Basic Leucine Zipper Domain Research Articles

Identification and characterization of MKK6 and AP-1 in Anodonta woodiana reveal their potential roles in the host defense response against bacterial challenge

Mitogen-activated protein kinase kinase 6 (MKK6) and activator protein-1 (AP-1) are two of the essential regulatory proteins in the p38 mitogen-activated protein kinase (MAPK) pathway, which participates in the innate immune response to bacterial infections. In this study, molluscan MKK6 (AwMKK6) and AP-1 (AwAP-1) genes were cloned and identified from Anodonta woodiana. The open reading frame (ORF) of AwMKK6 encodes for a putative polypeptide sequence of 345 amino acids containing a conserved serine/threonine protein kinase (S_TKc) domain, a SVAKT motif and a DVD domain. AwAP-1 consists of 294 amino acids including a typical nuclear localization signal (NLS), a Jun domain and a basic region leucine zipper (BRLZ) domain. Quantitative real-time PCR analysis showed that both AwMKK6 and AwAP-1 were widely expressed in all selected tissues of A. woodiana and their transcript levels in hemocytes were significantly upregulated when challenged with Aeromonas hydrophila and lipopolysaccharide (LPS). Additionally, the signaling molecules of the AwMKK6/AwAP-1 pathway including AwTLR4, AwMyD88, AwTRAF6, AwMEKK1, AwMEKK4, AwASK1, AwTAK1 and Awp38 mRNA expression showed a stronger responsiveness to LPS challenge in hemocytes of A. woodiana. RNA interference (RNAi) experiments indicated that the silencing of AwMKK6 or AwAP-1 could decrease the mRNA expression levels of immune effectors (AwTNF, AwLYZ and AwDefense). Subcellular localization studies suggested that AwMKK6 and AwAP-1 were distributed throughout the cells and nucleus, respectively, and their overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. These findings suggest that MKK6 and AP-1 play a major role in the host defense response to bacterial injection, which may make contributions to a better understanding of the immune function of the p38 MAPK pathway in mollusks.

Joint actions of diverse transcription factor families establish neuron-type identities and promote enhancer selectivity.

To systematically investigate the complexity of neuron specification regulatory networks, we performed an RNA interference (RNAi) screen against all 875 transcription factors (TFs) encoded in Caenorhabditis elegans genome and searched for defects in nine different neuron types of the monoaminergic (MA) superclass and two cholinergic motoneurons. We identified 91 TF candidates to be required for correct generation of these neuron types, of which 28 were confirmed by mutant analysis. We found that correct reporter expression in each individual neuron type requires at least nine different TFs. Individual neuron types do not usually share TFs involved in their specification but share a common pattern of TFs belonging to the five most common TF families: homeodomain (HD), basic helix loop helix (bHLH), zinc finger (ZF), basic leucine zipper domain (bZIP), and nuclear hormone receptors (NHR). HD TF members are overrepresented, supporting a key role for this family in the establishment of neuronal identities. These five TF families are also prevalent when considering mutant alleles with previously reported neuronal phenotypes in C. elegans, Drosophila, and mouse. In addition, we studied terminal differentiation complexity focusing on the dopaminergic terminal regulatory program. We found two HD TFs (UNC-62 and VAB-3) that work together with known dopaminergic terminal selectors (AST-1, CEH-43, CEH-20). Combined TF binding sites for these five TFs constitute a cis-regulatory signature enriched in the regulatory regions of dopaminergic effector genes. Our results provide new insights on neuron-type regulatory programs in C. elegans that could help better understand neuron specification and evolution of neuron types.

