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, 24 (8), 1285-95

Nucleosome Repositioning Links DNA (De)methylation and Differential CTCF Binding During Stem Cell Development

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Nucleosome Repositioning Links DNA (De)methylation and Differential CTCF Binding During Stem Cell Development

Vladimir B Teif et al. Genome Res.

Abstract

During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type-specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning, and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we found that the interplay between these factors depends on their genomic context. The mostly unmethylated CpG islands have reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated, and the average methylation density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands, binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the TET1 and 5hmC levels go down. This process regulates a class of CTCF binding sites outside CpG islands that are occupied by CTCF in ESCs but lose the protein during differentiation. We rationalize this cell type-dependent targeting of CTCF with a quantitative biophysical model of competitive binding with the histone octamer, depending on the TET1, 5hmC, and 5mC state.

Figures

Figure 1.
Figure 1.
DNA methylation patterns relative to nucleosomes. (A) Average CpG density of methylated (>50% 5mC) and unmethylated (<10% 5mC) mononucleosome sequences in ESCs for the complete genome. Most of the sequences reside outside of CGIs because the latter represent only a fraction of ∼1% of the total pool of sequences mapped by MNase-seq. (Black line) DNA methylation along the nucleosomes obtained at low MNase digestion; (blue line) high MNase digestion. The nucleosome dyad is the middle of the nucleosomal DNA fragment. (B) Same as in panel A but for the dinucleosome fraction. The linker is defined as the middle of the dinucleosome. (C) CpG density for mononucleosomes and associated linker DNA inside CGIs. Putative CGIs were taken from the coordinates of 125,303 CpG clusters defined by the proximity of neighboring CpGs with the CpGcluster2 algorithm (Hackenberg et al. 2006). (D) Same as in panel C but for the dinucleosome fraction.
Figure 2.
Figure 2.
CTCF binding in ESCs can be explained solely by the DNA sequence. (A) Average nucleosome occupancy profiles for genomic regions with different levels of DNA methylation. For fully methylated regions (FMR), the occupancy remains flat as compared to genome-average levels. Low methylated regions (LMR) and unmethylated regions (UMR) were nucleosome-depleted by ∼30% and ∼60%, respectively. (B) Average CTCF enrichment calculated from ChIP-seq data for the three different classes of 5mC density. (C) Average CTCF enrichment predicted for the same regions based on the DNA sequence preferences given by the TRANSFAC PWM, without taking into account nucleosomes and DNA methylation. (D) Receiver operator curves calculated for the TFnuc model, taking into account only the CTCF weight matrix without nucleosomes and DNA methylation (black). In addition to PWM, competition with nucleosomes (red) or DNA methylation (blue) was considered. The area under the curve (AUC) reflects both the sensitivity and specificity and thus determines the goodness of the model.
Figure 3.
Figure 3.
Distribution of 5mC and 5hmC around CTCF sites in ESCs, NPCs, and MEFs. (A) 5mC density calculated around constitutive (“ESC and MEF”), variable (“ESC not MEF”), and weak (“MEF not ESC”) CTCF sites in ESCs from the published CTCF ChIP-seq data (Shen et al. 2012). (B) Same as panel A but for the hydroxymethylation modification at CpGs. (C) 5mC density in ESCs at “ESC and NPC,” “ESC not NPC,” and “NPC not ESC” CTCF sites calculated with CTCF ChIP-seq data in ESCs (Shen et al. 2012) and NPCs (Phillips-Cremins et al. 2013). (D) Same as panel C but for 5mC density in NPCs.
Figure 4.
Figure 4.
