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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.


Related in: MedlinePlus

Comparison of nucleosome occupancy in mouse pluripotent and somatic cells(a) Nucleosome occupancy around transcription start and end sites computed for mouse ESCs, iPSCs and somatic tail tip fibroblasts (TTFs). We note that after normalizing the occupancy for the total number of tags in each library the profiles remain different, even between replicates of the same cell type. (b) The same profiles after normalization of the GC-content distribution in each sample with the target mean GC content of 50% (see Methods for more detail). (c) Comparison of the GC-normalized profiles for all genes and genes stratified by their expression status. (d). Boxplot showing nucleosome density distributions in TSS-proximal regions (+/−2 Kb) stratified by the enrichment in H3K4me3 and K3K27me3 marks in ESCs. Notches at boxes provide reference to 95% confidence intervals. (e) Normalized nucleosome occupancy signal around scaled ESC enhancer regions computed for replicate sets in three cell types. (f) Comparison of gene expression and nucleosome occupancy changes. The two left bars show the expression changes computed for genes assigned to enhancers that have either lower (LND, pink) or higher (HND, purple) nucleosome occupancy in ESCs as compared to somatic TTFs; the two right bars depict the same for genes where nucleosome occupancy loss or gain occurs in the TSS proximal regions. The 95% confidence intervals are shown with vertical arrows. (g) Comparison of the different chromatin properties (measured in ESCs40) for the LND and HND enhancers. As in (D) notches provide 95% confidence intervals.
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Figure 1: Comparison of nucleosome occupancy in mouse pluripotent and somatic cells(a) Nucleosome occupancy around transcription start and end sites computed for mouse ESCs, iPSCs and somatic tail tip fibroblasts (TTFs). We note that after normalizing the occupancy for the total number of tags in each library the profiles remain different, even between replicates of the same cell type. (b) The same profiles after normalization of the GC-content distribution in each sample with the target mean GC content of 50% (see Methods for more detail). (c) Comparison of the GC-normalized profiles for all genes and genes stratified by their expression status. (d). Boxplot showing nucleosome density distributions in TSS-proximal regions (+/−2 Kb) stratified by the enrichment in H3K4me3 and K3K27me3 marks in ESCs. Notches at boxes provide reference to 95% confidence intervals. (e) Normalized nucleosome occupancy signal around scaled ESC enhancer regions computed for replicate sets in three cell types. (f) Comparison of gene expression and nucleosome occupancy changes. The two left bars show the expression changes computed for genes assigned to enhancers that have either lower (LND, pink) or higher (HND, purple) nucleosome occupancy in ESCs as compared to somatic TTFs; the two right bars depict the same for genes where nucleosome occupancy loss or gain occurs in the TSS proximal regions. The 95% confidence intervals are shown with vertical arrows. (g) Comparison of the different chromatin properties (measured in ESCs40) for the LND and HND enhancers. As in (D) notches provide 95% confidence intervals.

Mentions: We first assessed the average nucleosome occupancy patterns at the TSSs for each cell type. As demonstrated previously16,17,19,26, a nucleosome depleted region (NDR) flanked by well-positioned +1 and −1 nucleosomes (relative to the TSS) is a characteristic feature of the occupancy profiles averaged across all genes (Figure 1). Indeed, we detected such a pattern across all samples (Figure 1A). However, we also observed high variability in the magnitude of the nucleosome occupancy for ESCs and iPSCs, which show nearly identical gene expression patterns in both the mouse and human data (Figure 1A and Supplementary Figure 1,4). Furthermore, such variation was observed even for biological replicates of the same cell type. This variability is not specific to our experimental protocol, as previous studies in mammalian genomes reported substantially different nucleosomal patterns at TSSs, ranging from an accumulation in tag counts greater than the surrounding regions to an apparent depletion in occupancy16-19,22,32. Thus, it likely originates from technical differences in experimental procedures, such as the extent of MNase digestion or chromatin isolation. This variability hinders direct comparisons of the nucleosome occupancy between cell types.


