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Single-Cell Analysis Elucidates Features of Nucleosome Organization

NEW YORK (GenomeWeb) – Using a single-cell micrococcal nuclease sequencing approach, researchers have begun to tease out additional features of nucleosome organization.

Nucleosome positioning is linked to both chromatin accessibility and gene expression, and while previous mapping methods have found nucleosomes to be positioned near the transcription start sites (TSSs) of active genes and DNaseI hypersensitivity sites (DHSs), these approaches reported the cell population average.

In a new study appearing in Nature today, researchers led by the National Heart, Lung, and Blood Institute's Keji Zhao used a single-cell approach called single-cell micrococcal nuclease sequencing (scMNase-seq) that simultaneously examined nucleosome positioning and chromatin accessibility. By applying this approach to three different cell types, they unearthed two principles of nucleosome organization and found that some cells' organizational states seemed to prime them to be ready to differentiate into certain cell types.

"Our application of scMNase-seq to three types of single cells revealed principles of nucleosome organization in different chromatin regions as well as heterogeneity of nucleosome positioning and spacing at DHSs," Zhao and his colleagues wrote in their paper.

The researchers used scMNase-seq to analyze 48 cells from the NIH3T3 cell line, 198 mouse embryonic stem cells, and 278 naïve mouse CD4 T cells. On average, they generated 3 million, 900,000, and 700,000 unique fragments per cell type, respectively. They considered fragments between 140 base pairs and 180 base pairs in length to be canonical nucleosomes, and those less than 80 base pairs in length to be subnucleosome-sized particles.

Nucleosome positioning and subnucleosome-sized particles at DHSs, TSSs, and CTCF-binding sites in both single-cell and bulk MNase-seq was consistent, the researchers noted. At the same time, density of subnucleosome-sized particles in pooled cells correlated with DNaseI tag density at DHSs and with gene expression at TSS, indicating these particles predict chromatin accessibility. This in combination with other data suggested to the researchers that their approach could measure both nucleosome positioning and chromatin accessibility in single cells, at the same time.

From their analyses, the researchers uncovered different rules governing nucleosome organization in different chromatin regions.

Promoters of silent genes had greater uniformity of nucleosome spacing than did those of active genes, and non-DHS regions had greater uniformity, as compared to DHS regions. In addition, nucleosome spacing in active chromatin regions associated with H3K4me1, H3K4me3, H3K27ac, H3K9ac and H2AZ exhibited lower uniformity that transcribed regions marked by H3K36me3.

These findings, the investigators said, reveal two organizational principles. Silent chromatin, including regions with repressed promoters and heterochromatic regions, contains highly uniformly spaced nucleosomes. But in these regions, the nucleosomes are not positioned relative to the underlying DNA. It's reversed, however, in active chromatin states such as transcribed promoters and DHS regions. There, nucleosomes are positioned but not uniformly spaced.

In addition, at DHS sites, they found that variation in nucleosome positioning correlated with variation in accessibility and that variation in positioning around TSSs was correlated with variation in gene expression. The underlying DNA sequence also influences nucleosome positioning, with high levels of CC, GG, and GC frequency at nucleosome-occupied locales.

At the same time, the researchers also noted that a sizable portion of naïve CD4 T cell and mouse embryonic stem cells had depleted nucleosome occupancy at certain enhancers. Some naïve CD4 T cells, for instance, had decreased occupancy at enhancers specific to T helper 1 or T helper 2 cells. Meanwhile, a portion of mouse ESCs had decreased nucleosome occupancy at embryoid body-specific enhancers. This suggested to the researchers that these cells were primed to differentiate into those cell types.

"Our data suggest that the cellular heterogeneity of undifferentiated cells is related to heterogeneous nucleosome organization in critical regulatory regions, which reflects the differentiation potential of these cells," the authors wrote.