Intricate coupling between the transactivation and basic-leucine zipper domains governs phosphorylation of transcription factor ATF4 by casein kinase 2

Most transcription factors possess at least one long intrinsically disordered transactivation domain that binds to a variety of coactivators and corepressors and plays a key role in modulating the transcriptional activity. Despite the crucial importance of these domains, the structural and functional basis of transactivation remains poorly understood. Here, we focused on activating transcription factor 4 (ATF4)/cAMP response element-binding protein-2, an essential transcription factor for cellular stress adaptation. Bioinformatic sequence analysis of the ATF4 transactivation domain sequence revealed that the first 125 amino acids have noticeably less propensity for structural disorder than the rest of the domain. Using solution nuclear magnetic resonance spectroscopy complemented by a range of biophysical methods, we found that the isolated transactivation domain is predominantly yet not fully disordered in solution. We also observed that a short motif at the N-terminus of the transactivation domain has a high helical propensity. Importantly, we found that the N-terminal region of the transactivation domain is involved in transient long-range interactions with the basic-leucine zipper domain involved in DNA binding. Finally, in vitro phosphorylation assays with the casein kinase 2 show that the presence of the basic-leucine zipper domain is required for phosphorylation of the transactivation domain. This study uncovers the intricate coupling existing between the transactivation and basic-leucine zipper domains of ATF4, highlighting its potential regulatory significance.

Involvement of the JNK signaling pathway in regulating yolk accumulation in the swimming crab, Portunus trituberculatus

The c-Jun N-terminal kinase (JNK) signaling pathway regulates the development of ovaries and synthesis of vitellogenin (Vg) in the swimming crab Portunus trituberculatus, but the underlying molecular mechanism remains unclear. In this study, we cloned the essential c-fos gene of the JNK signaling pathway using the rapid amplification of complementary DNA end method. The full-length cDNA of c-fos was 2829bp long and had a 1497bp open reading frame encoding a 498 amino acid protein and containing a basic leucine zipper domain. After injecting c-jun-double stranded (ds) RNA to silence the JNK signaling pathway target gene c-jun, the expression levels of key JNK signaling pathway genes (c-jun, JNK, Vg, and c-fos) in the hepatopancreas were significantly down-regulated (P < 0.05). In ovarian tissue, the expression levels of c-jun, c-fos, and Vg genes were significantly down-regulated (P < 0.05), but no significant change was detected for JNK gene expression. Hematoxylin-eosin staining indicated that the oocytes in the c-jun-dsRNA group and the Vg-dsRNA group developed slowly, and an enzyme-linked immunosorbent assay confirmed that the accumulation of vitellin in the ovary was significantly reduced by 56% and 52% in the two groups, respectively. Co-immunoprecipitation results showed that c-Jun and c-fos proteins play a regulatory role by forming a heterodimeric transcription factor activator protein 1. These results indicated that the JNK signaling pathway of P. trituberculatus regulated the synthesis of Vg through the transcription factor activator protein 1, thereby regulating the accumulation of yolk. The results of this study provided a theoretical basis and technical guidance for P. trituberculatus production.

CCAAT/Enhancer-Binding Proteins in Fibrosis: Complex Roles Beyond Conventional Understanding.

CCAAT/enhancer-binding proteins (C/EBPs) are a family of at least six identified transcription factors that contain a highly conserved basic leucine zipper domain and interact selectively with duplex DNA to regulate target gene expression. C/EBPs play important roles in various physiological processes, and their abnormal function can lead to various diseases. Recently, accumulating evidence has demonstrated that aberrant C/EBP expression or activity is closely associated with the onset and progression of fibrosis in several organs and tissues. During fibrosis, various C/EBPs can exert distinct functions in the same organ, while the same C/EBP can exert distinct functions in different organs. Modulating C/EBP expression or activity could regulate various molecular processes to alleviate fibrosis in multiple organs; therefore, novel C/EBPs-based therapeutic methods for treating fibrosis have attracted considerable attention. In this review, we will explore the features of C/EBPs and their critical functions in fibrosis in order to highlight new avenues for the development of novel therapies targeting C/EBPs.

