Mediator and cohesin connect gene expression and chromatin architecture

Abstract

Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.

Figure 1. Mediator and Cohesin Contribute to ES Cell State

a, Mediator and Cohesin components were highly represented in an shRNA screen for regulators of ES cell state. Complete results are listed in Supplementary Tables 1, 2. b, Knockdown of Mediator (Med12), Cohesin (Smc1a) or Nipbl caused reduced Oct4 protein levels and changes in ES cell colony morphology. Murine ES cells were infected with GFP control, Med12, Smc1a or Nipbl shRNAs, and stained for Oct4 and with Hoechst. Scale bar = 100μM. c, Mediator, Cohesin and Nipbl knockdowns all cause reduced expression of ES cell regulators and increased expression of developmental regulators. ES cells were infected with the indicated shRNA and gene expression levels relative to a control GFP infection were determined with microarrays. Log₂ fold expression changes were rank ordered from lowest to highest for all genes.

Figure 2. Genome-wide Occupancy of Mediator and Cohesin in ES cells

a, Binding profiles for ES cell transcription factors (Oct4, Nanog and Sox2), Mediator (Med1 and Med12), Cohesin (Smc1a, Smc3 and Nipbl), CTCF and components of the transcription apparatus (Pol2 and TBP) at the Oct4 (Pou5f1) and Nanog loci. ChIP-Seq data is shown in reads/million with the y-axis floor set to 0.5 reads/million. Oct4/Sox2, CTCF and TBP (TATA Box) sequence motifs are indicated. b, Venn diagram showing the overlap of high confidence (P-val <10⁻⁹) Cohesin (Smc1a) occupied sites with those bound by CTCF, Mediator (Med12) and Nipbl. c, Region map showing that Smc1a, Nipbl and Med12 co-occupied sites generally occur in close proximity to Pol2 and in the absence of CTCF. For each Smc1a occupied region, the occupancy of Med12, Nipbl, Pol2 and CTCF is indicated within a 10kb window centered on the Smc1a region. d, Heat map indicating that regions co-occupied by Smc1a, Med12 and Nipbl, which are associated with active genes, exhibit similar expression changes with knockdown of Smc1a, Med12 or Nipbl. Log₂ expression data was ordered based on the Smc1a knockdown data and is shown for all Smc1a, Med12 and Nipbl co-occupied regions that could be mapped to a gene, as described in supplementary information.

Figure 3. Mediator and Cohesin Interact

a, Mediator (Med23) is detected by western blot when crosslinked, sheared chromatin is subjected to immunoprecipitation with antibodies against Mediator (Med1 or Med12) or Cohesin (Smc1a or Smc3). b, Cohesin (Smc1a, Smc3) and Mediator (Med23) are detected by western blot following immunoprecipitation of uncrosslinked ES cell nuclear extracts with a Nipbl antibody. c, Cohesin (Smc3) and Nipbl co-purify with Mediator. The Input fractions and IP Eluate were examined by western blot and silver staining.

Figure 4. Mediator and Cohesin Binding Profiles Predict Enhancer-Promoter Looping Events

a-d, A looping event was detected between the upstream enhancer and the core promoter of Nanog, Phc1, Oct4(Pou5f1) and Lefty1 by Chromosome Conformation Capture (3C) in ES cells, but not in MEFs. ES cell and MEF crosslinked chromatin was digested by MspI or HaeIII and religated under conditions that favor intramolecular ligation events. The interaction frequency between the anchoring point and distal fragments was determined by PCR and normalized to BAC templates and control regions. Error bars represent the standard error of the average of 3 independent PCR reactions. The ChIP-Seq binding profiles for Med12, Smc1a and Nipbl are shown as in Fig. 2a. Restriction enzyme sites are indicated above the 3C graph. Biological replicates of the 3C experiments and the full 3C profile are presented in Supplementary Fig. 7.

Figure 5. Cell Type Specific Occupancy of Mediator and Cohesin

a, Region map of a 10kb window around Mediator and Cohesin co-occupied sites for ES cells (Smc1a and Med12) and MEFs (Smc1a and Med1) indicates that co-occupied regions are different between the cell types b, Region map of a 10kb window around Cohesin (Smc1a) and CTCF co-occupied sites indicates that many of these regions are co-occupied in ES cells and in MEFs. c, Western blot of ES and MEF cell extracts indicates that Cohesin protein levels are similar for both cell types, whereas Mediator protein levels are substantially lower in MEFs.