Extensive chromatin remodelling and establishment of transcription factor 'hotspots' during early adipogenesis

Abstract

Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPβ and -δ) to transcription factor 'hotspots'. Our results demonstrate that C/EBPβ marks a large number of these transcription factor 'hotspots' before induction of differentiation and chromatin remodelling and is required for their establishment. Furthermore, a subset of early remodelled C/EBP-binding sites persists throughout differentiation and is later occupied by PPARγ, indicating that early C/EBP family members, in addition to their well-established role in activation of PPARγ transcription, may act as pioneering factors for PPARγ binding.

Figure 1

DNase I HyperSensitive (DHS) site analysis during 3T3-L1 adipogenesis. (A) Experimental outline. Nuclei from 3T3-L1 cells were isolated at the indicated time points of adipocyte differentiation and subsequently subjected to a limited DNase I digestion. Small DNA fragments where DNase I cut twice (i.e., DHS sites) were purified over a sucrose gradient and subsequently subjected to deep sequencing using the Illumina platform. (B) Venn diagram representing the overlap between DHS sites in pre-adipocytes immediately before induction of differentiation (day 0; red), in adipocytes (day 6; green), and in cells stimulated for 4 h with the differentiation cocktail (blue). Sizes of the circles are proportional to the number of sites. (C) DHS-seq data at the PPARγ locus viewed in the UCSC genome browser.

Figure 2

Cluster analysis of DHS sites reveals four clusters with distinct temporal profiles (A–D). A K-means cluster analysis was performed on DHS sites to classify the temporal profiles of the magnitudes of their DNase I hypersensitivity. Cluster plots show the relative tag densities of DHS sites for each time point during differentiation (left). Yellow line represents the mean and red line represents the medoid for each cluster. Representative examples of DHS sites in each cluster are indicated by red arrows in screen shots from the UCSC genome browser (middle). Genes were grouped based on their RNA Pol II ChIP-seq tag counts during 3T3-L1 differentiation (Nielsen et al, 2008). A group of induced genes (>3-fold increase from day 0 to day 4 and not repressed at 4 h; 429 genes), repressed genes (>3-fold decrease from day 0 to day 4 and not induced at 4 h; 640 genes), transient genes (>3-fold increase from day 0 to day 1 and <2-fold increase from day 0 to day 4; 104 genes), and constitutive genes (expression >50th percentile at day 0 and <1.5-fold change from day 0 to day 1 and day 4, and neither transiently repressed nor induced at 4 h; 2692 genes) was defined. The number of DHS sites within 100 kb (divided into 10 kb bins) of the TSS of genes in each group was determined and the number of DHS sites per gene was visualized in a bar plot (right).

Figure 3

PPARγ target sites that are remodelled early in differentiation are occupied by C/EBPβ. (A) A subset of PPARγ target sites is open before PPARγ binding. PPARγ-binding sites in mature adipocytes (day 6) displaying a positional overlap with a DHS site at any given time point were assigned to the corresponding DHS cluster (Figure 2). Pie charts indicate the percentage of PPARγ-binding sites in a given cluster out of all the target sites that could be assigned to the four clusters. (B) PPARγ, C/EBPβ, and C/EBPδ ChIP-PCR for selected sites. Results are representative of at least three independent experiments. (C) Early binding of C/EBPβ and -δ at several PPARγ sites at the caveolin 1 (Cav1) and Cav2 locus viewed in the UCSC genome browser. (D) Classification of PPARγ-binding sites at day 6 based on their status at 4 h. PPARγ target sites in mature adipocytes were divided based on their positional overlap with DHS sites at 4 h of differentiation. The PPARγ sites overlapping with open chromatin at 4 h were further subdivided based on the presence of C/EBPβ at these sites at the 4-h time point.

