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• W2942734585 abstract "Scientific Report29 April 2019Open Access Transparent process Nucleosome dynamics of human iPSC during neural differentiation Janet C Harwood Janet C Harwood orcid.org/0000-0002-3225-0069 MRC Centre for Neuropsychiatric Genetics & Genomics, Cardiff University, Cardiff, UK Search for more papers by this author Nicholas A Kent Nicholas A Kent orcid.org/0000-0002-4114-1307 School of Biosciences, Cardiff University, Cardiff, UK Search for more papers by this author Nicholas D Allen Nicholas D Allen orcid.org/0000-0003-4009-186X School of Biosciences, Cardiff University, Cardiff, UK Search for more papers by this author Adrian J Harwood Corresponding Author Adrian J Harwood [email protected] orcid.org/0000-0003-3124-5169 School of Biosciences, Cardiff University, Cardiff, UK Neuroscience and Mental Health Research Institute (NMHRI), Cardiff University, Cardiff, UK Search for more papers by this author Janet C Harwood Janet C Harwood orcid.org/0000-0002-3225-0069 MRC Centre for Neuropsychiatric Genetics & Genomics, Cardiff University, Cardiff, UK Search for more papers by this author Nicholas A Kent Nicholas A Kent orcid.org/0000-0002-4114-1307 School of Biosciences, Cardiff University, Cardiff, UK Search for more papers by this author Nicholas D Allen Nicholas D Allen orcid.org/0000-0003-4009-186X School of Biosciences, Cardiff University, Cardiff, UK Search for more papers by this author Adrian J Harwood Corresponding Author Adrian J Harwood [email protected] orcid.org/0000-0003-3124-5169 School of Biosciences, Cardiff University, Cardiff, UK Neuroscience and Mental Health Research Institute (NMHRI), Cardiff University, Cardiff, UK Search for more papers by this author Author Information Janet C Harwood1, Nicholas A Kent2, Nicholas D Allen2 and Adrian J Harwood *,2,3 1MRC Centre for Neuropsychiatric Genetics & Genomics, Cardiff University, Cardiff, UK 2School of Biosciences, Cardiff University, Cardiff, UK 3Neuroscience and Mental Health Research Institute (NMHRI), Cardiff University, Cardiff, UK *Corresponding author. Tel: +44 2920688492; E-mail: [email protected] EMBO Reports (2019)20:e46960https://doi.org/10.15252/embr.201846960 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nucleosome positioning is important for neurodevelopment, and genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. To investigate changes in nucleosome positioning during neural differentiation, we generate genome-wide nucleosome maps from an undifferentiated human-induced pluripotent stem cell (hiPSC) line and after its differentiation to the neural progenitor cell (NPC) stage. We find that nearly 3% of nucleosomes are highly positioned in NPC, but significantly, there are eightfold fewer positioned nucleosomes in pluripotent cells, indicating increased positioning during cell differentiation. Positioned nucleosomes do not strongly correlate with active chromatin marks or gene transcription. Unexpectedly, we find a small population of nucleosomes that occupy similar positions in pluripotent and neural progenitor cells and are found at binding sites of the key gene regulators NRSF/REST and CTCF. Remarkably, the presence of these nucleosomes appears to be independent of the associated regulatory complexes. Together, these results present a scenario in human cells, where positioned nucleosomes are sparse and dynamic, but may act to alter gene expression at a distance via the structural conformation at sites of chromatin regulation. Synopsis Repositioning of nucleosomes occurs as human iPSC develop into neural progenitor cells, but a small number of nucleosome arrays remain in place at regulatory sites that control long-range chromatin organisation. An 8-fold increase and re-distribution of positioned nucleosomes occurs during human iPSC neural differentiation. No correlation is observed between positioned nucleosomes and gene activity at the genome-wide level. Positioned nucleosomes are retained at NRSF/REST and CTCF binding sites during cell differentiation, and in the absence of bound regulatory complexes. Introduction Placement of nucleosomes has been implicated in gene regulation from Saccharomyces cerevisiae 1 to human cells 2, and mutations in chromatin remodelling enzymes that control nucleosome positioning have a demonstrable role in gene regulation. Chromatin remodelers of the SNF2 protein family have strong associations with human disease, including mental health and cancer biology 3, 4. The SNF2 protein Brg-1 is associated with a range of psychiatric disorders and intellectual disabilities (ID), such as Coffin–Siris syndrome 5. More strikingly, the chromodomain-helicase-DNA binding (CHD) sub-family proteins are intimately associated with neuropsychiatric or neurodevelopmental disorders 3. CHD2 and CHD4 increase the risk of epileptic encephalopathies, and CHD2, along with CHD5 