Solution Nuclear Magnetic Resonance Studies of the Ligand-Binding Domain of an Orphan Nuclear Receptor Reveal a Dynamic Helix in the Ligand-Binding Pocket.

The ligand-binding domains (LBDs) of the NR5A subfamily of nuclear receptors activate transcription via ligand-dependent and ligand-independent mechanisms. The Drosophila Ftz-F1 receptor (NR5A3) belongs to the latter category, and its ligand independence is attributed to a short helical segment (α6) within the protein that resides in the canonical ligand-binding pocket (LBP) in the crystalline state. Here, we show that the α6 helix is dynamic in solution when Ftz-F1 is bound to the LxxLL motif of its cofactor Ftz, undergoing motions on the fast (picosecond to nanosecond) as well as slow (microsecond to millisecond) time scales. Motions on the slow time scale (∼10-3 s) appear to pervade throughout the domain, most prominently in the LBP and residues at or near the cofactor-binding site. We ascribe the fast time scale motions to a solvent-accessible conformation for the α6 helix akin to those described for its orthologs in higher organisms. We assign this conformation where the LBP is "open" to a lowly populated species, while the major conformer bears the properties of the crystal structure where the LBP is "closed". We propose that these conformational transitions could allow binding to small molecule ligands and/or play a role in dissociation of the cofactor from the binding site. Indeed, we show that the Ftz-F1 LBD can bind phospholipids, not unlike its orthologs. Our studies provide the first detailed insights into intrinsic motions occurring on a variety of time scales in a nuclear receptor LBD and reveal that potentially functionally significant motions pervade throughout the domain in solution, despite evidence to the contrary implied by the crystal structure.

Quantitative and Structural Assessment of Histone Methyllysine Analogue Engagement by Cognate Binding Proteins Reveals Affinity Decrements Relative to Those of Native Counterparts.

Methyllysine analogues (MLAs), furnished by aminoethylation of engineered cysteine residues, are widely used surrogates of histone methyllysine and are considered to be effective proxies for studying these epigenetic marks in vitro. Here we report the first structure of a trimethyllysine MLA histone in complex with a protein binding partner, quantify the thermodynamic distinctions between MLAs and their native methyllysine counterparts, and demonstrate that these differences can compromise qualitative interpretations of binding at the nucleosome level. Quantitative measurements with two methyllysine binding protein modules reveal substantial affinity losses for the MLA peptides versus the corresponding native methyllysine species in both cases, although the thermodynamic underpinnings are distinct. MLA and methyllysine adopt distinct conformational geometries when in complex with the BPTF PHD finger, a well-established H3K4me3 binding partner. In this case, an ∼13-fold Kd difference at the peptide level translates to nucleosomal affinities for MLA analogues that fall outside of the detectable range in a pull-down format, whereas the methyllysine species installed by native chemical ligation demonstrates robust binding. Thus, despite their facile production and commercial availability, there is a significant caveat of potentially altered binding affinity when MLAs are used in place of native methyllysine residues.

Kinetic and high-throughput profiling of epigenetic interactions by 3D-carbene chip-based surface plasmon resonance imaging technology.

Chemical modifications on histones and DNA/RNA constitute a fundamental mechanism for epigenetic regulation. These modifications often function as docking marks to recruit or stabilize cognate "reader" proteins. So far, a platform for quantitative and high-throughput profiling of the epigenetic interactome is urgently needed but still lacking. Here, we report a 3D-carbene chip-based surface plasmon resonance imaging (SPRi) technology for this purpose. The 3D-carbene chip is suitable for immobilizing versatile biomolecules (e.g., peptides, antibody, DNA/RNA) and features low nonspecific binding, random yet function-retaining immobilization, and robustness for reuses. We systematically profiled binding kinetics of 1,000 histone "reader-mark" pairs on a single 3D-carbene chip and validated two recognition events by calorimetric and structural studies. Notably, a discovery on H3K4me3 recognition by the DNA mismatch repair protein MSH6 in Capsella rubella suggests a mechanism of H3K4me3-mediated DNA damage repair in plant.

YEATS Domain-A Histone Acylation Reader in Health and Disease.

