Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1

Abstract

Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3-binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3-binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.epigenetics | multidomain structure | posttranslational modification | X-ray crystallography

Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation

“…2A). These results agree with those of Arita et al (2012), in which no measurable binding of wild-type UHRF1 TTD-PHD was detected by ITC to H3K9me3 peptides acetylated at their N-terminal amino group. Furthermore, binding of the UHRF1 TTD-PHD to H3 1-20 K9me3 peptides fluorescently labeled at the C terminus was outcompeted by unlabeled H3 1-5 peptides containing either L-alanine or L-valine at position 1 but not with unlabeled H3 1-5 peptides containing either D-alanine or D-valine, demonstrating that stereochemistry at the first position is critical for PHD binding and that the functional PHD is likely able to accommodate more bulky side chains (Supplemental Fig.…”

“…2A; Supplemental Fig. S1C), consistent with K d measurements for H3K9me3 peptides by ITC (Arita et al 2012). Perturbing the TTD interaction with H3K9 methylation (Y188A; TTD mutant) diminished the affinity for these peptides 10-fold to 15-fold (K d ;2.5 and 4.5 mM, respectively) ( Fig Many effector modules that recognize the extreme N terminus of H3 (residues 1-4)-including the UHRF1 PHD-specifically interact with the N-terminal amino group of Ala 1.…”

“…S1E; Nady et al 2011;Arita et al 2012;Rothbart et al 2012a;Cheng et al 2013), but our data demonstrate that in the context of the dual domain, the TTD has no measurable affinity for H3K9me2/3 peptides in the absence of PHD function. This led us to question why the physical linkage of the TTD to the PHD finger renders this H3K9 reader domain incapable of engaging H3K9 methylated histone tails in the absence of PHD coordination of the H3 N terminus.…”

“…Recent structural studies indicated that the linked TTD and PHD domains of UHRF1 simultaneously bind H3K9me3 peptides on a single histone tail in vitro ( Fig. 1C; Arita et al 2012;Cheng et al 2013). We therefore sought to characterize this multivalent cis interaction further in the context of the ''histone code'' and asked whether such binding is biologically important for UHRF1 function in the cell.…”

“…Crystal structures of the linked TTD-PHD bound to H3K9me3 peptides show that UHRF1 can simultaneously engage the unmodified H3 N terminus and H3K9me3 on a single H3 tail (cis recognition) through this linked recognition module ( Fig. 1C; Arita et al 2012;Cheng et al 2013), although the UHRF1 TTD and PHD are able to bind histone peptides indepen- (Nady et al 2011) and UHRF1 PHD (right; PDB: 3SOU) (Rajakumara et al 2011) bound to H3 1-11 K9me3 and H3 1-8 peptides, respectively. This H3K9me3-TTD interaction is referred to throughout the manuscript as the alternative H3-binding pose with respect to the TTD.…”