Article

Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation

1] Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. [2] Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 09/2012; 19(11). DOI: 10.1038/nsmb.2391
Source: PubMed

ABSTRACT

A fundamental challenge in mammalian biology has been the elucidation of mechanisms linking DNA methylation and histone post-translational modifications. Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has multiple domains that bind chromatin, and it is implicated genetically in the maintenance of DNA methylation. However, molecular mechanisms underlying DNA methylation regulation by UHRF1 are poorly defined. Here we show that UHRF1 association with methylated histone H3 Lys9 (H3K9) is required for DNA methylation maintenance. We further show that UHRF1 association with H3K9 methylation is insensitive to adjacent H3 S10 phosphorylation-a known mitotic 'phospho-methyl switch'. Notably, we demonstrate that UHRF1 mitotic chromatin association is necessary for DNA methylation maintenance through regulation of the stability of DNA methyltransferase-1. Collectively, our results define a previously unknown link between H3K9 methylation and the faithful epigenetic inheritance of DNA methylation, establishing a notable mitotic role for UHRF1 in this process.

Download full-text

Full-text

Available from: Brian D Strahl
  • Source
    • "Pull-down assays were performed in peptide binding buffer (PBB) [50mM Tris-HCl pH 8.0, 300mM NaCl, 0.1% NP-40 v/ v] based on previously described protocols [23] [24]. Biotinylated peptides (500 pmol) were immobilized on Streptavidin MagneSphereV R Paramagnetic Particles (Promega) for 30 min at 258C. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Course-based undergraduate research experiences (CUREs) provide an opportunity for students to engage in experiments with outcomes that are unknown to both the instructor and students. These experiences allow students and instructors to collaboratively bridge the research laboratory and classroom, and provide research experiences for a large number of students relative to traditional individual mentored research. Here, we describe a molecular biology CURE investigating the impact of clinically relevant mutations found in the bromodomain of the p300 transcriptional regulator on acetylated histone interaction. In the CURE, students identified missense mutations in the p300 bromodomain using the Catalogue of Somatic Mutations in Cancer (COSMIC) database and hypothesized the effects of the mutation on the acetyl-binding function of the domain. They cloned and purified the mutated bromodomain and performed peptide pulldown assays to define its potential to bind to acetylated histones. Upon completion of the course, students showed increased confidence performing molecular techniques and reported positively on doing a research project in class. In addition, results generated in the classroom were further validated in the research laboratory setting thereby providing a new model for faculty to engage in both course-based and individual undergraduate research experiences. © 2015 The International Union of Biochemistry and Molecular Biology.
    Full-text · Article · Nov 2015 · Biochemistry and Molecular Biology Education
  • Source
    • "UHRF1 ubiquitylates histone H3 at lysine 23, and this mark is required for the recruitment of DNMT1 to DNA replication sites (Nishiyama et al. 2013). Therefore, UHRF1 links two layers of epigenetic information; the methylation of DNA associated with transcriptional silencing and histone methylation marks associated with chromatin condensation and inhibition of gene expression (Rothbart et al. 2012; Liu et al. 2013). The tandem Tudor domains of UHRF1 (UHRF1-TT) mediate the recognition of histone H3. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein domains of the Royal Family were the first methyllysine binding domains to be discovered. Here, we review what was learned from the structural studies of Royal Family members including chromo, Tudor, MBT, chromo barrel, and PWWP domains. Our main focus is on methyllysine reader domains for which three-dimensional structures are available in the ligand-bound state.
    Full-text · Article · Jan 2015
  • Source
    • "designed or naturally occurring phosphorylation-insensitive H3K9me3 HMIDs (Rothbart et al. 2012). Additionally, there is an increased interest in the readout of complex histone PTM patterns. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
    Full-text · Article · Oct 2014 · Genome Research
Show more