Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

Cancer chemoresistance is regulated by complex genetic and epigenetic networks. In this study, the features of gene expression, methylation, and microRNA (miRNA) expression were investigated with high-throughput sequencing in human breast cancer MCF-7 cells resistant to adriamycin (MCF-7/ADM) and paclitaxel (MCF-7/PTX). We found that: ① both of the chemoresistant cell lines had similar, massive changes in gene expression, methylation, and miRNA expression versus chemosensitive controls. ② Pairwise integration of the data highlighted sets of genes that were regulated by either methylation or miRNAs, and sets of miRNAs whose expression was controlled by DNA methylation in chemoresistant cells. ③ By combining the three sets of high-throughput data, we obtained a list of genes whose expression was regulated by both methylation and miRNAs in chemoresistant cells; ④ Expression of these genes was then validated in clinical breast cancer samples to generate a 17-gene signature that showed good predictive and prognostic power in triple-negative breast cancer patients receiving anthracycline-taxane-based neoadjuvant chemotherapy. In conclusion, our results have generated a new workflow for the integrated analysis of the effects of miRNAs and methylation on gene expression during the development of chemoresistance.

PhoH is a host-derived auxiliary metabolic gene that can be used as a new biomarker for surveying phage diversity in marine and paddy waters. However, the applicability of this gene in other environments has not been addressed. In this paper, we surveyed the phoH gene in four wetland sediments in northeast China. DNA was extracted directly from sediments and used for PCR amplification with the degenerate primers vPhoHf and vPhoHr. In total, 44 and 58 phoH sequences were identified as belonging to bacteria and phages, respectively, suggesting that this primer set is not highly specific to the phage phoH gene. A BLASTp search showed that the 58 phage phoH sequences had the highest identity to the known viral sequences, ranging from 48% to 100%. Phylogenetic analysis showed that all phage sequences from wetlands distributed into the previously designated Groups 2, 3, 4 and 6. In addition, two new subgroups, Groups 2c and 4c, which contained sequences exclusively from wetlands, were detected in this study. Nonmetric multidimensional scaling analysis showed that the phage phoH assemblage from a coastal wetland was similar to that in marine environments, while the phage phoH assemblage from a lake wetland was similar to that in paddy waters. These findings indicated that different types of wetlands had distinct phage phoH compositions.

Sugarcane-soybean intercropping has been widely used to control disease and improve nutrition in the field. However, the response of the soil microbial community diversity and structure to intercropping is not well understood. Since microbial diversity corresponds to soil quality and plant health, a pot experiment was conducted with sugarcane intercropped with soybean. Rhizosphere soil was collected 40 days after sowing, and MiSeq sequencing was utilized to analyze the soil microbial community diversity and composition. Soil columns were used to assess the influence of intercropping on soil microbial activity (soil respiration and carbon-use efficiency: nitrogen-use efficiency ratio). PICRUSt and FUNGuild analysis were conducted to predict microbial functional profiling. Our results showed that intercropping decreased pH by approximately 8.9% and enhanced the soil organic carbon, dissolved organic carbon, and available nitrogen (N) by 5.5%, 13.4%, and 10.0%, respectively. These changes in physicochemical properties corresponded to increased microbial diversity and shifts in soil microbial communities. Microbial community correlated significantly (p < 0.05) with soil respiration rates and nutrient use efficiency. Furthermore, intercropping influenced microbial functions, such as carbon fixation pathways in prokaryotes, citrate cycle (TCA cycle) of bacteria and wood saprotrophs of fungi. These overrepresented functions might accelerate nutrient conversion and control phytopathogens in soil.

The human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. Research reports on the mechanism of HEF1 in gastric cancer (GC) differentiation are limited. In this study, we reveal that HEF1 plays an essential role in regulating of differentiation in human GC. HEF1 was found to be highly expressed in GC tissues. Besides, In GC tissues or cells, cellular level of HEF1 negatively correlated with tumor differentiation. In addition, we showed that upregulation of HEF1 increased Wnt5a expression and the nuclear translocation of β-catenin, thereby resulting in poor differentiation in GC. Notably, GC patients with a higher expression of HEF1 showed significantly poorer disease-free and overall survival. Thus, our findings suggest that HEF1 reduces differentiation through the Wnt5a/β-catenin signaling pathway and that HEF1 is an independent unfavorable prognostic death factor in GC.

MYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

Eukaryotic elongation factor 1 alpha (eEF1A) delivers aminoacyl-tRNA to the ribosome and thereby plays a key role in protein synthesis. Human eEF1A is subject to extensive post-translational methylation, but several of the responsible enzymes remain unknown. Using a wide range of experimental approaches, we here show that human methyltransferase (MTase)-like protein 13 (METTL13) contains two distinct MTase domains targeting the N terminus and Lys55 of eEF1A, respectively. Our biochemical and structural analyses provide detailed mechanistic insights into recognition of the eEF1A N terminus by METTL13. Moreover, through ribosome profiling, we demonstrate that loss of METTL13 function alters translation dynamics and results in changed translation rates of specific codons. In summary, we here unravel the function of a human MTase, showing that it methylates eEF1A and modulates mRNA translation in a codon-specific manner.

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.

Tacrolimus (TAC)-induced pancreatic islet injury is one of the important causes of new-onset diabetes in transplant recipients. This study was performed to evaluate whether a dipeptidyl peptidase IV (DPP IV) inhibitor is effective in improving TAC-induced diabetes mellitus by reducing pancreatic islet injury.

Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.

Numerous studies have revealed the high diversity of cyanophages in marine and freshwater environments, but little is currently known about the diversity of cyanophages in paddy fields, particularly in Northeast (NE) China. To elucidate the genetic diversity of cyanophages in paddy floodwaters in NE China, viral capsid assembly protein gene (g20) sequences from five floodwater samples were amplified with the primers CPS1 and CPS8. Denaturing gradient gel electrophoresis (DGGE) was applied to distinguish different g20 clones. In total, 54 clones differing in g20 nucleotide sequences were obtained in this study. Phylogenetic analysis showed that the distribution of g20 sequences in this study was different from that in Japanese paddy fields, and all the sequences were grouped into Clusters α, β, γ and ε. Within Clusters α and β, three new small clusters (PFW-VII∼-IX) were identified. UniFrac analysis of g20 clone assemblages demonstrated that the community compositions of cyanophage varied among marine, lake and paddy field environments. In paddy floodwater, community compositions of cyanophage were also different between NE China and Japan.

Mammalian members of the forkhead box protein O (FoxO) class of transcription factors are implicated in the regulation of oxidative stress, and FoxO proteins are negatively regulated by the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway. We examined the effect of Klotho on the PI3K/AKT pathway and manganese superoxide dismutase (MnSOD) during tacrolimus (Tac)-induced oxidative stress. Klotho-treated mice showed decreased Tac-induced oxidative stress accompanied by functional and histological improvements. Klotho inhibited the PI3K/AKT-mediated phosphorylation of FoxO3a and enhanced FoxO3a binding to the MnSOD promoter. Klotho increased MnSOD mRNA and protein expression in mitochondria. In addition, Klotho reduced Tac-induced mitochondrial dysfunction and decreased mitochondrial reactive oxygen species production, and these effects were enhanced by blocking PI3K activity with LY294002. Collectively, our data showed that Klotho protects Tac-induced oxidative stress by negatively regulating the PI3K/AKT pathway and subsequently enhancing FoxO3a-mediated MnSOD expression.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

Small molecule tool compounds have enabled profound advances in life science research. These chemicals are potent, cell active, and selective, and, thus, are suitable for interrogating biological processes. For these chemicals to be useful they must be correctly characterized and researchers must be aware of them. We mined the ChEMBL bioactivity database to identify high quality tool compounds in an unbiased way. We identified 407 best-in-class compounds for 278 protein targets, and these are reported in an annotated data set. Additionally, we developed informatics functions and a web application for data visualization and automated pharmacological hypothesis generation. These functions were used to predict inhibitors of the Chromobox Protein Homologue 5 (CBX5) mediated gene repression pathway that currently lacks appropriate inhibitors. The predictions were subsequently validated by a highly specific cell based assay, revealing new chemical modulators of CBX5-mediated heterochromatin formation. This data set and associated functions will help researchers make the best use of these valuable compounds.

Adriamycin is a first-line chemotherapy agent against cancer, but the development of resistance has become a major problem. Although autophagy is considered to be an adaptive survival response in response to chemotherapy and may be associated with chemoresistance, its inducer and the underlying molecular mechanisms remain unclear. Here, we demonstrate that adriamycin up-regulates the both levels of TRPC5 and autophagy, and the increase in autophagy is mediated by TRPC5 in breast cancer cells. Blockade of TRPC5 or autophagy increased the sensitivity to chemotherapy in vitro and in vivo. Notably, we revealed a positive correlation between TRPC5 and the autophagy-associated protein LC3 in paired patients with or without anthracycline-taxane-based chemotherapy. Furthermore, pharmacological inhibition and gene-silencing showed that the cytoprotective autophagy mediated by TRPC5 during adriamycin treatment is dependent on the CaMKKβ/AMPKα/mTOR pathway. Moreover, adriamycin-resistant MCF-7/ADM cells maintained a high basal level of autophagy, and silencing of TRPC5 and inhibition of autophagy counteracted the resistance to adriamycin. Thus, our results revealed a novel role of TRPC5 as an inducer of autophagy, and this suggests a novel mechanism of drug resistance in chemotherapy for breast cancer.

Multidrug resistance (MDR) is one of the major problems in the treatment of cancer. Overcoming it is therefore expected to improve clinical outcomes for cancer patients. MDR is usually characterized by overexpression of ABC (ATP-binding cassette) protein transporters such as P-gp, MRP1, and ABCG2. Though the importance of ABC transporters for cancer cells is recognized, few studies have looked at its implications for the endothelial cells that are essential to tumor angiogenesis. This study investigated the expression and functions of these ABC transporters in endothelial cells in vitro and their potential contribution to cancer growth in mice.

The cellular prion protein (PrP(C)) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrP(C) and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein. When recombinantly expressed, the PrP-like domain of ZIP5 could be obtained with yields and levels of purity sufficient for structural analyses but it tended to aggregate, thereby precluding attempts to study its structure. These obstacles were overcome by moving to a mammalian cell expression system. The subsequent biophysical characterization of a homogeneous preparation of the ZIP5 PrP-like ectodomain shows that this protein acquires a dimeric, largely globular fold with an α-helical content similar to that of mammalian PrP(C). The use of a mammalian cell expression system also allowed for the expression and purification of stable preparations of Takifugu rubripes PrP-1, thereby overcoming a key hindrance to high-resolution work on a fish PrP(C).

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.