ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation.

Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

STAT3-induced upregulation of circCCDC66 facilitates the progression of non-small cell lung cancer by targeting miR-33a-5p/KPNA4 axis.

Emerging evidence has manifested the critical effect of abnormally expressed circular RNAs (circRNAs) on the initiation and progression of non-small cell lung cancer (NSCLC). Although circRNA circCCDC66 has been revealed to elicit facilitating impact on cell growth and metastasis in colon cancer, the potential biological function and regulatory mechanism of it in NSCLC still require to be explored. In this study, circCCDC66 in NSCLC cells was highly expressed. Downregulation of circCCDC66 impaired cell proliferation, migration and invasion whereas boosted cell apoptosis in NSCLC. Data from molecular mechanism assays testified that circCCDC66 bound with miR-33a-5p in NSCLC cells. And miR-33a-5p inhibition could rescue the suppressive effect of circCCDC66 knockdown on NSCLC progression. In addition, karyopherin subunit alpha 4 (KPNA4) in NSCLC cells was proofed to be directly targeted by miR-33a-5p. Moreover, through rescued-function assays, we observed that upregulating KPNA4 expression could countervail the restraining function of silenced circCCDC66 on NSCLC progression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was validated to activate CCDC66 transcription and thereby promote circCCDC66 expression in NSCLC cells. Briefly, STAT3-induced circCCDC66 upregulation accelerates NSCLC progression via miR-33a-5p/KPNA4 axis, suggesting circCCDC66 as a promising biomarker in NSCLC treatment.

Chemical and Light Inducible Epigenome Editing.

The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers a revolutionary tool for mechanistic studies in epigenome regulation as well as the development of novel epigenome therapies. Inducible and reversible epigenome editing provides unique temporal control critical for understanding the dynamics and kinetics of epigenome regulation. This review summarizes the progress in the development of spatiotemporal-specific tools using small molecules or light as inducers to achieve the conditional control of epigenome editing and their applications in epigenetic research.

A successful bronchial thermoplasty procedure in a "very severe" asthma patient with rare complications: a case report.

Introduction: Bronchial thermoplasty (BT) is a unique bronchoscopic treatment for severe asthma that utilizes radiofrequency ablation to reduce smooth muscle in the bronchial walls. Current studies mainly focused on uncontrolled severe asthma with a forced expiratory volume in 1 second (FEV1) above 60% and associated complications, no human studies have performed on "very severe" asthma as well as its complications. Case study: We present a 60-year-old male with more than 15 years history of very severe asthma, who underwent BT. His FEV1 was only 20.4% predicted, which would have excluded him from all prior clinical trials of BT. The first BT procedure occurred without an issue. After the second BT procedure, he experienced severe dyspnea due to an infection with a non-flu respiratory virus. This illness was complicated by the formation of a pulmonary cyst. During recovering from the third procedure, he developed stomach stones. This is mainly related with taking large amounts of hawthorn previously, also cannot exclude the role of thermal energy injury on gastrointestinal nerve function. Results: Despite these unexpected complications, his quality of life greatly improved after BT, yet his lung function did not improve. Conclusion: This case is the first to describe BT procedures in patient with this level of lung function compromise, although accompanied with rare complications; our report indicates BT may be an opportunity and choice for the "very severe" asthma patients.

A chemically induced proximity system engineered from the plant auxin signaling pathway.

Methods based on chemically induced proximity (CIP) serve as powerful tools to control cellular processes in a temporally specific manner. To expand the repertoire of CIP systems available for studies of cellular processes, we engineered the plant auxin signaling pathway to create a new indole-3-acetic acid (IAA) based CIP method. Auxin-induced protein degradation that occurs in the natural pathway was eliminated in the system. The new IAA based method is both readily inducible and reversible, and used to control the production of therapeutic proteins that induced the apoptosis of cancer cells. The approach is also orthogonal to existing CIP systems and used to construct a biological Boolean logic gate controlling gene expression system. We believe that the new CIP method will be applicable to the artificial control and dissection of complex cellular functions.

Salt-tolerant and -sensitive alfalfa (Medicago sativa) cultivars have large variations in defense responses to the lepidopteran insect Spodoptera litura under normal and salt stress condition.