Histone post-translational modifications (PTMs) carry an epigenetic layer of message to regulate diverse cellular processes at the chromatin level. Many of these PTMs are selectively recognized by dedicated effector proteins for normal cell growth and development, while dysregulation of these recognition events is often implicated in human diseases, notably cancer. Thus, it is fundamentally important to elucidate the regulatory mechanism(s) underlying the readout of PTMs on histones. The Yaf9, ENL, AF9, Taf14, Sas5 (YEATS) domain is an emerging reader module that selectively recognizes histone lysine acylation with a preference for crotonylation over acetylation. In the review, we discuss the recognition of histone acylations by the YEATS domain and the biological significance of this readout from multiple perspectives.

YEATS domain: Linking histone crotonylation to gene regulation.

Recent research reveals that the YEATS domains preferentially recognize crotonylated lysines on histones. Here, we discuss the molecular mechanisms that enable this recognition and the biological significances of this interaction. The dynamics of histone crotonylation and its potential roles in the regulation of gene expression will also be discussed.

Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex.

Acetylation is correlated with chromatin decondensation and transcriptional activation, but its regulation by histone deacetylase (HDAC)-bearing corepressor complexes is poorly understood. Here, we describe the mechanism of assembly of the mammalian Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit deemed critical for proper assembly. Sds3 engages a globular, helical region of the HDAC interaction domain (HID) of the scaffolding protein Sin3A through a bipartite motif comprising a helix and an adjacent extended segment. Sds3 dimerizes through not only one of the predicted coiled-coil motifs but also, the segment preceding it, forming an ∼ 150-Å-long antiparallel dimer. Contrary to previous findings in yeast, Sin3A rather than Sds3 functions in recruiting HDAC1 into the complex by engaging the latter through a highly conserved segment adjacent to the helical HID subdomain. In the resulting model for the ternary complex, the two copies of the HDACs are situated distally and dynamically because of a natively unstructured linker connecting the dimerization domain and the Sin3A interaction domain of Sds3; these features contrast with the static organization described previously for the NuRD (nucleosome remodeling and deacetylase) complex. The Sds3 linker features several conserved basic residues that could potentially maintain the complex on chromatin by nonspecific interactions with DNA after initial recruitment by sequence-specific DNA-binding repressors.

Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide.

Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison among experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), wherein a native chromatin sample is spiked with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons, and reveals unique insight into the nature of bivalent domains. This technology provides in situ assessment of the immunoprecipitation step, accommodating for many experimental pitfalls as well as providing a critical examination of untested assumptions inherent to conventional ChIP.

Traceless semisynthesis of a set of histone 3 species bearing specific lysine methylation marks.

Considerable mechanistic insight into the function of histone post-translational modifications and the enzymes that install and remove them derives from in vitro experiments with modified histones, often embedded in nucleosomes. We report the first semisyntheses of native-like histone 3 (H3) bearing tri- and dimethyllysines at position 79 and trimethyllysine at position 36, as well as more facile and traceless semisyntheses of K9 and K27 trimethylated species. These semisyntheses are practical on a multi-milligram scale and can also generate H3 with combinations of marks. Each of these modifications has distinct functional consequences, although the pathways by which H3K36me3 and H3K79me2/3 act have not been entirely mapped. To this end, we demonstrated that our semisynthetic histones, when reconstituted into nucleosomes, are valuable affinity reagents for unbiased binding partner discovery and compare them to their methyllysine analogue (MLA) counterparts at the nucleosome level.

Rationally simplified bistramide analog reversibly targets actin polymerization and inhibits cancer progression in vitro and in vivo.

We describe structure-based design and chemical synthesis of a simplified analog of bistramide A, which potently and reversibly binds monomeric actin with a K(d) of 9.0 nM, depolymerizes filamentous actin in vitro and in A549 (nonsmall cell lung cancer) cells, inhibits growth of cancer cell lines in vitro at submicromolar concentrations, and significantly suppresses proliferation of A549 cells in a nude mice tumor xenograft model in terms of both tumor growth delay and average tumor volume. This study provides a conceptual framework for the design and development of new antiproliferative compounds that target cytoskeletal organization of cancer cells in vivo by a combination of reversible G-actin binding and effective F-actin severing.