differentiation (FPKM > 5). The tables show the top 3 (non-redundant) enriched GO categories at individual stages. See S2 Table for the full list. (C) BA and WA lineage-specific coding genes. The list highlights genes showing consistent lineage specific expression between this dataset and primary cells as well as in vivo tissues. Several established lineage markers are highlighted in red. (D) Pim1 gene expression. Left panel: expression pattern of the Pim1 kinase gene determined via RNA-seq in mature BA and WA, differentiated primary SVF cells derived from BAT (pri-BA) and WAT (pri-WA), as well as brown adipose tissue (BAT) and white adipose tissue (WAT). Right panel: Pim1 expression in BAT tissue isolated from male C57BL/6 mice (BAT); in SVF cells isolated from the posterior subcutaneous WAT and differentiated into adipocytes (SVF scWAT); in C3H10T1/2 cells differentiation into BAs (C3H10T1/2). Mice were housed either at RT or 4°C for 7days before isolation of BAT tissue. n = 3 for each group, error bars indicate standard deviation. p-values (paired student’s t-test): * < 0.05. Cells were treated either with 10μM Forskolin or DMSO for 3h before harvesting. Graphs represent the average of three independent experiments, error bars indicate standard deviation. p-values (paired student’s t-test): * <0.05. (E) Heatmaps showing the expression patterns of BA-specific, WA-specific and common adipogenic genes, as well as the status of chromatin modifications at corresponding promoters throughout the differentiation of BAs and WAs.
Fig 3.
TF binding motif analysis of stage specific enhancers and identification of super-enhancer associated genes during brown adipogenesis.
(A) and (B) Differentiation stage-specific enhancers (defined as H3K27ac positive and H3K4me3 negative regions) are also marked by H3K4me1 and associate with genes reflecting their developmental stages. (C) and (D) TF binding motif analysis of stage specific enhancers with HOMER. “Site” indicates the bound TF(s), “Gene” indicates the corresponding gene coding for (one of) the TF(s). Expression of genes was determined using RNA-seq data. Sites with enriched motifs and correspondingly expressed genes were shown in the list. In differentiated BAs and WAs, the binding motifs for PPARγ, RXR and C/EBP were enriched, whereas the SIX1 motif was only enriched in BAs at late stage. (E) Genes associated with super-enhancers (SEs) in mature BAs and WAs. A subset of SE associated genes is shared between BAs and WAs, including established adipogenic markers such as Cebpa and Fabp4, whereas known brown and brite selective genes are specifically associated with SEs in BAs. (F) The Rreb1 gene was significantly up-regulated during brown adipogenesis and is associated with a SE in BAs.
Fig 4.
BMP7 triggers a transcriptional response during BA lineage commitment largely dependent on p38 signaling.
(A) Heatmap of coding genes transiently up-regulated upon BMP7 treatment. C3H10T1/2 cells were treated with or without BMP7 for 3 days before differentiation was induced at d0 (BA). Global gene expression profiles were generated by RNA-seq at the indicated time points. 3T3-L1 cells were also included in the analysis (WA). (B) Five members of the Sox gene family were transiently induced by BMP7 treatment. (C) Gene ontology analysis of the 89 BMP7 induced coding genes from (A). (D) and (E) Blocking the p38 signaling pathway by inhibitors PD169316 (PD) or SB202190 (SB) abolished the BMP7 induced transcriptional activation of all five Sox genes (D) as well as 27 other BMP7 target genes (E). Values represent the average of three biological replicates. Error bars indicate standard deviation. p-values: (paired student’s t-test) * <0.05; ** <0.01.
Fig 5.
Over-expression of Six1, Sox13, Pim1 and Rreb1 promotes BA differentiation and function.
(A) Oil-Red-O staining of C3H10T1/2 cells differentiated for 7 days without BMP7 treatment. Cells were lenti-virally transduced to over-express the indicated genes tagged with tGFP before differentiation. Cells over-expressing tGFP only or the known brown adipogenic regulator Ebf2 served as negative and positive controls, respectively. (B) Expression of brown / mitochondrial marker genes was measured by qRT-PCR on day 7 of differentiation and found to be up-regulated upon over-expression of Six1, Sox13, Pim1, Rreb1, and Ebf2. (C) Expression of Ucp1 gene in cells treated with or without forskolin. (D) Protein levels of CIDEA and PPARα were examined by Western blot at day 7 of differentiation. Calnexin (CNX) was used as a loading control. (E) Expression of general adipogenic marker genes on day 7 of adipogenesis. (F) Oxygen consumption rates (OCR) measured in mature BAs over-expressing the indicated genes. (G) Basal respiration, proton leak, ATP production and maximal respiration were determined according to the OCR values in (F). (H) Uncoupled respiration was enhanced by over-expression of Six1, Sox13, Pim1, Rreb1, and Ebf2. (I) Over-expression of the indicated genes enhanced the response to Norepinephrine (NE) treatment in mature BAs. (J) qRT-PCR analysis confirmed the over-expression of the transduced genes using a primer pair targeting the tGFP coding sequence. Data in panel (B), (C), (E) and (J) represent the average of three independent experiments. Panel (F)—(I) show the representative result of two independent biological replicates (assayed in quadruplets). Error bars represent standard deviation. p-values (paired student’s t-test): * <0.05; ** <0.01; *** <0.001
Fig 6.
Knock-down of Six1, Sox13, Pim1 and Rreb1 impairs brown adipogenesis.
(A) Oil-Red-O staining of SVF cells isolated from BAT after in vitro differentiation for 7 days. Before adipogenic induction, cells were transfected with two independent (#1 and #2) locked nucleic acid (LNA) longRNA GapmeRs targeting Six1, Sox13, Pim1, or Rreb1. Scrambled (Scr) or Pparg-targeting LNA GapmeRs served as negative and positive controls, respectively. (B) Expression of brown / mitochondrial marker genes was measured by qRT-PCR on day 7 of differentiation and found to be down-regulated upon knock-down of Six1, Sox13, Pim1, Rreb1 and Pparg. (C) Expression of Ucp1 was down-regulated upon knock-down of the indicated genes. (D) Expression of general adipogenic genes on day 7 of differentiation. (E) mRNA levels of the targeted genes were assayed 24-hour post transfection. Panel (B)-(E) summarize the average of three independent experiments. Error bars represent standard deviation. p-values (paired student’s t-test): * <0.05.
Fig 7.
Genome-wide SIX1 binding analysis in mature BAs.
(A) ChIP-seq analysis of SIX1 binding revealed that it binds preferentially to intergenic regions, promoters and introns. (B) Gene ontology analysis of genes associated with SIX1 binding peaks. (C) SIX1 binds to the cis-regulatory regions of the brown marker genes Cidea and Ucp1. PPARγbinding, H3K27ac and mRNA expression profiles are also shown at these loci. (D) Box plots comparing SIX1 binding signals (RPKM) at BA-specific, WA-specific and commonly expressed genes, and distances of the closest SIX1 binding site to the transcription start site of BA-specific, WA-specific and commonly expressed genes. (E) Motif analysis of SIX1 binding sites revealed enrichment of C/EBP, EBF and NF1 binding motifs. (F) Co-immunoprecipitation experiments confirmed physical interactions between SIX1 and C/EBPα, C/EBPβ, as well as EBF2 in HEK293 cells. (G) Luciferase assay in HEK293 cells transfected with a plasmid carrying the luciferase gene driven by a portion of the Cidea distal enhancer (see S12 Fig for details) with or without SIX1 over-expression. n = 3, Error bars represent standard deviation. p-values were calculated using the paired student’s t-test.