Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

Understanding salt tolerance mechanism using transcriptome profiling and de novo assembly of wild tomato Solanum chilense.

Soil salinity affects the plant growth and productivity detrimentally, but Solanum chilense, a wild relative of cultivated tomato (Solanum lycopersicum L.), is known to have exceptional salt tolerance. It has precise adaptations against direct exposure to salt stress conditions. Hence, a better understanding of the mechanism to salinity stress tolerance by S. chilense can be accomplished by comprehensive gene expression studies. In this study 1-month-old seedlings of S. chilense and S. lycopersicum were subjected to salinity stress through application of sodium chloride (NaCl) solution. Through RNA-sequencing here we have studied the differences in the gene expression patterns. A total of 386 million clean reads were obtained through RNAseq analysis using the Illumina HiSeq 2000 platform. Clean reads were further assembled de novo into a transcriptome dataset comprising of 514,747 unigenes with N50 length of 578 bp and were further aligned to the public databases. Genebank non-redundant (Nr), Viridiplantae, Gene Ontology (GO), KOG, and KEGG databases classification suggested enrichment of these unigenes in 30 GO categories, 26 KOG, and 127 pathways, respectively. Out of 265,158 genes that were differentially expressed in response to salt treatment, 134,566 and 130,592 genes were significantly up and down-regulated, respectively. Upon placing all the differentially expressed genes (DEG) in known signaling pathways, it was evident that most of the DEGs involved in cytokinin, ethylene, auxin, abscisic acid, gibberellin, and Ca2+ mediated signaling pathways were up-regulated. Furthermore, GO enrichment analysis was performed using REVIGO and up-regulation of multiple genes involved in various biological processes in chilense under salinity were identified. Through pathway analysis of DEGs, "Wnt signaling pathway" was identified as a novel pathway for the response to the salinity stress. Moreover, key genes for salinity tolerance, such as genes encoding proline and arginine metabolism, ROS scavenging system, transporters, osmotic regulation, defense and stress response, homeostasis and transcription factors were not only salt-induced but also showed higher expression in S. chilense as compared to S. lycopersicum. Thus indicating that these genes may have an important role in salinity tolerance in S. chilense. Overall, the results of this study improve our understanding on possible molecular mechanisms underlying salt tolerance in plants in general and tomato in particular.

Transcriptional regulation-mediating ROS homeostasis and physio-biochemical changes in wild tomato (Solanum chilense) and cultivated tomato (Solanum lycopersicum) under high salinity.

Salinity intrusion is one of the biggest problems in the context of sustainable agricultural practices. The major concern and challenge in developing salt-resistance in cultivated crops is the genetic complexity of the trait and lack of natural variability for stress-responsive traits. In this context, tomato wild relatives are important and have provided novel alleles for breeding abiotic stress tolerance including salt tolerance. We provide here a case study, involving tomato wild relative Solanum chilense and cultivated variety Solanum lycopersicum, carried out under high salt stress to investigate comparative transcriptional regulation mediating ROS homeostasis and other physiological attributes. Salt dependent oxidative stress in S. lycopersicum was characterized by a relatively higher H2O2 content, generation of O2 •-, electrolytic leakage and lipid peroxidation whereas reduced content of both ascorbate and glutathione. On the contrary, the robust anti-oxidative system in the S. chilense particularly counteracted the salt-induced oxidative damages by a higher fold change in expression profile of defense-related salt-responsive genes along with the increased activities of anti-oxidative enzymes. We conclude that S. chilense harbours novel genes or alleles for salt stress-related traits that could be identified, characterized, and mapped for its possible introgression into cultivated tomato lines.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

The roles of ubiquitin modifying enzymes in neoplastic disease.

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFß, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy.

Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers.

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress.

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both ß-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress.

Somatic Embryogenesis and Plant Regeneration in Sapindus mukorossi Gaertn. from Leaf-Derived Callus Induced with 6-Benzylaminopurine.

A somatic embryogenesis system was developed for Sapindus mukorossi Gaertn. from leaf explants obtained from fresh flushes of a mature tree. Callus was induced from the midrib region of leaf explants on Murashige and Skoog (MS) medium containing different concentrations of 2,4-dichlorophenoxyacetic acid or 6-benzylaminopurine. Callus induction and somatic embryogenesis was significantly influenced by the size, physiological age, and orientation of leaf explants on the culture medium and plant growth regulators. Adaxial-side-up orientation of leaf explants significantly promoted embryogenesis in comparison with abaxial-side-up orientation. Maximum number of somatic embryos was induced on MS medium supplemented with 8.88 µM 6-benzylaminopurine. Scanning electron microscopy of embryogenic callus revealed somatic embryo origin and the development of globular-, heart-, and cotyledonary-stage somatic embryos. The frequency of maturation as well as germination of somatic embryos was higher on MS medium containing 8.88 µM 6-benzylaminopurine than on medium without 6-benzylaminopurine. Plantlets which developed from somatic embryos were acclimatized successfully with 90 % survival.

Overexpression of WsSGTL1 gene of Withania somnifera enhances salt tolerance, heat tolerance and cold acclimation ability in transgenic Arabidopsis plants.

BACKGROUND: Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant's adaptation to abiotic stress. METHODOLOGY: The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses--salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5. RESULTS: The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana. CONCLUSIONS: Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.