In nature, plants are often exposed to multiple stress factors at the same time. Yet, little is known about how plants modulate their physiology to counteract simultaneous abiotic and biotic stresses, such as soil salinity and insect herbivory. In this study, insect performance bioassays, phytohormone measurements, quantification of transcripts, and protein determination were employed to study the phenotypic variations of two alfalfa (Medicago sativa) cultivars in response to insect Spodoptera litura feeding under normal and salt stress condition. When being cultivated in normal soil, the salt-tolerant alfalfa cultivar Zhongmu-1 exhibited lower insect resistance than did the salt-sensitive cultivar Xinjiang Daye. Under salinity stress, the defense responses of Xinjiang Daye were repressed, whereas Zhongmu-1 did not show changes in resistance levels. It is likely that salinity influenced the resistance of Xinjiang Daye through suppressing the accumulation of jasmonic acid-isoleucine (JA-Ile), which is the bioactive hormone inducing herbivore defense responses, leading to attenuated trypsin proteinase inhibitor (TPI) activity. Furthermore, exogenous ABA supplementation suppressed the insect herbivory-induced JA/JA-Ile accumulation and levels of JAR1 (jasmonate resistant 1) and TPI, and further decreased the resistance of Xinjiang Daye, whereas Zhongmu-1 showed very little response to the increased ABA level. We propose a mechanism, in which high levels of abscisic acid induced by salt treatment may affect the expression levels of JAR1 and consequently decrease JA-Ile accumulation and thus partly suppress the defense of Xinjiang Daye against insects under salt stress. This study provides new insight into the mechanism by which alfalfa responds to concurrent abiotic and biotic stresses.

COI1-Regulated Hydroxylation of Jasmonoyl-L-isoleucine Impairs Nicotiana attenuata's Resistance to the Generalist Herbivore Spodoptera litura.

The phytohormone jasmonoyl-L-isoleucine (JA-Ile) is well-known as the key signaling molecule that elicits plant defense responses after insect herbivory. Oxidation, which is catalyzed by the cytochrome P450s of the CYP94 family, is thought to be one of the main catabolic pathways of JA-Ile. In this study, we identified four CYP94B3 homologues in the wild tobacco plant Nicotiana attenuata. Individually silencing the four homologues revealed that NaCYP94B3 like-1 and NaCYP94B3 like-2, but not NaCYP94B3 like-3 and NaCYP94B3 like-4, are involved in the C-12-hydroxylation of JA-Ile. Simultaneously silencing three of the NaCYP94B3 like genes, NaCYP94B3 like-1, -2, and -4, in the VIGS-NaCYP94B3s plants doubled herbivory-induced JA-Ile levels and greatly enhanced plant resistance to the generalist insect herbivore, Spodoptera litura. The poor larval performance was strongly correlated with the high concentrations of several JA-Ile-dependent direct defense metabolites in VIGS-NaCYP94B3s plants. Furthermore, we show that the abundance of 12-hydroxy-JA-Ile was dependent on JA-Ile levels as well as COI1, the receptor of JA-Ile. COI1 appeared to transcriptionally control NaCYP94B3 like-1 and -2 and thus regulates the catabolism of its own ligand molecule, JA-Ile. These results highlight the important role of JA-Ile degradation in jasmonate homeostasis and provide new insight into the feedback regulation of JA-Ile catabolism. Given that silencing these CYP94 genes did not detectably alter plant growth and highly increased plant defense levels, we propose that CYP94B3 genes can be potential targets for genetic improvement of herbivore-resistant crops.

[In vitro antimicrobial activity of defensins from rabbit neutrophils against Pseudomonas aeruginosa and its multiple-drug-resistance strains].

OBJECTIVE: To investigate the role and mechanism of defensins from rabbit neutrophils against the multi-drug-resistant (MDR) strains of Pseudomonas aeruginosa. METHODS: Defensins were extracted from rabbit peritoneal neutrophil granules with citric acid and purified by acid urea polyacrylamide gel electrophoresis (AU-PAGE). An agrose gel radial diffusion assay for bactericidal activity in vitro was used and ultrastructural changes of the killed Pseudomonas aeruginosa were examined by use of transmission electron microscope. RESULTS: NP2 was isolated and purified successfully by AU-PAGE. NP2 demonstrated concentration-dependent killing of Pseudomonas aeruginosa and its MDR strain. When the two kinds of Pseudomonas aeruginosa cells exposed to NP2 were examined by transmission electron microscope, we found that the cell membrane was incomplete, the components of cells condensed into pyknotic bodies accompanied by vacuolization, and there was some transudate between the cells. CONCLUSION: Defensin from the rabbit neutrophils exerts potent bactericidal activity against the MDR strain of Pseudomonas aeruginosa. The findings under electron microscope suggest the theory that defensins are capable of destabilizing the cell membrane permeability and causing the death of microorganism.