DNaseI HS determination by QCP. (A) Genomic structure of the Nanog locus. The intergenic cluster region is shown boxed. (B) Nanog regulatory elements are indicated with four peaks of HS located at the −5-kb region. A novel regulatory element is also seen at +9 kb. (C,D) QCP profiles for Dppa3 and GDF3 are shown. Note the large tandem HS peak in the GDF3 intron located −99 kb from the start of Nanog transcription. Arrows at the promoters indicate direction of transcription. Genomic positions relative to the Nanog start site are shown above each HS. Data were mapped to the March 2005 (mm6) genome assembly and are visualized within the University of California at Santa Cruz genome browser (Kent et al. 2002).

Oct4 ablation disrupts gene expression throughout the Nanog locus. ChIP was used to analyze chromatin occupancy within HSs and at selected genomic positions in wild-type or Oct4-depleted ES cells during the time course indicated after doxycycline induction. Wild-type ES cells are indicated in blue and Oct4-depleted cells are in red. (A,B) Chromatin occupancy of RNA polymerase II and the transcriptional cofactor p300 are shown throughout the Nanog locus. (C) Enrichment of histone H3K4me3 is indicated. Values of antibody-bound and IgG control ChIPs are normalized to an HPRT control.

Transcription factors involved in ES cell self-renewal bind HS throughout the Nanog locus. ChIP was used to analyze enrichment of nuclear factors on chromatin within the Nanog locus in wild-type ES cells. A schematic of the Nanog locus located on murine chromosome 6 is shown at the top, with boxed lines depicting the exon structure of the genes and noncoding sequences in green. HSs are indicated as solid boxes at the positions noted relative to Nanog transcriptional initiation. Light-red boxes indicate binding at the genomic positions shown above. Gray boxes mark HSs where chromatin occupancy was not observed. Fold enrichment of chromatin occupancy by biotagged transcription factors over the BirA background control are indicated above each bar, and all values were normalized to an HPRT control.

Long-range chromosomal contacts connect HSs throughout the Nanog locus. (A) A schematic of the Nanog locus, with HS indicated by light-red boxes. The region of chromosomal contact, relative to the Nanog start site, is shown below each graph. The red bar shows normalization against a nonchimeric amplification product at the specified region of analysis. HS up to 150 kb away make contact with the Nanog proximal promoter. (B,C) Chromosome conformation at the Nanog proximal promoter and −5-kb HS. Wild-type ES cells are indicated in blue and 72-h Oct4-depleted cells are in red. Higher-order structure is lost throughout the locus following Oct4 depletion. (D) A two-dimensional rendering of chromatin looping in three-dimensional space between distant regulatory elements (red ovals) and Nanog locus genes (green and black rectangles indicate active and repressed states, respectively). The model depicts DNA-bound factors within the proximal promoters of GDF3, Dppa3, and Nanog initiating contact (green arrows) with an active transcriptional node formed by RNA polymerase II (large purple oval); accessory DNA-binding or bridging transcription factors p300, ZFP281, Nac1, and CTCF (smaller purple ovals); and essential DNA-bound transcription factors Oct4 and Nanog (green ovals).