Characterizing the role of Tupaia DNA damage inducible transcript 3 (DDIT3) gene in viral infections

DNA damage inducible transcript 3 (DDIT3, also known as CHOP) belongs to the CCAAT/enhancer-binding protein (C/EBP) family and plays an essential role in endoplasmic reticulum stress. Here, we characterized the potential role of the Chinese tree shrew (Tupaia belangeri chinensis) DDIT3 (tDDIT3) in viral infections. The tDDIT3 protein is highly conserved and has a species-specific insertion of the SQSS repeat upstream of the C-terminal basic-leucine zipper (bZIP) domain. Phylogenetic analysis of DDIT3 protein sequences of tree shrew and related mammals indicated a closer genetic affinity between tree shrew and primates than between tree shrew and rodents. Three positively selected sites (PSSs: Glu83, Pro93, and Ser172) were identified in tDDIT3 based on the branch-site model. Expression analysis of tDDIT3 showed a constitutively expressed level in different tissues and a significantly increased level in tree shrew cells upon herpes simplex virus type 1 (HSV-1) and Newcastle disease virus (NDV) infections. Overexpression of tDDIT3 significantly increased the production of HSV-1 and vesicular stomatitis virus (VSV) in tree shrew primary renal cells (TSPRCs), whereas tDDIT3 knockout in tree shrew stable cell line (TSR6 cells) had an inhibitory effect on virus production. The enhanced effect on viral infection by tDDIT3 was not associated with the three PSSs. Mechanistically, tDDIT3 overexpression inhibited type I IFN signaling. tDDIT3 interacted with tMAVS through CARD and PRR domains, but not with other immune-related factors such as tMDA5, tSTING and tTBK1. Collectively, our results revealed tDDIT3 as a negative regulator for virus infection.

De novo transcriptome sequencing, assembly and characterization of Heliopsis longipes roots vs. leaves to discover putative genes involved in specialized metabolites biosynthesis

Heliopsis longipes is a valuable source of specialized metabolites (or secondary metabolites) with medicinal properties mainly in roots. However, little is known about genes involved in the biosynthesis of these metabolites, primarily due to the lack of genome or transcriptome resources. In this work, the genes of the biosynthetic pathway of the specialized metabolism from H. longipes roots and leaves through de novo RNA sequencing (RNA-Seq) using the platform of Illumina paired-end sequencing were studied. After de novo transcriptome assembly using the software Newbler, a total of 172,342 non-redundant transcripts with an N50 value of 816 bp was obtained. Further functional classification and annotation with Gene Ontology (GO), BLAST2GO, Kyoto Encyclopedia of Genes and Genome (KEGG), and KEGG automatic annotation server (KAAS), revealed that active genes in tissues are predominately involved in the metabolic process and biosynthesis of specialized metabolite pathways. Differential expression analysis of roots vs. leaves using Cuffdiff software (p-value ≤0.05 and log-fold change ratio (log2) ≥1) revealed that differentially expressed genes (DEGs) were in an organ-specific manner, such as in leaf, DEGs were significantly enriched in photosynthesis, while in roots, were a higher enriched function of plant hormone signal transduction. A total of 63 transcripts DEGs were related to 9 specialized metabolites pathways, in roots the most abundant was the phenylpropanoid biosynthesis, and in leaves was the carotenoids biosynthesis. Several regulatory genes including the basic-helix-loop-helix and basic leucine zipper domain, transcriptions factor families involved in the regulation of phenylpropanoids and carotenoid biosynthesis, respectively, were discovered. This study established a global transcriptome dataset for H. longipes. Data shall be useful to study the functional genomics or genetic engineering of this specie. These results will promote the understanding of the genetic mechanism involved in the biosynthesis of specialized metabolites in H. longipes

OMICS approaches towards understanding plant's responses to counterattack heavy metal stress: An insight into molecular mechanisms of plant defense

