Cdyl Deficiency Brakes Neuronal Excitability and Nociception through Promoting Kcnb1 Transcription in Peripheral Sensory Neurons.

Epigenetic modifications are involved in the onset, development, and maintenance of pain; however, the precise epigenetic mechanism underlying pain regulation remains elusive. Here it is reported that the epigenetic factor chromodomain Y-like (CDYL) is crucial for pain processing. Selective knockout of CDYL in sensory neurons results in decreased neuronal excitability and nociception. Moreover, CDYL facilitates histone 3 lysine 27 trimethylation (H3K27me3) deposition at the Kcnb1 intron region thus silencing voltage-gated potassium channel (Kv ) subfamily member Kv 2.1 transcription. Loss function of CDYL enhances total Kv and Kv 2.1 current density in dorsal root ganglia and knockdown of Kv 2.1 reverses the pain-related phenotypes of Cdyl deficiency mice. Furthermore, focal administration of a novel potent CDYL antagonist blunts nociception and attenuates neuropathic pain. These findings reveal that CDYL is a critical regulator of pain sensation and shed light on the development of novel analgesics targeting epigenetic mechanisms.

Discovery of Potent Peptidomimetic Antagonists for Heterochromatin Protein 1 Family Proteins.

The heterochromatin protein 1 (HP1) sub-family of CBX chromodomains are responsible for the recognition of histone H3 lysine 9 tri-methyl (H3K9me3)-marked nucleosomal substrates through binding of the N-terminal chromodomain. These HP1 proteins, namely, CBX1 (HP1ß), CBX3 (HP1γ), and CBX5 (HP1α), are commonly associated with regions of pericentric heterochromatin, but recent literature studies suggest that regulation by these proteins is likely more dynamic and includes other loci. Importantly, there are no chemical tools toward HP1 chromodomains to spatiotemporally explore the effects of HP1-mediated processes, underscoring the need for novel HP1 chemical probes. Here, we report the discovery of HP1 targeting peptidomimetic compounds, UNC7047 and UNC7560, and a biotinylated derivative tool compound, UNC7565. These compounds represent an important milestone, as they possess nanomolar affinity for the CBX5 chromodomain by isothermal titration calorimetry (ITC) and bind HP1-containing complexes in cell lysates. These chemical tools provide a starting point for further optimization and the study of CBX5-mediated processes.

A Peptidomimetic Ligand Targeting the Chromodomain of MPP8 Reveals HRP2's Association with the HUSH Complex.

The interpretation of histone post-translational modifications (PTMs), specifically lysine methylation, by specific classes of "reader" proteins marks an important aspect of epigenetic control of gene expression. Methyl-lysine (Kme) readers often regulate gene expression patterns through the recognition of a specific Kme PTM while participating in or recruiting large protein complexes that contain enzymatic or chromatin remodeling activity. Understanding the composition of these Kme-reader-containing protein complexes can serve to further our understanding of the biological roles of Kme readers, while small molecule chemical tools can be valuable reagents in interrogating novel protein-protein interactions. Here, we describe our efforts to target the chromodomain of M-phase phosphoprotein 8 (MPP8), a member of the human silencing hub (HUSH) complex and a histone 3 lysine 9 trimethyl (H3K9me3) reader that is vital for heterochromatin formation and has specific roles in cancer metastasis. Utilizing a one-bead, one-compound (OBOC) combinatorial screening approach, we identified UNC5246, a peptidomimetic ligand capable of interacting with the MPP8 chromodomain in the context of the HUSH complex. Additionally, a biotinylated derivative of UNC5246 facilitated chemoproteomics studies which revealed hepatoma-derived growth factor-related protein 2 (HRP2) as a novel protein associated with MPP8. HRP2 was further shown to colocalize with MPP8 at the E-cadherin gene locus, suggesting a possible role in cancer cell plasticity.

Getting a handle on chemical probes of chomatin readers.

The dynamic nature of histone post-translational modifications such as methylation or acetylation makes possible the alteration of disease associated epigenetic states through the manipulation of the associated epigenetic machinery. One approach is through small molecule perturbation. Chemical probes of epigenetic reader domains have been critical in improving our understanding of the biological consequences of modulating their targets, while also enabling the development of novel probe-based reagents. By appending a functional handle to a reader domain probe, a chemical toolbox of reagents can be created to facilitate chemiprecipitation of epigenetic complexes, evaluate probe selectivity, develop in vitro screening assays, visualize cellular target localization, enable target degradation and recruit epigenetic machinery to a site within the genome in a highly controlled fashion.

A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.

A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-ß3 by Gαq Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution.

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes.

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.