and CHD6, confer risk for ID. CHD7 is causative of CHARGE syndrome 6, 7, a multi-system, developmental disorder, but also imparts genetic risk for autism spectrum disorder (ASD) as does CHD1, CHD2 and CHD3. Finally, CHD8 has a strong association with both ASD and schizophrenia 8, 9. Ultimately to understand why aberrant chromatin remodelling is important for disease risk, we need to establish the underlying principles that determine the relationship between nucleosome positioning and gene regulation. Nucleosome interactions within chromatin are likely to be complex but can be described by the following quantitative parameters: position, the sequence coordinates where nucleosomes occur across the cell population; occupancy, the frequency that a nucleosome is present at an individual site; and accessibility, the degree to which the presence of nucleosomes restricts access to the DNA. In organisms with small genomes, such as in the yeasts and Dictyostelium, nearly 80% of nucleosomes are positioned in the same place in the genome across the cell population, forming arrays that span gene bodies 10-13. There are distinct regions of low occupancy and increased accessibility, for example in the nucleosome-free region (NFR) found at the transcriptional start site (TSS) of genes. Finally, there is evidence for mutual exclusivity between a positioned nucleosome and transcription factor (TF) binding at the same site 14. Nucleosomes at active genes appear to be more positionally organised than at inactive genes and changes in nucleosome positioning, such as the specific increase in nucleosome spacing seen in a Dictyostelium mutant lacking the CHD8 homologue, ChdC, alter gene expression. Nonetheless, even in these organisms the actual correlation between positioning and gene expression can be low; for example, only 15% of genes with altered nucleosome spacing in a chdC mutant show corresponding changes in gene expression 12. In mammalian cells, including human cells, with large and complex genomes, non-coding and intragenic DNA sequences can make up approximately 98% of the genome. In these larger and more complex genomes, the pattern of positioned nucleosomes appears to be radically different. Studies in human cells have shown that only a very small proportion of the total nucleosome number are strongly positioned 15, 16. Although the nucleosome patterns flanking TSSs can conform to those of non-metazoan organisms, arrays within gene bodies have not been widely reported. Studies also suggest that occupancy and accessibility may be uncoupled. Mapping DNA accessibility in chromatin by its sensitivity to DNase digestion can reveal hypersensitive sites, which are often found at gene regulatory elements distant from the gene body. Other regions, although not hypersensitive, show increased sensitivity to MNase digestion; hence, accessibility of proteins is higher in these regions of DNA. These MNase-sensitive regions coincide with regions of open chromatin as defined by ATAC-seq, certain chromatin marks and transcriptional activity 17. Interestingly, such MNase accessibility maps (MACC) show that increased accessibility can occur without changes of nucleosome occupancy 18. Here, we focus on the relationship between gene expression and nucleosome positioning, rather than accessibility, in the context of human neural differentiation. Unlike the relationship of MACC to open chromatin and gene expression, the relationship of these positioned nucleosomes to gene expression is unclear. By comparing maps of well-positioned nucleosomes with low MNase sensitivity for the same human iPSC line in the pluripotent state and following differentiation to neural progenitor cells (NPCs), we have directly examined the developmental dynamics of nucleosome positioning during early neural differentiation and its relationship to gene expression. Results and Discussion Positional mapping of nucleosomes during human neural differentiation To generate nucleosome maps based on positioning, we used a modified MNase-seq methodology, where chromatin is rapidly digested with MNase in situ in permeabilised cells without cross-linking 11. Bulk chromatin of pluripotent iPSC, and following their differentiation to NPCs, was digested with MNase to an equivalent degree, giving near identical levels of mono-nucleosome fragments (Fig EV1A). Size-fractionated, MNase-protected DNA digestion fragments were paired-end-sequenced to generate between 2.25 and 2.5 × 109 paired-end reads per digest, and the positions of the fragment mid-points were mapped to the genome. To monitor the initial digestions, the abundance of fragment sizes was plotted for both cell types (Fig EV1B), and directly compared in the size classes that span the range of fragment sizes seen for human nucleosomes (Fig EV1C). This confirmed comparable degrees of digestion between the two cell states in the mono-nucleosome size range, but