In addition to recognizing histone tails, several reports have implicated PHD fingers in binding to non-histone proteins, expanding their role as transcriptional regulators and signaling components.
Pygopus (Pygo) and a co-factor BCL9 control β-catenin-mediated transcription within the Wnt-signaling pathway. The PHD finger of homologous human PYGO1 and PYGO2 can simultaneously bind H3K4me2/3 and homology domain 1 (HD1) of BCL9 (B9L in the case of PYGO2) (21
). These concomitant interactions of the PYGO1/2 PHD finger are essential for Wnt responses during development. The crystal structure of the ternary complex, PYGO1 PHD finger bound to H3K4me2 and HD1, reveals that the PHD–HD1 interface involves two sets of contacts. A loop connecting the sixth and the seventh zinc-coordinating Cys residues is unusually long in the PYGO1 PHD finger. In the complex, this loop folds into an α-helix (α1) and a β-strand (β5) with the latter forming a parallel β-sheet with the only β-strand of HD1, and the former making hydrogen bonding and hydrophobic contacts with the α-helix of HD1.
The H3K4me2-binding pocket is located on the opposite side of the PYGO1 PHD finger (a). It consists of four aromatic and hydrophobic residues but also contains an aspartate that forms a hydrogen bond with the proton of the dimethylammonim group of Lys4. This additional contact may account for a ~2-fold increase in affinity toward H3K4me2 versus H3K4me3. There is no discernible pocket for Arg2; however, Ala1 of the peptide is bound by a typical backbone carbonyl net, whereas the side chain of Ala1 is involved in the interactions with hydrophobic residues in the α1 helix and the β5-strand of the PHD finger, exactly the same elements that contact BCL9 HD1. Association of BCL9 HD1 with PYGO1 PHD drives a short loop between the α1 helix and the β5-strand out, opening up the binding cavity for Ala1 of the H3K4me2 peptide (21
). Likewise in the homologous PYGO2 complex, binding of B9L HD1 triggers allosteric remodeling of the binding channel for Thr3 of the peptide (34
). Such allosteric effects enhance affinities of the HD1-bound PHD fingers of PYGO1 and PYGO2 to H3K4me2 ~2- to 3-fold. The PYGO1/2 PHD–H3K4me2-HD1 assembly represents the first example of the histone-binding function of a PHD finger being modulated by a co-factor.
The structural basis of non-histone recognition by PHD fingers. (a) The ternary complex of the PHD finger of PYGO1 (2VPG). (b) The PHD finger of MLL1 binds to H3K4me3 (3LQJ) and the RRM domain of Cyp33 (2KU7).
MLL1 methyltransferase is a member of the trithorax family of evolutionarily conserved proteins required for maintaining the expression levels of HOX
and other developmental genes. Although MLL1 generally promotes gene expression, it associates with numerous co-factors that activate or suppress transcription. MLL1 contains three consecutive PHD fingers, the third of which has been shown to bind H3K4me3, the product of the enzymatic activity of the MLL1 SET domain that methylates Lys4 (30–32
). Additionally, the PHD3 finger was found to interact with the RNA recognition motif (RRM) of a nuclear cyclophilin Cyp33, an MLL1-associated co-repressor (64
). Several groups revealed the molecular basis of these interactions (30–32
) and Wang et al.
) uncovered the mechanism of the PHD3–H3K4me3–RRM assembly by determining the crystal structure of the PHD3 finger and adjacent bromodomain in complex with the H3K4me3 peptide and the solution structure of an α-helix of the PHD3 finger in complex with the Cyp33 RRM domain.
The structure of the MLL1 PHD3–bromodomain cassette in the apo-state demonstrates that the two modules are in close contact involving the C-terminal α-helix of the PHD3 finger (30
). A proline residue in the linker connecting the PHD3 finger with the bromodomain adopts a cis
conformation, facilitating the formation of a pair of salt bridges between the two modules. Although this bromodomain does not bind acetylated lysine residues, it plays a significant role in modulating the function of the PHD3 finger, affecting binding to both H3K4me3 and Cyp33. In the presence of the bromodomain, the binding affinity of the PHD finger for H3K4me3 is augmented by ~20-fold, whereas association with an isolated Cyp33 RRM domain is abrogated because the RRM-binding site is blocked by the bromodomain. Remarkably, when full-length Cyp33 was tested, the interaction between the PHD3 finger and Cyp33 RRM was restored. Cyp33 is a peptidyl–prolyl isomerase (PPIase) that generally catalyzes cis–trans
isomerization of a proline residue and acts on specific proline residues in the histone H3 tail (32
). Here, the Cyp33 PPIase domain converts a Pro–His peptide bond in the MLL1 PHD3–bromodomain linker from a cis
conformation disrupting the PHD3–bromodomain contacts and freeing the previously occluded Cyp33 RRM-binding site allowing PHD3 to interact with RRM (30
Binding of PHD3 to RRM involves a C-terminal α-helix of the PHD finger, the same helix that associates with the bromodomain. This α-helix interacts with one face of the anti-parallel β-sheet and a loop connecting two of the β-strands of the RRM domain (b). The RRM-binding site is adjacent to and does not visibly overlap with the H3K4me3-binding site, however the PHD–bromodomain cassette binds to H3K4me3 2.7-fold weaker in the presence of full-length Cyp33 (30
). Likewise binding of a single PHD3 finger is reduced by 5.7-fold in the presence of the RRM domain of Cyp33, and interaction of the PHD3 finger with Cyp33 RRM is decreased by 4.4-fold in the presence of H3K4me3 (32
The recognition of H3K4me3 and Cyp33 by the MLL1 PHD finger is a striking example of the context dependent function of a PHD finger, aiding to interpret the different regulatory environments of MLL1 and facilitating the switch from transcriptional activation to repression. Overall, these comprehensive studies uncovered a remarkably complex mechanism of functioning and regulation of the MLL1 PHD3 finger. However, several remaining questions need to be addressed. For example whether the PHD3 finger is able to concomitantly recognize both binding partners, how the bromodomain enhances binding of the PHD3 finger to H3K4me3, and what the role is of the interaction of the Cyp33 RRM domain with an RNA (65