Recognition of Multivalent Histone States Associated with Heterochromatin by UHRF1 Protein

Nataliya Nady,Alexander Lemak,John R Walker,George V Avvakumov,Michael S Kareta,Mayada Achour,Sheng Xue,Shili Duan,Abdellah Allali-Hassani,Xiaobing Zuo,Yun-Xing Wang,Christian Bronner,Frédéric Chédin,Cheryl H Arrowsmith,Sirano Dhe-Paganon

doi:10.1074/jbc.m111.234104

Abstract

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16(INK4A), when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.

Highlights

France, the **Protein Nucleic Acid Interaction Section, Structural Biophysics Laboratory, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, and the ‡‡Department of Physiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada
UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are recognized through its SET- and RING-associated domain (SRA) domain and interacts with histone H3 trimethylated on lysine 9 (H3K9me3)
We show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3)

Summary

EXPERIMENTAL PROCEDURES

Protein Expression, and Purification—The cDNAencoding residues 121–286 of the human UHRF1 protein was cloned into a modified pET28a bacterial expression vector encoding an N-terminal hexahistidine fusion protein with a tobacco etch virus protease cleavage site. Data from crystals of selenomethione labeled UHRF1 TTD that had been soaked with a six-residue peptide from H3 (TARKme3ST) were collected at SBC-CAT 19ID at the Advanced Photon Source at 0.99987 Å and processed using HKL2000. Close examination of the Np95Ϫ/Ϫ cell line revealed that an unexpected splicing event from exons 1 to 8, which immediately flank the region that was targeted for gene replacement, drives the production of a truncated UHRF1 protein lacking coding exons 2 to 7 (supplemental Fig. S9) This truncated UHRF1 remnant, referred to as UHRF1⌬(2–7), lacks the ubiquitin-like domain, TTD, and PHD domains, but still carries intact SRA and RING domains. Signals were detected by chemiluminescence using the ECL detection system (Amersham Biosciences)

RESULTS

Data collection Space group Cell dimensions

Total RDCs

DISCUSSION