with a slight bias towards larger fragments in the pluripotent cell samples. Click here to expand this figure. Figure EV1. Size distribution of MNase-digested fragments Agarose gel (1%) images showing matched MNase digests from bulk human chromatin from pl-iPSC (left panel) and NPC (right panel), and the subsequent size-selected DNA fragment range used for paired-end sequencing. Gels were scanned and the mono-nucleosome peak quantified as a fraction of the total chromatin DNA. MNase digestion released near equivalent amounts of mono-nucleosome DNA, pl-iPSC (3% of total DNA) and NPC (2.4% of total DNA). This indicates approximately equal numbers of nucleosomes in both cell types, but we subsequently show that only a small fraction of these are positioned within the chromatin. The size distribution of fragments calculated from the pl-iPSC and NPC paired-end sequencing (from a total of 3.4 and 3.0 billion paired-end reads for pl-iPSC and NPC, respectively). NPC samples had a slightly smaller fragment size distribution, with no evidence for loss of 139–161 fragments (corresponding to core nucleosomes) from pl-iPSC samples. Histogram to compare the key size ranges of 112–137 bp (sub-nucleosome), 138–161 bp (core nucleosome) and 162–188 bp (large nucleosome footprint) for pl-iPSC and NPC samples (delineated by grey lines in B). Download figure Download PowerPoint To map nucleosome positions in the genome, we plotted the sequence read mid-point frequency distribution of fragments in the 138–161 bp range, corresponding to the nucleosome footprint size (Fig 1A). We developed a heuristic, peak-finding algorithm (PeakFinder) to identify highly positioned nucleosomes, based on peak shape and read depth. This identified more than 400,000 highly positioned nucleosomes in NPCs (Fig 1B). For brevity, we will refer to these nucleosomes as “positioned”. Using this tool on published MNase-seq datasets from the human cell lines, K562 and GM12878 19, we identified broadly similar numbers of positioned nucleosomes to those reported in their associated publication (Table EV1). Thus, we estimate that 2.7, 2.4 and 1.6% of nucleosomes are positioned in human NPC, K562 and GM12878 cells, respectively. Based on a theoretical total of 15 million nucleosomes in the human genome, assuming 1 nucleosome per 200 bp. This is consistent with observations from other human cells 15, 16. Figure 1. Nucleosome dynamics of human pluripotent stem cell and differentiated cells Genome-wide nucleosome maps were generated from the frequency distribution of the read mid-point positions of paired-end sequenced MNase-resistant fragments in the size range 138–161 bp (spanning the nucleosome footprint). Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC (pl-iPSC) to show examples of nucleosomes selected as highly positioned, marked*. The distribution of positioned nucleosomes (derived 138–161 bp size class) and sub-nucleosome fragments (derived 112–137 bp size class) for pl-iPSC- and iPSC-derived NPC (NPC). Actual calculated numbers are presented above each column. Venn diagram showing the overlap in the genomic location of positioned nucleosomes in pl-iPSC and in the same cell line differentiated into neural progenitor cells (NPCs). Average frequency distribution of nucleosome positions relative to each other. Data were aligned to each mapped nucleosome in the genome and plotted for a 600-bp window for pl-iPSC, NPC and the chronic myelogenous leukaemia (Cml)-derived cell line, K562. In all cases, a prominent single nucleosome is presented with only minor flanking peaks, indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells. Distribution of nucleosome array sizes for pl-iPSC, NPC and K562, calculated as the number of nucleosomes within a distance of 150-200 bp of each other. Bars show the number of nucleosomes arrayed as singletons (1), pairs (2), triplets (3) and greater than 3 (> 3), displayed as log10 values, and the percentage distribution within a cell line is shown above the column. Download figure Download PowerPoint In contrast, there were 8.4-fold fewer positioned nucleosomes (0.33% of the total nucleosome number) in the pluripotent cell state, indicating that a substantial increase in positioned nucleosomes occurs during differentiation from pluripotent cells to NPC. We recognise that it may be difficult to detect positioned nucleosomes that have high MNase sensitivity using our methodology. To investigate the impact of this possibility on the detected nucleosome number, we mapped the mid-points of DNA fragments in the 112–137 bp sub-nucleosome size range (Fig 1B). We found no evidence for increased cleavage of nucleosomes or accumulation of sub-nucleosome size fragments in pluripotent iPSC. Furthermore, in the total population of DNA fragments, there was no accumulation of small DNA fragments (Fig EV1B). Comparison of both pluripotent iPSC and NPC maps indicated that