ABSTRACT
The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.
Subject(s)
Gene Expression Regulation, Neoplastic , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Binding Sites , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Chromatin Assembly and Disassembly , Histone Deacetylase 1/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Binding , Repressor Proteins/chemistry , Repressor Proteins/geneticsABSTRACT
The production of biofuels from cellulosic biomass is a promising technology for developing a renewable source of energy. Efforts to produce ethanol from cellulosic biomass using microbes, such as the yeast Saccharomyces cerevisiae, face major challenges, including the need for detoxification. Here, we apply a strategy to discover genetic alterations that lead to improved robustness of S. cerevisiae in the presence of acetate, which is present at toxic concentrations in hemicellulose hydrolysates. Acetate in its protonated form (acetic acid) enters the cell through passive diffusion and dissociates into a proton and acetate, acidifying the cytosol and inhibiting cell function, an effect that is exacerbated in the presence of sodium. Through flow cytometry analysis, implemented as part of a novel cell culture technique, the Cytostat, we characterized the deleterious effects of sodium acetate on growth and on cell size homeostasis. Further, using the Cytostat to screen a genome-wide, gene overexpression library, we identified that overexpressing the ENA2 gene, a P-type sodium pump ATPase, provides a significant growth improvement in the presence of sodium acetate. Together, our data support the proposed mechanism for the synergistic growth inhibition exerted by acetate and sodium, as well as the mechanism that develops resistance.
Subject(s)
Genome, Fungal , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Sodium Acetate/pharmacology , Sodium Chloride/pharmacology , Stress, Physiological/genetics , Cell Size , Flow Cytometry , Gene Library , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sodium Chloride/chemistry , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis.
Subject(s)
Histones/chemistry , Homeodomain Proteins/chemistry , Tumor Suppressor Proteins/chemistry , Amino Acid Motifs , Animals , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Reference Standards , Sensitivity and Specificity , Tumor Suppressor Protein p53/chemistryABSTRACT
Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 A resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.
