Department of Chemistry, University of Washington, Seattle, United States
Miho Shimada
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York City, United States
Caroline E Weller
Department of Chemistry, University of Washington, Seattle, United States
Tomoyoshi Nakadai
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York City, United States; Project for Cancer Epigenomics, Cancer Institute of JFCR, Tokyo, Japan
Peter L Hsu
Department of Pharmacology, University of Washington, Seattle, United States; Howard Hughes Medical Institute, University of Washington, Seattle, United States
Elizabeth L Tyson
Department of Chemistry, University of Washington, Seattle, United States
Arpit Mishra
Department of Genome Sciences, Department of Medicine, University of Washington, Seattle, United States
Patrick MM Shelton
Department of Chemistry, University of Washington, Seattle, United States
Martin Sadilek
Department of Chemistry, University of Washington, Seattle, United States
Department of Genome Sciences, Department of Medicine, University of Washington, Seattle, United States
Ning Zheng
Department of Pharmacology, University of Washington, Seattle, United States; Howard Hughes Medical Institute, University of Washington, Seattle, United States
The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.
