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Elife. 2017 Sep 12;6. pii: e28836. doi: 10.7554/eLife.28836.

A synthetic biology approach to probing nucleosome symmetry.

Author information

1
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States.
2
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
3
School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
4
The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
5
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
6
National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States.
#
Contributed equally

Abstract

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the 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 also 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 out by effector proteins in the cell.

KEYWORDS:

S. cerevisiae; chromatin; chromosomes; genes; synthetic biology; transcription

PMID:
28895528
PMCID:
PMC5626479
DOI:
10.7554/eLife.28836
[Indexed for MEDLINE]
Free PMC Article

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