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. 2011 Dec 9;147(6):1283-94.
doi: 10.1016/j.cell.2011.10.040.

Polycomb-repressed genes have permissive enhancers that initiate reprogramming

Phillippa C Taberlay  1 , Theresa K Kelly, Chun-Chi Liu, Jueng Soo You, Daniel D De Carvalho, Tina B Miranda, Xianghong J Zhou, Gangning Liang, Peter A Jones
Affiliations

Polycomb-repressed genes have permissive enhancers that initiate reprogramming

Phillippa C Taberlay et al. Cell. .

Abstract

Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome-depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer, leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer but has a different effect on the cognate promoter, where the monovalent H3K27me3 marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting that they may provide a widespread avenue for the initiation of cell-fate reprogramming.

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Figures

Figure 1
Figure 1. MYOD1 exhibits a multivalent epigenotype when repressed by PRC
(A) RNA was isolated, reverse transcribed and analyzed by qPCR using primers detecting MYOD1. Data is expressed as copies of MYOD1 relative to GAPDH expression (green bars). Average DNA methylation (%) was determined by bisulfite sequencing and is expressed as the average of 3 CpG dinucleotides across the enhancer (black bars) and 33 CpG dinucleotides across the promoter (hatched bars). (B–G) ChIP assays were performed with antibodies detecting (B) H3K4me3, (C) RNA PolII-P, (D) EZH2, (E) H3K27me3, (F) H2A.Z and (G) H3K4me1 on chromatin from RD (green bars) and LD419 (red bars) cells. Data are presented as percent total input. Bars, mean ± SEM of 3 biological experiments.
Figure 2
Figure 2. Nucleosome depleted regions characterize permissive enhancers
Nuclei were extracted from (A) RD and (B) LD419 cells and treated with M.CviPI GpC methyltransferase and subjected to bisulfite conversion and cloning. Horizontal lines represent individual MYOD1 enhancers (left) or promoters (right). Circles represent GpC dinucleotides (white, unmethylated and inaccessible to M.CviPI; teal, methylated and accessible to M.CviPI). Pink bars are ≥146bp, representing sites associated with nucleosomes. Regions accessible to M.CviPI (teal) indicate NDRs. Arrow denotes the TSS. Diagram is drawn to scale.
Figure 3
Figure 3. The enhancer NDR underlies MYOD1 transcriptional competence
(A–C) ChIP assays were performed with an anti-TAP antibody on chromatin from LD419 cells transfected with Myod1-TAP for (A) 6hr, (B) 24hr or (C) 48hr. Data are presented as percent total input. (D) Nuclei were extracted from LD419 cells transfected with Myod1-TAP for 48hr then treated with M.CviPI as described previously (see Figure 2). (E) ChIP assays were performed with an anti-TAP antibody on chromatin from LD419 (black bars) or RKO (grey bars) cells transfected with Myod1-TAP for 48hr. The data are presented as percent total input. (F) Nuclei were extracted from RKO cells transfected with Myod1-TAP for 48hr then treated with M.CviPI as described for LD419 cells. See also Figure S1. Bars, mean ± SEM of 3 biological experiments.
Figure 4
Figure 4. MYOD1 gains a bivalent state after OCT4 initiates reprogramming through the enhancer
(A–D) ChIP assays were performed with (A) anti-OCT4 (black), (B) anti-H3K4me3 (green), (C) anti-H3K27me3 (red) and (D) anti-H3K4me1 (grey) antibodies on chromatin from LD419 cells transfected with OCT4 for 0hr (mock), 24hr or 72hr (left, center and right panels, respectively). Data are presented as percent total input. Bars, mean ± SEM of 3 biological experiments. See also Figure S2.
Figure 5
Figure 5. Enhancer/promoter interactions underlie reprogramming events
ChIP assays were performed with an anti-RAD21 antibody on untransfected LD419 cells (0hr, black bars) and LD419 cells transfected with Myod1-TAP for 24hr (white bars) or 48hr (red bars), as indicated. The data are presented as percent total input. Bars, mean ± SEM of 3 biological experiments. See also Figure S3.
Figure 6
Figure 6. H3K4me1 marks enhancers of transcriptionally active, as well as PRC repressed genes
For each cell line, genes with the histone modifications (A) H3K4me3 or (B) H3K27me3 at promoters (defined as ±500 bases around the TSS) were identified, and the percentage of those promoters with putative H3K4me1 enhancer regions (defined to be the −2kB to −12kB window located upstream of the TSS) were calculated (green/red bars, left y-axes). Similarly, DNase hypersensitivity (black bars, right y-axes) was examined and are expressed as a percentage of those promoters (A) H3K4me3 or (B) H3K27me3 with H3K4me1 marked enhancers that exhibit DNase hypersensitivity (DHS). kB, kilobase; arrow, TSS. See also Figure S4.
Figure 7
Figure 7. Polycomb repressed promoters maintain regulatory flexibility through epigenetic crosstalk with their permissive enhancers
A model shows a permissive enhancer is highly comparable to the active enhancer, exemplified by the NDR and H3K4me1 despite the presence of promoter nucleosomes carrying H3K27me3. Reprogramming can be orchestrated from this permissive state by master transcription factors such as Myod1 or OCT4 that access their target through the enhancer. Myod1 over-expression activates a self-regulatory mechanism by binding first to its own enhancer, resulting in recruitment of RAD21 (the cohesin complex), consistent with enhancer/promoter interactions. Promoter changes follow, such as nucleosome eviction and transcription factor occupancy near the TSS. Enhancer-mediated reprogramming is also observed after over-expression of OCT4, resulting in the generation of a bivalent promoter state. Together, these data demonstrate that reprogramming can be forced in multiple directions yet is initiated from the enhancer. NDR, nucleosome depleted region; small white circle, unmethylated CpG site; small black circle, methylated CpG site; large circle, nucleosome; 4me1 (dark green), H3K4me1; 4me3 (light green), H3K4me3; 27me3 (red), H3K27me3; AcH3, acetylated H3; X, inactive TSS.
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