A synthetic biology approach to probing nucleosome symmetry
Yuichi Ichikawa,
Caitlin F Connelly,
Alon Appleboim,
Thomas CR Miller,
Hadas Jacobi,
Nebiyu A Abshiru,
Hsin-Jung Chou,
Yuanyuan Chen,
Upasna Sharma,
Yupeng Zheng,
Paul M Thomas,
Hsuiyi V Chen,
Vineeta Bajaj,
Christoph W Müller,
Neil L Kelleher,
Nir Friedman,
Daniel NA Bolon,
Oliver J Rando,
Paul D Kaufman
Affiliations
Yuichi Ichikawa
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
Caitlin F Connelly
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
Alon Appleboim
School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel; The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
Thomas CR Miller
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
Hadas Jacobi
School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel; The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
Nebiyu A Abshiru
National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
Hsin-Jung Chou
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
Yuanyuan Chen
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
Upasna Sharma
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
Yupeng Zheng
National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
Paul M Thomas
National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
Hsuiyi V Chen
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
Vineeta Bajaj
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
Christoph W Müller
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel; The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
Daniel NA Bolon
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
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.