only 32% of the positioned nucleosomes present in the pluripotent state retain their position during differentiation to NPC (Fig 1C). This indicates that there are considerable changes in nucleosome positioning during human neural differentiation due to both re-positioning and the de novo formation of positioned nucleosomes. Importantly, there is a small population of nucleosomes that retain their positions in both cell states, albeit corresponding to only approximately 0.1% of the total nucleosome population. Global organisation of nucleosome positioning To determine how human nucleosomes are distributed throughout the genome, the organisation of positioned nucleosomes was analysed in pluripotent iPSC and NPC states, and for the K562 cell line. A frequency distribution was plotted for all nucleosome positions and their surrounding window (± 300 bp) centred on the nucleosome peak (Fig 1D). This showed that very few nucleosomes appear in evenly spaced arrays. To quantify the number and length of positioned nucleosome arrays, we calculated the frequency that positioned nucleosomes occur with a spacing of 50 bp or less (Fig 1E) and the distribution of their inter-nucleosome spacing from 0 to 100 kb (Fig EV2). Approximately 90% of positioned nucleosomes are present as singletons, and few nucleosomes (7.1, 7.0 and 13.1%, respectively) occur in pairs or nucleosome arrays. The same basic pattern of nucleosome positioning is seen across the different human cell types tested here (Fig 1D and E). This distribution contrasts with the arrays of nucleosomes that are seen across gene bodies in organisms with small genomes. Nonetheless, in total there are in fact more positioned nucleosomes in the human genome than in the yeast genome. This apparent sparsity of positioned human nucleosomes may arise from the need to precisely position nucleosomes at key regulatory sites rather than organise nucleosome arrays on active genes. Click here to expand this figure. Figure EV2. Distribution of inter-nucleosome spacingThe distance between nucleosomes (also known as linker length) was plotted between each adjacent nucleosome for pl-iPSC, NPC and K562 cells. In all three cases, the distribution of inter-nucleosome spacing distances is bimodal, with a small peak at < 50 bp, representing only a small proportion on nucleosomes, with a second peak in the range of 10–20 kb for pl-IPSC or 550–700 bp range for the differentiated NPC and K562 cells, which possess eightfold more positioned nucleosomes. Download figure Download PowerPoint To probe the relationship between nucleosome positions and gene activity further, we examined the distribution of positioned nucleosomes across different chromatin states. Using the 15-state model of Ernst and Kellis 20, 21, which classifies chromatin from highly active to repressed states based on modifications, functions and associated proteins, we investigated whether positioned nucleosomes partition into particular chromatin states in pluripotent cells. We found no significant enrichment for the major chromatin states associated with transcriptionally active, repressed or heterochromatin. An exception was for chromatin state 8, where we observed an approximate sixfold enrichment of nucleosome positioning over that expected for random placement in the genome (Fig 2A). This state is characterised by its association with the chromatin architectural protein CCCTC-binding factor (CTCF) 22 (Fig 2B). Analysis of our NPC data showed that despite an eightfold increase in the number of positioned nucleosomes, the increase was distributed evenly across all states, with no particular state showing a strong proportional increase (Table EV2). Figure 2. Positioned nucleosomes, chromatin states and transcriptional activity Bar chart showing the number of nucleosomes that map to selected chromatin states 20. Active (state 1); Insulator (state 8); Repressed, Rep (state 12) and heterochromatin, Hetero (state 13), and to open chromatin (based on ATAC-seq). Shown are the observed number of nucleosomes per chromatin state (black) and the expected number calculated assuming a random distribution across the genome (grey). Bar chart showing the number of CTCF sites that map to different chromatin states and open chromatin, using the same methodology as (A). Download figure Download PowerPoint Finally, we examined the relationship between nucleosome positioning and open and closed chromatin. Using the map coordinates for open chromatin regions (based on high ATAC-seq accessibility) for pluripotent human embryonic stem cells (H9), 23 we calculated that 5.7% of positioned nucleosomes fall within regions of open chromatin, but given that only 0.72% of chromatin is in the open state, this is an eightfold enrichment over that expected by random placement (Fig 2A). 