Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells

The histone methyltransferase G9a is overexpressed in a variety of cancer types, including pancreatic adenocarcinoma, and promotes tumor invasiveness and metastasis. We recently reported the discovery of BRD4770, a small-molecule inhibitor of G9a that induces senescence in PANC-1 cells. We observed that the cytotoxic effects of BRD4770 were dependent on genetic background, with cell lines lacking functional p53 being relatively resistant to compound treatment. To understand the mechanism of genetic selectivity, we used two complementary screening approaches to identify enhancers of BRD4770. The natural product and putative BH3 mimetic gossypol enhanced the cytotoxicity of BRD4770 in a synergistic manner in p53-mutant PANC-1 cells but not in immortalized non-tumorigenic pancreatic cells. The combination of gossypol and BRD4770 increased LC3-II levels and the autophagosome number in PANC-1 cells, and the compound combination appears to act in a BNIP3 (B-cell lymphoma 2 19-kDa interacting protein)-dependent manner, suggesting that these compounds act together to induce autophagy-related cell death in pancreatic cancer cells.     

ALG1-CDG: A new case with early fatal outcome

Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases.     

Crystal Structure of the C-Terminal Domain of Human DPY-30-Like Protein: A Component of the Histone Methyltransferase Complex

The conserved DPY-30 is an essential component of the dosage compensation complex that balances the X-linked gene expression by regulation of the complex formation in Caenorhabditis elegans. The human DPY-30-like protein (DPY-30L) homolog is a conserved member of certain histone methyltransferase (HMT) complexes. In the human MLL1 (mixed-lineage leukemia-1) HMT complex, DPY-30L binds to the BRE2 homolog ASH2L in order to regulate histone 3-lysine 4 trimethylation. We have determined the 1.2-Å crystal structure of the human DPY-30L C-terminal domain (DPY-30L_C). The DPY-30L_C structure, harboring the conserved DPY-30 motif, is composed of two α-helices linked by a sharp loop and forms a typical X-type four-helix bundle required for dimer formation. DPY-30L_C dimer formation is largely mediated by an extensive hydrophobic interface with some additional polar interactions. The oligomerization of DPY-30L_C in solution, together with its reported binding to ASH2L, leads us to propose that the hydrophobic surface of the dimer may provide a platform for interaction with ASH2L in the MLL1 HMT complex.     

SIR–nucleosome interactions: Structure–function relationships in yeast silent chromatin

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression, referred to as heterochromatin or “silent” chromatin. Constitutively silent chromatin is found in subtelomeric domains in a number of species, ranging from yeast to man. In addition, chromatin-dependent repression of mating type loci occurs in both budding and fission yeasts, to enable sexual reproduction. The silencing of chromatin in budding yeast is characterized by an assembly of Silent Information Regulatory (SIR) proteins—Sir2, Sir3 and Sir4—with unmodified nucleosomes. Silencing requires the lysine deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions among the SIR proteins, to generate the Sir2–3–4 or SIR complex. Results from recent structural and reconstitution studies suggest an updated model for the ordered assembly and organization of SIR-dependent silent chromatin in yeast. Moreover, studies of subtelomeric gene expression reveal the importance of subtelomeric silent chromatin in the regulation of genes other than the silent mating type loci. This review covers recent advances in this field.     

A 2x folic acid treatment affects epigenetics and dendritic spine densities in SHSY5Y cells

Many diseases are now associated with aberrant epigenetics and gene expression changes. Epigenetics can be modified by factors like diet. One dietary factor, folic acid, is consumed in various forms including supplements, energy drinks, and fortified grains. It was hypothesized high levels of folic acid would affect gene expression and enzyme activity of chromatin modifying enzymes as well as dendritic spine densities in a commonly utilized neuron model, the SHSY5Y cell. Decreased MBD2 and MECP2 were discovered upon treatment of SHSY5Y cells with a 2x folic acid dose. Corresponding decreases in dendritic spines were apparent in the 2x folic acid treated cells as well. Activity of DNMTs and H3K4 HMTs was altered. Further, H3K4me1, H3K4me3, H3K9Ac, and global DNA methylation were decreased in the 2x folic acid treated cells. Further studies are warranted to determine if the effects of excess folic acid are detrimental to organismal physiology.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.     

Histone modifying enzymes and cancer: going beyond histones

Mutations in the molecular pathways that regulate cell proliferation, differentiation, and cell death all contribute to cancer formation. Enzymes that covalently modify histones affect these pathways by controlling the dynamic remodeling of chromatin structure. This article reviews several connections between histone modifying enzymes and cancer that are likely mediated via both histone and non-histone substrates. We propose that multiple protein modifications, including phosphorylation, methylation, and acetylation, cross regulate one another to coordinate intermolecular signaling, and that miscues in this regulation can lead to oncogenesis.     

Repair of psoralen and acetylaminofluorene DNA adducts by ABC excinuclease

Escherichia coli UvrA, UvrB and UvrC proteins acting in concert remove the major ultraviolet light-induced photoproduct, the pyrimidine dimer, from DNA in the form of a 12 to 13-nucleotide long single-stranded fragment. In vivo data indicate that the UvrABC enzyme is also capable of removing other nucleotide diadducts as well as certain nucleotide monoadducts from DNA and initiating the repair process that leads to removal of interstrand crosslinks caused by some bifunctional chemical agents. We have determined the action mechanism of the enzyme on nucleotide monoadducts produced by 4'-hydroxymethyl-4,5',8-trimethylpsoralen and N-acetoxy-N-2-acetylaminofluorene. In both cases we find that the enzyme hydrolyzes the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the modified base. This cutting pattern is similar to that observed with diadduct substrate, the only difference being that while the enzyme incises the fourth or fifth phosphodiester bond 3' to the pyrimidine dimer it always hydrolyzes the fifth bond relative to monoadducts. Our results also suggest that ABC excinuclease cuts the same two phosphodiester bonds on both sides of a T whether that T has a psoralen monoadduct or is involved in psoralen-mediated interstrand crosslink.