Massively multiplex single-molecule oligonucleosome footprinting
Abstract
Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure.
Data availability
All raw data will be made available at GEO Accession GSE162410; processed data is available at Zenodo (https://doi.org/10.5281/zenodo.3834705). All scripts and notebooks for reproducing analyses in the paper are available at https://github.com/RamaniLab/SAMOSA.
-
Massively multiplex single-molecule oligonucleosome footprintingZenodo , zenodo.3834706.
-
Massively multiplex single-molecule oligonucleosome footprintingNCBI Gene Expression Omnibus, GSE162410.
Article and author information
Author details
Funding
Sandler Foundation
- Vijay Ramani
American Cancer Society
- Laura J Hsieh
National Institutes of Health (R01GM123977)
- Hani Goodarzi
National Institutes of Health (R35GM127020)
- Geeta J Narlikar
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Job Dekker, University of Massachusetts Medical School, United States
Version history
- Received: May 28, 2020
- Accepted: November 24, 2020
- Accepted Manuscript published: December 2, 2020 (version 1)
- Version of Record published: December 14, 2020 (version 2)
Copyright
© 2020, Abdulhay et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,189
- views
-
- 522
- downloads
-
- 53
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
- Chromosomes and Gene Expression
Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.
-
- Chromosomes and Gene Expression
Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.