Nucleosome dynamics of human iPSC during the early stages of neurodevelopment

Abstract

Regulation of nucleosome positioning is important for neurodevelopment, and mutation of genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. Unicellular organisms possess arrays of highly positioned nucleosomes within their chromatin, occupying up to 80% of their genomes. These span gene-coding and regulatory regions, and can be associated with local changes of gene transcription. In the much larger genome of human cells, the roles of nucleosome positioning are less clear, and this raises questions of how nucleosome dynamics interfaces with human neurodevelopment.We have generated genome-wide nucleosome maps from an undifferentiated human induced pluripotent stem cell (hiPSC) line and after its differentiation to the neuronal progenitor cell (NPC) stage. We found that approximately 3% of nucleosomes are highly positioned in NPC. In contrast, there are 8-fold less positioned nucleosomes in pluripotent cells, with the majority arising de novo or relocating during cell differentiation. Positioned nucleosomes do not directly correlate with active chromatin or gene transcription, such as marking Transcriptional Start Sites (TSS).Unexpectedly, we find a small population of nucleosomes that remain positioned after differentiation, occupying similar positions in pluripotent and NPC cells. They flank the binding sites of the key gene regulators NRSF/REST and CTCF, but remain in place whether or not their regulatory complexes are present. Together, these results present an alternative view in human cells, where positioned nucleosomes are sparse and dynamic, but act to alter gene expression at a distance via structural conformation at sites of chromatin regulation, not local changes in gene organisation.3 Main Nucleosome maps generated by MNase-seq show the dynamic nature of nucleosome positioning. Narrow sequence read mid-point distribution profiles are indicative of highly positioned nucleosomes present at the same position in all cells of the population, whereas broader distributions occur where there is substantial variation in nucleosome positioning between individual cells ( Figure 1A). By mapping nucleosomes in the same human induced pluripotent stem cell (hiPSC) line in both its undifferentiated, pluripotent cell state and following differentiation to the neural progenitor cell (NPC) stage, we followed the changes in nucleosome positioning during early stages of neurodevelopment. We found that only 2.7% of nucleosomes are highly positioned in human NPC (408,152 of a theoretical total of 15 million based on an assumption of 1 nucleosome per 200 bp). This is consistent with observations from other human and mammalian cells [1,2], but contrasts with the yeasts and Dictyostelium genomes where positioned nucleosomes occupy approximately 80% of the genome [3][4][5][6]. Using the same analysis on data generated from the human lymphoblastoid cell lines, K562 and GM12878 [7] we calculated that 2.4% and 1.6% of nucleosomes respectively were highly positioned in these cell line...