TET1 binding outside CGIs is linked with labile MNase-sensitive nucleosomes. (A) Average TET1 enrichment calculated from ChIP-seq data in ESCs (Williams et al. 2011) around CTCF binding sites (Shen et al. 2012). (B) Enrichment of 5hmC calculated from hMeDIP data in ESCs and NPCs (Tan et al. 2013) around CTCF binding sites (Shen et al. 2012; Phillips-Cremins et al. 2013). (C) Average nucleosome occupancy profiles around 5(h)mC in the absence of TET1 within CGIs for low (black line), medium (red line), and high (blue line) MNase digestion in ESCs. Note that bisulfite sequencing does not distinguish between 5mC and 5hmC. (D) Same as panel C but for 5(h)mC regions enriched with TET1. (E) Nucleosome occupancy at 5(h)mC without TET1 outside of CGIs. Same color-coding as in panel C. (F) Same as panel E but for 5(h)mC regions with TET1. (G) Same as in panel F but only for (hydroxy)methylated CpGs that were within a 500 bp distance of bound CTCF. (H) Nucleosome occupancy around CTCF sites and occupied by CTCF in ESCs but not in MEFs. Same color-coding as in panel C.
Figure 5.
Figure 5.
Nucleosome occupancy around TET1 binding sites and in relation to 5hmC levels. (A) Average nucleosome occupancy (top panel) and k-means cluster plots showing nucleosome occupancy around each of 92,888 TET1 ChIP-seq peaks in ESCs (Yu et al. 2012) at low MNase digestion (bottom panel). (B) Same as panel A but for high MNase digestion. (C) Aggregate plot of nucleosome occupancy around 5hmC sites in ESCs (Yu et al. 2012) grouped according to their 5hmC levels as >25% 5hmC (black line), >50% 5hmC (red line), and > 90% 5hmC (blue line). Upon increasing the 5hmC level, the nucleosome density changed from slight enrichment to nucleosome depletion, which corresponds to the nucleosome removal at a subset of these sites. (D) Changes of the nucleosome occupancy during cell development (ESCs, NPCs, and MEFs) around hydroxymethylated sites in ESCs (>50% 5hmC).
Figure 6.
Figure 6.
Quantitative model to predict CTCF occupancy changes due to competition with nucleosomes. (A) Experimentally observed occupancy of CTCF binding sites in ESCs and MEFs derived from ChIP-seq peak heights (Shen et al. 2012). Three subsets of CTCF sites can be distinguished: constitutive sites bound in both cell types (ESC and MEF), variable sites predominantly bound by CTCF in ESCs (ESC not MEF), and weak sites which are slightly more strongly bound in differentiated cells (MEF not ESC). (B) Predicted CTCF binding site occupancy in ESCs and MEFs when accounting for nucleosome positioning for the three binding site classes shown in panel A. (C) Conservation score of CTCF DNA binding sequence motifs for constitutive, variable, and weak binding sites. (D) Average CpG density at CTCF sites. The variable sites in the “ESC not MEF” class are located preferentially outside of CGIs.
Figure 7.
Figure 7.
Model rationalizing the linkages between 5mC, 5hmC, TET1-, and CTCF-binding with nucleosome positioning inside and outside of CpG islands. (A) Inside CpG islands, most CpGs are unmethylated and have low nucleosome occupancy. The small fraction of CpGs in CGIs that are methylated has a nucleosome positioned preferably within the DNA methylation sites. CTCF binding inside CGIs is mostly invariant and determined by the DNA sequence: Strong constitutive CTCF sites stay unmethylated and bound by CTCF and its cobinders during the cell development, while weak sites in these regions are mostly not bound by CTCF in both ESCs and differentiated cells. (B) Outside of CGIs, the genomic DNA is mostly methylated at CpGs. In relation to nucleosome positioning, the following features were found: DNA methylation density is lowest in the middle of the nucleosome, smoothly increases toward the nucleosome entry/exit, and reaches a maximum between nucleosomes. In these regions, TET1 binding creates MNase-sensitive labile nucleosomes, which are being removed/translocated during the process of 5mC to 5hmC conversion. Variable CTCF sites are found preferentially outside CGIs, where active, TET1-dependent hydroxymethylation and associated nucleosome repositioning promotes CTCF binding. As CpGs in these regions change to a methylated state during stem cell differentiation, the formation of stably bound nucleosomes leads to a loss of CTCF at these sites.

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