The PHD Finger of Human UHRF1 Reveals a New Subgroup of Unmethylated Histone H3 Tail Readers

Abstract

The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.

Conserved linker regions and their regulation determine multiple chromatin-binding modes of UHRF1

“…16 Remarkably, the first zinc atom coordinates a loop, the so-called prePHD that precedes the canonical PHD-fold. This structural feature was first identified in the UHRF1 PHD domain and its detailed function still needs to be determined.…”

“…This structural feature was first identified in the UHRF1 PHD domain and its detailed function still needs to be determined. 16,17 It was suggested that the prePHD might be essential for the right orientation of residue C316, which makes contact with H3K4. 16 However, if analyzed in isolation the PHD does not much discriminate the modification status of H3K4.…”

“…16,17 It was suggested that the prePHD might be essential for the right orientation of residue C316, which makes contact with H3K4. 16 However, if analyzed in isolation the PHD does not much discriminate the modification status of H3K4. In contrast, phosphorylation of H3 Threonine 3 (H3T3ph), symmetric as well as asymmetric dimethylation of H3R2 (R2me2s/a) and H3A1 N-terminal acetylation (H3A1ac) strongly interfere with binding of the PHD to the H3-tail.…”

PHD Fingers as Histone Readers

“…The non-canonical (a C4C4 topology) PHD fingers of DNA (cytosine-5)-methyltransferases recognize unmodified H3, coupling histone tail association with DNA methylation, necessary for gene repression (Ooi et al 2007). A more complex interplay between the unmodified H3-binding PHD finger and the neighboring H3K9me3-binding tandem Tudor domain (TTD) of UHRF1 is required for epigenetic inheritance of DNA methylation (Arita et al 2012;Cheng et al 2013;Lallous et al 2011;Liu et al 2013;Rajakumara et al 2011;Rothbart et al 2012Rothbart et al , 2013Wang et al 2011;Xie et al 2012). Binding of the BPTF PHD finger to H3K4me3 stabilizes the nucleosomeremodeling NURF complex at chromatin, enhancing NURF-catalyzed nucleosome sliding and activation of developmental genes Wysocka et al 2006).…”

“…The extended PHD finger of UHRF1 utilizes a distinct mechanism to bind unmodified H3 (Arita et al 2012;Cheng et al 2013;Lallous et al 2011;Liu et al 2013;Rajakumara et al 2011;Rothbart et al 2012Rothbart et al , 2013Wang et al 2011; Xie Fig. 2.3 The molecular mechanism of binding of the PHD finger to unmodified H3 tail.…”