Kyoko L. Yap

Molecular Interplay of the Noncoding RNA ANRIL and Methylated Histone H3 Lysine 27 by Polycomb CBX7 in Transcriptional Silencing of INK4a

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Calmodulin target database.

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for a -helix formation.

Diversity of conformational states and changes within the EF-hand protein superfamily

The EF‐hand motif, which assumes a helix‐loop‐helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF‐hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF‐hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF‐hand proteins that this “closed‐to‐open” conformational transition is not the sole model for EF‐hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF‐hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF‐hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF‐hand proteins and its correlation with target recognition. Proteins 1999;37:499–507. ©1999 Wiley‐Liss, Inc.

Keeping it in the family: diverse histone recognition by conserved structural folds.

Epigenetic regulation of gene transcription relies on an array of recurring structural domains that have evolved to recognize post-translational modifications on histones. The roles of bromodomains, PHD fingers, and the Royal family domains in the recognition of histone modifications to direct transcription have been well characterized. However, only through recent structural studies has it been realized that these basic folds are capable of interacting with increasingly more complex histone modification landscapes, illuminating how nature has concocted a way to accomplish more with less. Here we review the recent biochemical and structural studies of several conserved folds that recognize modified as well as unmodified histone sequences, and discuss their implications on gene expression.

Structural Basis for Simultaneous Binding of Two Carboxy-terminal Peptides of Plant Glutamate Decarboxylase to Calmodulin

Activation of glutamate decarboxylase (GAD) by calcium-bound calmodulin (CaM) is required for normal plant growth through regulation of gamma-aminobutyrate and glutamate metabolism. The interaction of CaM with the C-terminal domain of GAD is believed to induce dimerization of the enzyme, an event implicated for Ca(2+)-dependent enzyme activation. Here, we present the solution structure of CaM in complex with a dimer of peptides derived from the C-terminus of Petunia hybrida GAD. The 23 kDa ternary complex is pseudo-symmetrical with each domain of CaM bound to one of the two antiparallel GAD peptides, which form an X-shape with an interhelical angle of 60 degrees. To accommodate the dimeric helical GAD target, the two domains of CaM adopt an orientation markedly different from that seen in other CaM-target complexes. Although the dimeric GAD domain is much larger than previously studied CaM-binding peptides, the two CaM domains appear closer together and make a number of interdomain contacts not observed in earlier complexes. The present structure of a single CaM molecule interacting with two target peptides provides new evidence for the conformational flexibility of CaM as well as a structural basis for the ability of CaM to activate two enzyme molecules simultaneously.

Structural insights into human KAP1 PHD finger–bromodomain and its role in gene silencing

The tandem PHD finger–bromodomain, found in many chromatin-associated proteins, has an important role in gene silencing by the human co-repressor KRAB-associated protein 1 (KAP1). Here we report the three-dimensional solution structure of the tandem PHD finger–bromodomain of KAP1. The structure reveals a distinct scaffold unifying the two protein modules, in which the first helix, αZ, of an atypical bromodomain forms the central hydrophobic core that anchors the other three helices of the bromodomain on one side and the zinc binding PHD finger on the other. A comprehensive mutation-based structure-function analysis correlating transcriptional repression, ubiquitin-conjugating enzyme 9 (UBC9) binding and SUMOylation shows that the PHD finger and the bromodomain of KAP1 cooperate as one functional unit to facilitate lysine SUMOylation, which is required for KAP1 co-repressor activity in gene silencing. These results demonstrate a previously unknown unified function for the tandem PHD finger–bromodomain as an intramolecular small ubiquitin-like modifier (SUMO) E3 ligase for transcriptional silencing.

Structure and Mechanisms of Lysine Methylation Recognition by the Chromodomain in Gene Transcription

Histone methylation recognition is accomplished by a number of evolutionarily conserved protein domains, including those belonging to the methylated lysine-binding Royal family of structural folds. One well-known member of the Royal family, the chromodomain, is found in the HP1/chromobox and CHD subfamilies of proteins, in addition to a small number of other proteins that are involved in chromatin remodeling and gene transcriptional silencing. Here we discuss the structure and function of the chromodomain within these proteins as methylated histone lysine binders and how the functions of these chromodomains can be modulated by additional post-translational modifications or binding to nucleic acids.

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding

Small‐angle X‐ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium‐bound calmodulin (Ca2+/CaM) in solution upon binding to its antagonist, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7). The radius of gyration was 17.4±0.3 Å for Ca2+/CaM‐W‐7 with a molar ratio of 1:5 and 20.3±0.7 Å for Ca2+/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca2+/CaM‐W‐7 complex with those of other complexes indicates that binding of two W‐7 molecules induces a globular shape for Ca2+/CaM, probably caused by an inter‐domain compaction. The results suggest a tendency for Ca2+/CaM to form a globular structure in solution, which is inducible by a small compound like W‐7.

The SUVR4 Histone Lysine Methyltransferase Binds Ubiquitin and Converts H3K9me1 to H3K9me3 on Transposon Chromatin in Arabidopsis

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation–dependent and –independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.

Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7

The chromobox 7 (CBX7) protein of the polycomb repressive complex 1 (PRC1) functions to repress transcription of tumor suppressor p16 (INK4a) through long noncoding RNA, ANRIL (antisense noncoding RNA in the INK4 locus) directed chromodomain (ChD) binding to trimethylated lysine 27 of histone H3 (H3K27me3), resulting in chromatin compaction at the INK4a/ARF locus. In this study, we report structure-guided discovery of two distinct classes of small-molecule antagonists for the CBX7ChD. Our Class A compounds, a series including analogues of the previously reported MS452, inhibit CBX7ChD/methyl-lysine binding by occupying the H3K27me3 peptide binding site, whereas our Class B compound, the newly discovered MS351, appears to inhibit H3K27me3 binding when CBX7ChD is bound to RNA. Our crystal structure of the CBX7ChD/MS351 complex reveals the molecular details of ligand recognition by the aromatic cage residues that typically engage in methyl-lysine binding. We further demonstrate that MS351 effectively induces transcriptional derepression of CBX7 target genes, including p16 (INK4a) in mouse embryonic stem cells and human prostate cancer PC3 cells. Thus, MS351 represents a new class of ChD antagonists that selectively targets the biologically active form of CBX7 of the PRC1 in long noncoding RNA- and H3K27me3-directed gene transcriptional repression.