Under constant climatic change and the existing environment, plants are exposed to several adverse factors such as extreme temperatures, high salt, heavy metals (HMs), drought, and pathogens. Prolonged exposure to such adverse conditions results in phytotoxicity and overall retardation of growth and development. Excess of HMs is one of the significant factors that affects plant growth significantly. Plants develop several defense mechanisms at the cellular and physiological levels to survive HMs stress. However, these internal defense mechanisms are not enough to overcome excessive HMs stress. Plants generate various secondary messengers to activate cell signaling, which results in the activation of several transcriptional responses associated with plant defense. Different transcription factors (TFs) gene families such as WRKY, my elob lastosis (MYB), basic leucine zipper domain (bZIP), basic helix-loop-helix (bHLH), APETALA2/ethylene-responsive element binding protein (AP2/EREBP), Cys2-His2 (C2H2), receptor genes/proteins, and kinases, like mitogen-activated protein kinase (MAPK), play an essential role in the regulation of stress using hormone-mediated pathways under HMs stress. Additionally, phytohormones and their signaling network also contribute to regulating cellular functions at the molecular level to combat HMs stress. Several mechanisms have been explored to understand the defense mechanism of plants under HMs stress. However, many more advanced molecular approaches are required to explore these defense mechanisms. Herein, we have described OMICS’ approaches to improve the plants' internal defense system to counteract HMs stress.

Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia

AbstractMutations of CCAAT/enhancer–binding protein alpha (CEBPAmu) are found in 10% to 15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not seem to improve prognosis. We investigated CEBPAmu for prognosis in 1028 patients with AML, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (P < .001), better overall survival (OS; P < .001) and a lower risk of relapse (P < .001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients: OS, P = .008; the cumulative incidence of relapse, P = .063; patients aged ≤70 years and with intermediate-risk karyotype: OS, P = .008; cumulative incidence of relapse, P = .026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio, 0.3287; P < .001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA-mutated AML.

LncRNA NEAT1 promotes glioma cancer progression via regulation of miR-98-5p/BZW1.

Background: Glioma is the most common malignant tumor in the human central nervous system. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes oncogenesis in various tumors. In the present study, we aimed to examine the role of NEAT1 in altering the properties of gliomas.Methods: Quantitative real-time PCR technology was used to determine the expression levels of relevant genes in tumor tissues and cell lines. The protein expression levels were validated by Western blotting. Cell counting kit-8 (CCK-8) and colony formation assays were used to test the cell proliferation ability. A luciferase reporter assay was used to determine the interactions of the genes. Tumor xenografts were used to detect the role of NEAT1 in gliomas in vivo.Results: We demonstrated that NEAT1 up-regulated glioma cells and negatively correlated with miR-98-5p in glioma tissues. A potential binding region between NEAT1 and miR-98-5p was confirmed by dual-luciferase assays. NEAT1 knockdown inhibited glioma cell proliferation. The inhibition of miR-98-5p rescued the knockdown of NEAT1 in glioma cells. Basic leucine zipper and W2 domain containing protein 1 (BZW1) was identified as a direct target of miR-98-5p. We also identified that BZW1 was positively correlated with NEAT1 in glioma tissues. NEAT1 knockdown inhibited glioma cell proliferation in vivo via miR-98-5p/BZW1.Conclusion: Our results suggest that NEAT1 plays an oncogenic function in glioma progression. Targeting NEAT1/miR-98-5p/BZW1 may be a novel therapeutic treatment approach for glioma patients.

Siberian Wildrye (Elymus sibiricus L.) Abscisic Acid-Insensitive 5 Gene Is Involved in Abscisic Acid-Dependent Salt Response.

Siberian wildrye (Elymus sibiricus L.) is a salt-tolerant, high-quality forage grass that plays an important role in forage production and ecological restoration. Abscisic acid (ABA)-insensitive 5 (ABI5) is essential for the normal functioning of the ABA signal pathway. However, the role of ABI5 from Siberian wildrye under salt stress remains unclear. Here, we evaluated the role of Elymus sibiricus L. abscisic acid-insensitive 5 (EsABI5) in the ABA-dependent regulation of the response of Siberian wildrye to salt stress. The open reading frame length of EsABI5 isolated from Siberian wildrye was 1170 bp, and it encoded a 389 amino acid protein, which was localized to the nucleus, with obvious coiled coil areas. EsABI5 had high homology, with ABI5 proteins from Hordeum vulgare, Triticum monococcum, Triticum aestivum, and Aegilops tauschii. The conserved domains of EsABI5 belonged to the basic leucine zipper domain superfamily. EsABI5 had 10 functional interaction proteins with credibility greater than 0.7. EsABI5 expression was upregulated in roots and leaves under NaCl stress and was upregulated in leaves and downregulated in roots under ABA treatment. Notably, tobacco plants overexpressing the EsABI5 were more sensitive to salt stress, as confirmed by the determining of related physiological indicators. EsABI5 expression affected the ABA and mitogen-activated protein kinase pathways. Therefore, EsABI5 is involved in antisalt responses in these pathways and plays a negative regulatory role during salt stress.