Figure 4

Extensive overlap between C/EBPβ, GR, and Stat5a binding during early adipocyte differentiation. (A) GR and Stat5a ChIP-PCR for a few selected sites. Results are representative of three independent experiments. (B) GR and Stat5a-binding sites at the 4-h time point displaying a positional overlap with a DHS site at any given time point were assigned to the corresponding DHS cluster (Figure 2). Pie charts indicate the percentage of GR and Stat5a-binding sites in a given cluster out of all the target sites that could be assigned to the four clusters. (C) Venn diagram representing the overlap between the C/EBPβ, GR, and Stat5a-binding profiles 4 h after induction of adipogenesis. Circles are proportional to the number of sites.

Figure 5

C/EBPβ binds to transcription factor ‘hotspots’ before induction of differentiation and chromatin remodelling. (A) Co-occurrence of early adipogenic transcription factors (TFs). The proportion of binding sites for the transcription factors on the y axis that overlaps with a binding site for the TFs on the x axis at the 4-h time point was calculated for each TF pair and visualized as a heat map. (B) Classification of transcription factor ‘hotspots’ by K-means cluster analysis based on C/EBPβ ChIP-seq data. The corresponding DHS profiles are shown below. Cluster plots show the relative tag densities for each time point during differentiation. Yellow line represents the mean and red line represents the medoid for each cluster. (C) Maximum DHS tag density at transcription factor-binding sites 4 h after induction of adipogenesis. Error bars represent the 25th and 75th quantiles, respectively. ^* denotes a statistically significant difference with a P-value <2.2E-16 as determined using a Wilcoxon's test.

Figure 6

Transcription factor ‘hotspots’ are enriched in the vicinity of early induced genes. RNA Pol II ChIP-seq profiles were determined for day 0 and 4 h as previously described (Nielsen et al, 2008) and genes were grouped as induced (795 genes), repressed (776), or constitutive (2414 genes) as described in Figure 2. The number of transcription factor-binding sites per 10 000 binding sites within 50 kb of the TSS of genes in each group was determined.

Figure 7

C/EBPβ is required for efficient binding of GR, Stat5a, and RXR, but not C/EBPδ, to shared target sites. (A) Lentiviral-mediated knockdown of C/EBPβ expression. 3T3-L1 cells were transduced with lentivirus expressing shRNA targeting LacZ (control) or C/EBPβ, respectively, and cells were induced to differentiate for 4 h. The three different isoforms of C/EBPβ (liver inhibitory protein (LIP), liver activating protein (LAP), and LAP^*) are indicated. TFIIB was used as a loading control. C/EBPβ (B), Stat5a (C), GR (D), RXR (E), and C/EBPδ (F) ChIP-PCR for a few selected shared sites 4 h after induction of differentiation of cells expressing shRNA targeting LacZ (control) or C/EBPβ, respectively. Control denotes sites that are occupied by the respective factors but not co-occupied by C/EBPβ. Results are representative of three independent experiments. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 8

GR and Stat5a are required for efficient binding of transcription factors to selected ‘hotspots’. 3T3-L1 cells were transduced with lentivirus expressing scrambled shRNA or shRNA targeting GR (A) or Stat5a (B), and cells were induced to differentiate using a standard hormone induction of adipogenesis. GR, Stat5a, C/EBPβ, C/EBPδ, and RXR ChIP-PCR for selected ‘hotspots’ 4 h after induction of differentiation. Control denotes sites that are occupied by the respective factors but not co-occupied by GR (A) or Stat5a (B). Results are representative of two independent experiments. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 9

Model illustrating C/EBPβ as a pioneering factor for adipogenic transcription factors and chromatin remodelling. C/EBPβ binds to closed chromatin in pre-adipocytes. Upon induction of differentiation, several other transcription factors are activated and recruited to C/EBPβ sites, resulting in remodelling of the chromatin structure and formation of transcription factor ‘hotspots’. Some of these are transient in nature, whereas others persist throughout differentiation and are later occupied by PPARγ and C/EBPα, which induce the mature adipocyte phenotype.