40% of CTCF sites are in the open state (Fig 2B), and the overlap between positioned nucleosomes with CTCF sites could substantially contribute to their partitioning into open chromatin. The relationship between nucleosome positioning and gene activity The poor correlation between the presence of positioned nucleosomes and transcriptionally active chromatin states was unexpected given the known association of nucleosome positioning within active gene loci of non-mammalian cells and the association of gene expression with SNF-2 family chromatin remodelers 24; therefore, we investigated the local distribution of nucleosome positioning at TSS. In non-mammalian cells, actively transcribed genes possess a positioned nucleosome at their TSS, termed the +1 nucleosome. In the human genome, we identified individual loci where positioned nucleosomes were present flanking the TSS. For example, a nucleosome was present at the −1 position in the pluripotent cell state but not in NPC at the TSS of the pluripotent-specific gene NANOG. (Fig 3A). Conversely, we found individual loci where a positioned nucleosome was present at the +1 position only in NPC, such as at the TSS of the ionotropic glutamate receptor subunit GRIA1 (Fig 3B). However, this correlation did not hold at the whole genome level. Figure 3. The relationship of positioned nucleosomes at the TSS and gene expression A, B. Nucleosome maps at and surrounding, the TSS region of the NANOG and GRIA1 genes in pl-iPSC (upper, black) and in NPCs (lower, grey). The TSS is indicated by a dashed line at chr 12: 7,941,991 for NANOG and chr5: 152,870,105 for GRIA1. * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene. C. Global frequency distribution of nucleosome distributions at TSS of protein-coding genes. Alignment of nucleosome maps centred on TSS using the non-redundant list of 66,047 mapped human protein-coding gene TSS. The features corresponding to the nucleosome-free region (NFR) and −1 to +2 nucleosomes are marked. D. Genome-wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene. The number of nucleosomes observed is plotted for both pl-iPSC and NPC types for gene sets selected either as uniquely expressed in pl-iPSC (n = 3,833) or NPC (n = 2,082). If positioned nucleosomes are a strong predictor of gene activity, high numbers of nucleosomes would be expected for pluripotent-specific genes in pl-iPSC, but not NPC stages, and for NPC-specific genes in NPC, but not pl-iPSC stages. No correlation between gene activity and positioned nucleosomes was observed. Download figure Download PowerPoint We conducted a global analysis of chromatin patterns at the TSS of protein-coding genes, using a non-redundant list of 66,047 mapped human TSS. The frequency distribution of MNase-protected fragments at and surrounding protein-coding TSS showed a distinct MNase-hypersensitive region at the TSS (Fig 3C), a NFR, as reported previously by others 25. We also noted distinct nucleosome peaks flanking the NFR and TSS, sitting at positions −1, +1 and +2. This demonstrates the presence of distinctive consensus nucleosome pattern at the TSS, to which our two gene examples, NANOG and GRIA1, conform. This raises the question of whether positioned nucleosomes in general are required for active gene expression. This can be addressed by comparison between our two developmental states by examining genes expressed only in the pluripotent or NPC state. By cross-comparison of published RNA-seq data from human cells 26 with the non-redundant TSS list of all human TSS (n = 83,179), we created datasets containing the locations of TSS of human genes that are transcribed only in pluripotent or NPCs. These datasets were then filtered for the presence or absence of a positioned nucleosome within ± 300 bp of the TSS. The results of this analysis show that for genes expressed only in pluripotent cells there were in fact more nucleosomes positioned at the TSS of genes inactive in the NPC state than for the same genes when they are actively expressed in pluripotent state (Figs 3D and EV3). For genes expressed only in NPCs, a substantial number of NPC-specific genes had nucleosomes associated with the gene TSS in the inactive, pluripotent cell state (Figs 3D and EV3). Click here to expand this figure. Figure EV3. Nucleosome distributions at the TSS of genes uniquely expressed in either pluripotent or NPC in both active and inactive cell statesGlobal frequency distribution of nucleosome distributions within ± 300 bp of a TSS of genes selected for expression exclusively in pluripotent (pl-iPSC, n = 3,833) or NPC (n = 2,082), shown for both active and inactive cell states. The features corresponding to the nucleosome-free region (NFR) and −1 to +2 nucleosomes are marked. No correlation between gene activity and positioned nucleosomes was observed. Download figure Download PowerPoint Overall, we did not see a strong correlation between the presence of positioned nucleosomes at the TSS and gene expression. In contrast to nucleosome modifications 