Abstract 2427: Identification of transcription factors activated in glioblastoma multiforme

Abstract Introduction Glioblastoma multiforme (GBM) is the most common intrinsic brain tumor in adults and is universally fatal. Our strategy to study the molecular mechanisms leading to GBM is to investigate the role of transcription factors (TFs) that are differentially expressed between normal and GBM. Previous studies have shown that ZFX is a key TF that shows increased expression in GBM. However, the role of ZFX, and the identity of TFs that may work with ZFX, in driving GBM carcinogenesis is not yet understood. Materials and methods: ChIP-seq was performed to determine ZFX binding sites in the U87 GBM cancer cell line. RNA-seq datasets generated from GBM tissues by The Cancer Genome Atlas (TCGA) were analyzed to identify TFs showing increased expression in GBM. Publicly available H3K27ac chromatin immunoprecipitation (ChIP-seq) and Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) datasets generated from GBM tissues were analyzed to identify active enhancers and TFs that bind to GBM enhancers. Results: We found that a majority of ZFX binding sites in GBM are located at promoters, with ~1000 binding sites being at non-promoter regions. An overlap analysis of ZFX binding sites in multiple cancer cell types showed that a high percentage of ZFX-binding sites at promoter regions are shared across cell types whereas hundreds of GBM-specific binding sites are located at non-promoter regions. Using TCGA GBM RNA-seq datasets, we next identified differentially expressed TFs. For example, we found that MTY1L and NEUROD2, which have been reported to be tumor suppressors, are downregulated while HOX family TFs are upregulated in GBM tissues, as compared to normal tissues. We also identified thousands of enhancers specifically active in GBM, as compared to normal cells, using H3K27ac ChIP-seq and ATAC-seq datasets. TF motif analysis of the identified enhancers revealed that motifs of Basic Leucine Zipper Domain (bZIP), CAAT box-binding transcription factor (CTF), homeobox (HOX), signal transducer and activator of transcription (STAT), and helix-turn-helix (HTH) family were enriched. Conclusion: By integrating transcriptomic and epigenomic datasets, we identified and characterized TFs activated in GBM. We are currently investigating how ZFX cooperates with the TFs we identified as bound to the GBM-specific enhancers. These studies will provide insight into the molecular mechanisms underlying GBM. Citation Format: Zexun Wu, Shannon Schreiner, Peggy J. Farnham, Suhn K. Rhie. Identification of transcription factors activated in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2427.

Venetoclax sensitivity in multiple myeloma is associated with B-cell gene expression

AbstractVenetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain–binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).

Expression and Functional Characterization of c-Fos Gene in Chinese Fire-Bellied Newt Cynops Orientalis.

c-Fos is an immediate-early gene that modulates cellular responses to a wide variety of stimuli and also plays an important role in tissue regeneration. However, the sequence and functions of c-Fos are still poorly understood in newts. This study describes the molecular cloning and characterization of the c-Fos gene (Co-c-Fos) of the Chinese fire-bellied newt, Cynops orientalis. The full-length Co-c-Fos cDNA sequence consists of a 1290 bp coding sequence that encoded 429 amino acids. The alignment and phylogenetic analyses reveal that the amino acid sequence of Co-c-Fos shared a conserved basic leucine zipper domain, including a nuclear localization sequence and a leucine heptad repeat. The Co-c-Fos mRNA is widely expressed in various tissues and is highly and uniformly expressed along the newt limb. After limb amputation, the expression of Co-c-Fos mRNA was immediately upregulated, but rapidly declined. However, the significant upregulation of Co-c-Fos protein expression was sustained for 24 h, overlapping with the wound healing stage of C. orientalis limb regeneration. To investigate if Co-c-Fos participate in newt wound healing, a skin wound healing model is employed. The results show that the treatment of T-5224, a selective c-Fos inhibitor, could largely impair the healing process of newt’s skin wound, as well as the injury-induced matrix metalloproteinase-3 upregulation, which is fundamental to wound epithelium formation. These data suggest that Co-c-Fos might participate in wound healing by modulating the expression of its potential target gene matrix metalloproteinase-3. Our study provides important insights into mechanisms that are responsible for the initiation of newt limb regeneration.

