A synthetic biology approach to probing nucleosome symmetry
- University of Massachusetts Medical School, United States
- The Hebrew University, Israel
- The Francis Crick Institute, United Kingdom
- Northwestern University, United States
- European Molecular Biology Laboratory (EMBL), Germany
Abstract
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read , we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read by effector proteins in the cell.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (R01GM100164)
- Yuichi Ichikawa
- Caitlin F Connelly
- Hsin-Jung Chou
- Hsuiyi V Chen
- Oliver J Rando
- Paul D Kaufman
European Commission (340712)
- Alon Appleboim
- Hadas Jacobi
- Nir Friedman
National Institute of General Medical Sciences (P41GM108569)
- Nebiyu A Abshiru
- Yupeng Zheng
- Paul M Thomas
- Neil L Kelleher
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jerry L Workman, Stowers Institute for Medical Research, United States
Version history
- Received: May 19, 2017
- Accepted: September 12, 2017
- Accepted Manuscript published: September 12, 2017 (version 1)
- Accepted Manuscript updated: September 13, 2017 (version 2)
- Version of Record published: October 3, 2017 (version 3)
- Version of Record updated: March 29, 2018 (version 4)
Copyright
© 2017, Ichikawa 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
-
- 2,693
- views
-
- 513
- downloads
-
- 16
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
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)
A synthetic biology approach to probing nucleosome symmetry
eLife 6:e28836.
https://doi.org/10.7554/eLife.28836
Further reading
-
- Chromosomes and Gene Expression
- Developmental Biology
Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.
-
- Chromosomes and Gene Expression
Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity-biotinylation method targeting the RNA and proteins constituents. The method that we termed Antibody-Mediated-Proximity-Labelling-coupled to Mass Spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. This analysis identified a number of known RNA binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.
