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Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

West JA, Cook A, Alver BH, Stadtfeld M, Deaton AM, Hochedlinger K, Park PJ, Tolstorukov MY, Kingston RE - Nat Commun (2014)

Bottom Line: RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation.Most changes are reset during reprogramming.We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA [3] [4].

ABSTRACT
Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

No MeSH data available.


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Distribution of the regions of difference (RoDs) detected in nucleosome occupancy profiles relative to annotated regions in the mouse genome(a) Chromosome wide snapshot of the normalized nucleosome occupancy and RoD occurrence. (b) Nucleosome occupancy at one of the super-enhancers identified in Whyte et al.40 shown as an example of multiple RoDs present in this class of enhancers. (c) The RoD frequencies in the regions encompassing TSSs and enhancers identified in ESCs40. The 95% confidence intervals are shown with the vertical arrows. The confidence intervals were estimated based on the variability of the frequency values in individual profiles used for averaging.
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Figure 4: Distribution of the regions of difference (RoDs) detected in nucleosome occupancy profiles relative to annotated regions in the mouse genome(a) Chromosome wide snapshot of the normalized nucleosome occupancy and RoD occurrence. (b) Nucleosome occupancy at one of the super-enhancers identified in Whyte et al.40 shown as an example of multiple RoDs present in this class of enhancers. (c) The RoD frequencies in the regions encompassing TSSs and enhancers identified in ESCs40. The 95% confidence intervals are shown with the vertical arrows. The confidence intervals were estimated based on the variability of the frequency values in individual profiles used for averaging.

Mentions: Our analysis showed that approximately 40% of RoDs are at gene regions annotated in the mouse genome (Figure 3B-D, see also Figure 4A, Supplementary Figure 8B), which is significantly more than expected for a randomized distribution (P = 10−12, see Methods for details on significance estimation). Around genes, TSS-proximal regions are specifically enriched in RoDs (Figure 4C, blue line), including the promoters of genes associated with pluripotency and transcription activation (as exemplified by Oct4 in Figure 2B). Indeed, in pluripotent versus somatic cell comparisons, between 7 to 16% of RoDs occur at TSSs, and these are enriched 2.4 to 5 fold over the genome average (Figure 3B,C). In addition to genes and their promoters, pluripotency-associated enhancers exhibited significant enrichment in RoDs (Figure 4C, orange line, and Supplementary Figure 8C). To our surprise, enhancers demonstrated differences with the same or greater magnitude as TSSs. In pluripotent versus somatic cell comparisons, between 5 to 7.4% of RoDs occurred at ESC-defined enhancers, which corresponds to a 10 to 15 fold enrichment over the genome-wide occurrence of these enhancers (Figure 3B,C). ‘Super-enhancers’ – large enhancer regions associated with a high density of regulatory protein binding40 –showed even stronger enrichment in RoDs (Figure 4C, red line). As an additional validation of this result, we identified RoDs between ESCs and another somatic cell type, mouse liver. This set of RoDs was also skewed towards LND enhancers in ESCs and showed enrichment at TSSs and ESC enhancers (Supplementary Figure 14), confirming that these effects are not specific to the somatic cell type to which ESCs are compared.


Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

West JA, Cook A, Alver BH, Stadtfeld M, Deaton AM, Hochedlinger K, Park PJ, Tolstorukov MY, Kingston RE - Nat Commun (2014)

Distribution of the regions of difference (RoDs) detected in nucleosome occupancy profiles relative to annotated regions in the mouse genome(a) Chromosome wide snapshot of the normalized nucleosome occupancy and RoD occurrence. (b) Nucleosome occupancy at one of the super-enhancers identified in Whyte et al.40 shown as an example of multiple RoDs present in this class of enhancers. (c) The RoD frequencies in the regions encompassing TSSs and enhancers identified in ESCs40. The 95% confidence intervals are shown with the vertical arrows. The confidence intervals were estimated based on the variability of the frequency values in individual profiles used for averaging.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4217530&req=5

Figure 4: Distribution of the regions of difference (RoDs) detected in nucleosome occupancy profiles relative to annotated regions in the mouse genome(a) Chromosome wide snapshot of the normalized nucleosome occupancy and RoD occurrence. (b) Nucleosome occupancy at one of the super-enhancers identified in Whyte et al.40 shown as an example of multiple RoDs present in this class of enhancers. (c) The RoD frequencies in the regions encompassing TSSs and enhancers identified in ESCs40. The 95% confidence intervals are shown with the vertical arrows. The confidence intervals were estimated based on the variability of the frequency values in individual profiles used for averaging.
Mentions: Our analysis showed that approximately 40% of RoDs are at gene regions annotated in the mouse genome (Figure 3B-D, see also Figure 4A, Supplementary Figure 8B), which is significantly more than expected for a randomized distribution (P = 10−12, see Methods for details on significance estimation). Around genes, TSS-proximal regions are specifically enriched in RoDs (Figure 4C, blue line), including the promoters of genes associated with pluripotency and transcription activation (as exemplified by Oct4 in Figure 2B). Indeed, in pluripotent versus somatic cell comparisons, between 7 to 16% of RoDs occur at TSSs, and these are enriched 2.4 to 5 fold over the genome average (Figure 3B,C). In addition to genes and their promoters, pluripotency-associated enhancers exhibited significant enrichment in RoDs (Figure 4C, orange line, and Supplementary Figure 8C). To our surprise, enhancers demonstrated differences with the same or greater magnitude as TSSs. In pluripotent versus somatic cell comparisons, between 5 to 7.4% of RoDs occurred at ESC-defined enhancers, which corresponds to a 10 to 15 fold enrichment over the genome-wide occurrence of these enhancers (Figure 3B,C). ‘Super-enhancers’ – large enhancer regions associated with a high density of regulatory protein binding40 –showed even stronger enrichment in RoDs (Figure 4C, red line). As an additional validation of this result, we identified RoDs between ESCs and another somatic cell type, mouse liver. This set of RoDs was also skewed towards LND enhancers in ESCs and showed enrichment at TSSs and ESC enhancers (Supplementary Figure 14), confirming that these effects are not specific to the somatic cell type to which ESCs are compared.

Bottom Line: RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation.Most changes are reset during reprogramming.We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA [3] [4].

ABSTRACT
Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

No MeSH data available.


Related in: MedlinePlus