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)

Comparison of nucleosome occupancy in mouse pluripotent and somatic cells(a) Nucleosome occupancy around transcription start and end sites computed for mouse ESCs, iPSCs and somatic tail tip fibroblasts (TTFs). We note that after normalizing the occupancy for the total number of tags in each library the profiles remain different, even between replicates of the same cell type. (b) The same profiles after normalization of the GC-content distribution in each sample with the target mean GC content of 50% (see Methods for more detail). (c) Comparison of the GC-normalized profiles for all genes and genes stratified by their expression status. (d). Boxplot showing nucleosome density distributions in TSS-proximal regions (+/−2 Kb) stratified by the enrichment in H3K4me3 and K3K27me3 marks in ESCs. Notches at boxes provide reference to 95% confidence intervals. (e) Normalized nucleosome occupancy signal around scaled ESC enhancer regions computed for replicate sets in three cell types. (f) Comparison of gene expression and nucleosome occupancy changes. The two left bars show the expression changes computed for genes assigned to enhancers that have either lower (LND, pink) or higher (HND, purple) nucleosome occupancy in ESCs as compared to somatic TTFs; the two right bars depict the same for genes where nucleosome occupancy loss or gain occurs in the TSS proximal regions. The 95% confidence intervals are shown with vertical arrows. (g) Comparison of the different chromatin properties (measured in ESCs40) for the LND and HND enhancers. As in (D) notches provide 95% confidence intervals.
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Figure 1: Comparison of nucleosome occupancy in mouse pluripotent and somatic cells(a) Nucleosome occupancy around transcription start and end sites computed for mouse ESCs, iPSCs and somatic tail tip fibroblasts (TTFs). We note that after normalizing the occupancy for the total number of tags in each library the profiles remain different, even between replicates of the same cell type. (b) The same profiles after normalization of the GC-content distribution in each sample with the target mean GC content of 50% (see Methods for more detail). (c) Comparison of the GC-normalized profiles for all genes and genes stratified by their expression status. (d). Boxplot showing nucleosome density distributions in TSS-proximal regions (+/−2 Kb) stratified by the enrichment in H3K4me3 and K3K27me3 marks in ESCs. Notches at boxes provide reference to 95% confidence intervals. (e) Normalized nucleosome occupancy signal around scaled ESC enhancer regions computed for replicate sets in three cell types. (f) Comparison of gene expression and nucleosome occupancy changes. The two left bars show the expression changes computed for genes assigned to enhancers that have either lower (LND, pink) or higher (HND, purple) nucleosome occupancy in ESCs as compared to somatic TTFs; the two right bars depict the same for genes where nucleosome occupancy loss or gain occurs in the TSS proximal regions. The 95% confidence intervals are shown with vertical arrows. (g) Comparison of the different chromatin properties (measured in ESCs40) for the LND and HND enhancers. As in (D) notches provide 95% confidence intervals.
Mentions: We first assessed the average nucleosome occupancy patterns at the TSSs for each cell type. As demonstrated previously16,17,19,26, a nucleosome depleted region (NDR) flanked by well-positioned +1 and −1 nucleosomes (relative to the TSS) is a characteristic feature of the occupancy profiles averaged across all genes (Figure 1). Indeed, we detected such a pattern across all samples (Figure 1A). However, we also observed high variability in the magnitude of the nucleosome occupancy for ESCs and iPSCs, which show nearly identical gene expression patterns in both the mouse and human data (Figure 1A and Supplementary Figure 1,4). Furthermore, such variation was observed even for biological replicates of the same cell type. This variability is not specific to our experimental protocol, as previous studies in mammalian genomes reported substantially different nucleosomal patterns at TSSs, ranging from an accumulation in tag counts greater than the surrounding regions to an apparent depletion in occupancy16-19,22,32. Thus, it likely originates from technical differences in experimental procedures, such as the extent of MNase digestion or chromatin isolation. This variability hinders direct comparisons of the nucleosome occupancy between cell types.

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