EZH2/G9a interact to mediate drug resistance in non-small-cell lung cancer by regulating the SMAD4/ERK/c-Myc signaling axis.

Drug resistance is the leading problem in non-small-cell lung cancer (NSCLC) therapy. The contribution of histone methylation in mediating malignant phenotypes of NSCLC is well known. However, the role of histone methylation in NSCLC drug-resistance mechanisms remains unclear. Here, our data show that EZH2 and G9a, two histone methyltransferases, are involved in the drug resistance of NSCLC. Gene manipulation results indicate that the combination of EZH2 and G9a promotes tumor growth and mediates drug resistance in a complementary manner. Importantly, clinical study demonstrates that co-expression of both enzymes predicts a poor outcome in patients with NSCLC. Mechanistically, G9a and EZH2 interact and promote the silencing of the tumor-suppressor gene SMAD4, activating the ERK/c-Myc signaling pathway. Finally, SU08, a compound targeting both EZH2 and G9a, is demonstrated to sensitize resistant cells to therapeutic drugs by regulating the SMAD4/ERK/c-Myc signaling axis. These findings uncover the resistance mechanism and a strategy for reversing NSCLC drug resistance.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae.

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

The COMPASS-like complex modulates fungal development and pathogenesis by regulating H3K4me3-mediated targeted gene expression in Magnaporthe oryzae.

Histone-3-lysine-4 (H3K4) methylation is catalysed by the multiprotein complex known as the Set1/COMPASS or MLL/COMPASS-like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS-like complex targets the H3K4 methylation of plant-pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome-wide approaches to characterize the roles of the COMPASS-like family in the rice blast fungus Magnaporthe oryzae, a model plant pathogen. We purified and identified six conserved subunits of COMPASS from M. oryzae: MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL), and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the sequence repeat in dual-specificity kinase splA and ryanodine receptors domain of MoBre2 can interact directly with the DPY30 domain of MoSdc1 in vitro. Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSPP1, and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome-wide profiling of H3K4me3 revealed that it has remarkable co-occupancy at the transcription start site regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion of MoBre2, MoSwd2, or MoSpp1 was highly correlated with a decrease in H3K4me3. These results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3-targeted genes in M. oryzae.

Enhancing the Anticancer Efficacy of Immunotherapy through Combination with Histone Modification Inhibitors.

In the nucleus of each cell, the DNA is wrapped around histone octamers, forming the so-called "nucleosomal core particles". The histones undergo various modifications that influence chromatin structure and function, including methylation, acetylation, ubiquitination, phosphorylation, and SUMOylation. These modifications, known as epigenetic modifications (defined as heritable molecular determinants of phenotype that are independent of the DNA sequence), result in alterations of gene expression and changes in cell behavior. Recent work has shown that epigenetic drugs targeting histone deacetylation or methylation modulate the immune response and overcome acquired resistance to immunotherapy. A number of combination therapies involving immunotherapy and epigenetic drugs, which target histone deacetylation or methylation, are currently under various clinical/pre-clinical investigations and have shown promising anticancer efficacy. These combination therapies may provide a new strategy for achieving sustained anticancer efficacy and overcoming resistance.