27, 28, the presence of highly positioned nucleosomes at TSS is not a genome-wide predictor of gene activity in human cells. Our findings also mirror the conclusions of 18 and co-workers, who found that nucleosome accessibility, not occupancy, is the predominant predictor of gene expression for UPR-associated gene activation in Drosophila. Nucleosome positioning at NRSF/REST-binding sites To widen our analysis further, we examined the patterns of nucleosome positions at selected transcription factor (TF) binding sites involved in neural differentiation, namely YY1, ATF2 and PAX6 29-31. We did not observe a pattern of highly positioned nucleosomes flanking these TF binding sites (Fig EV4). In contrast, well-positioned nucleosomes were present flanking the RE1 binding site 32 of NRSF/REST (Fig 4). NRSF/REST promotes epigenetic repression of gene activity by binding to DNA and acting as a scaffold for a protein complex containing enzymes mediating repressive nucleosome modifications, including the H3K9 dimethyltransferase G9a/EHMT2, the histone deacetylase complex Sin3/HDAC1/2 and the H3K4 demethylase LSD1 as well as the chromatin remodeller Brg1/SMARCA4 33. Click here to expand this figure. Figure EV4. Nucleosomes are not positioned at several transcription factor binding sites that are involved in neurodevelopmentAverage frequency distributions for sequence mid-point data at and surrounding transcription factor binding sites (± 600 bp) for nucleosomes at ATF2 (n = 9,881), YY1 (n = 39,945) and PAX6 (n = 1,432) sites. Download figure Download PowerPoint Figure 4. Nucleosome patterning associated with the RE1 NRSF/REST-binding motif A. The average distribution of nucleosomes centred on the RE1 site (n = 871), spanning 2 kb of flanking genomic sequence. B, C. Aligned paired-end sequence read data were stratified into two size classes, (B) 122–137 bp and (C) 162–188 bp for pl-iPSC and NPC. Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site (n = 871). Peaks correspond to the NRSF/REST complex, and nucleosomes in (B) and (C), respectively. D. Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp. RE1 sites were clustered based on the 122–137 bp mid-point sequence read values (NRSF/REST protein complex) from pl-iPSC, shown as a frequency distribution in upper left panel. The frequency distribution data for the same sites were plotted using 122–137 bp data from NPC (upper right panel) and 162–188 bp data (nucleosomes; bottom panels) for the strongest (solid line) and weakest (dotted line) clusters. E. Schematic of nucleosome patterning at the RE1 site in pl-iPSC and NPC. Nucleosomes are represented by filled black circles. Download figure Download PowerPoint RE1 sites were selected by mapping the consensus sequence of RE1 sites to NRSF/REST ChIP-seq data 34 creating a dataset of 871 well-characterised RE1 sites. Aligning nucleosome positioning data to these RE1 sites across the genome revealed a consensus pattern comprising an array of nucleosomes flanking the RE1 site (Fig 4A). The NRSF/REST complex is present at RE1 sites in mouse pluripotent cells 35, but it is lost upon neuronal differentiation. It was therefore unexpected to find that the pattern of nucleosomes surrounding the RE1 site in human pluripotent cells was still present in NPC (Fig 4A). To analyse this pattern in more detail, we stratified sequencing reads into the fragments in the 122–137 bp range for protection by transcription factors or regulatory complexes with large DNA footprints and identified a distinct peak of protected fragments in this smaller size-class at the RE1 site in pluripotent cells corresponding to the NRSF/REST complex. This peak is reduced by more than 90% in NPCs (Fig 4B). To separate the NRSF/REST footprint from that of the flanking nucleosomes, we constructed a nucleosome map using only larger fragment sizes (162–188 bp), minimising the overlap with the smaller 122- to 137-bp fragments (Fig 4C). This shows a complete separation of NRSF/REST binding from nucleosome positions and significantly that the average pattern of nucleosome positioning is unchanged in NPC in the absence of the NRSF/REST complex. To ensure that genome averaging did not mask any differences at these sites, we carried out cluster analysis (Fig 4D) to establish that 64% of the selected RE1 sites had detectable peaks, corresponding to NRSF/REST complex in undifferentiated pluripotent cells. These were absent at the same sites at the NPC stage. In pluripotent cells, there was a strong correlation between the presence of NRSF/REST at the RE1 binding site and a" @default.
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• W2942734585 title "Nucleosome dynamics of human iPSC during neural differentiation" @default.
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• W2942734585 hasConceptScore W2942734585C169760540 @default.
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