Peach PpSnRK1α interacts with bZIP11 and maintains trehalose balance in plants

The nonreducing disaccharide trehalose is widespread in nature. It plays a very important role in plant growth and development. In plants, trehalose is present in trace amounts. High concentration of trehalose disrupts energy balance and inhibits normal growth and development. Studies have shown that high levels of trehalose and trehalose-6-phosphate (T6P), the metabolic precursor of trehalose, inhibit sucrose non-fermenting-1-related protein kinase1 (SnRK1) activity, which affect plant growth and development. However, the role of SnRK1, the energy balance center, in the regulation of trehalose metabolism in plants is unknown. In this study, exogenous trehalose at higher concentrations inhibited the expression of SnRK1 genes, especially PpSnRK1α in peach (Prunus persica) seedlings. This change in gene expression was dependent on trehalose concentration. Furthermore, overexpression of peach PpSnRK1α in Arabidopsis thaliana significantly promoted trehalase activity, reduced T6P content, and suppressed the trehalose synthesis related genes (TPSs, TPPB) expression, promoted the trehalose metabolism of gene expression (TRE1), in addition the transgenic plants alleviated photosynthetic product distribution imbalance (aboveground and underground parts), and enhanced root growth. Yeast two-hybrid and bimolecular fluorescence assays revealed the interaction between PpSnRK1α and peach basic domain leucine zipper transcription factor 11 (PpbZIP11), a key transcription factor of trehalose metabolism, in the nucleus. To summarize, PpSnRK1α overexpression improved bZIP11 transcriptional activity and regulated trehalose metabolism to protect the plants against trehalose-induced damage. This study preliminarily explained the mechanism of SnRK1 regulating trehalose metabolism balance in plants, which laid a foundation for further understanding of energy metabolism and function of SnRK1 in plants.

Marek\u2019s disease virus Meq oncoprotein interacts with chicken HDAC 1 and 2 and mediates their degradation via proteasome dependent pathway

Marek’s disease virus (MDV) encodes a basic-leucine zipper (BZIP) protein, Meq, which is considered the major MDV oncoprotein. It has been reported that the oncogenicity of Meq is associated with its interaction with C-terminal binding protein 1 (CtBP), which is also an interaction partner of Epstein-Barr virus encoded EBNA3A and EBNA3C oncoproteins. Since both EBNA3C and CtBP interact with histone deacetylase 1 (HDAC1) and HDAC2, we examined whether Meq shares this interaction with chicken HDAC1 (chHDAC1) and chHDAC2. Using confocal microscopy analysis, we show that Meq co-localizes with chHDAC1 and chHDAC2 in the nuclei of MDV lymphoblastoid tumor cells. In addition, immunoprecipitation assays demonstrate that Meq interacts with chHDAC1 and chHDAC2 in transfected cells and MDV lymphoblastoid tumor cells. Using deletion mutants, interaction domains were mapped to the N-terminal dimerization domain of chHDAC1 and chHDAC2, and the BZIP domain of Meq. Our results further demonstrate that this interaction mediates the degradation of chHDAC1 and chHDAC2 via the proteasome dependent pathway. In addition, our results show that Meq also induces the reduction of global ubiquitinated proteins through a proteasome dependent pathway. In conclusion, our results provide evidence that Meq interacts with chHDAC1 and chHDAC2, and induces their proteasome dependent degradation.

BZW2/5MP1 acts as a promising target in hepatocellular carcinoma.

Basic leucine zipper and W2 domain 2 (BZW2), also known as 5MP1, is a protein related to translation regulation. Evidence from previous studies indicates that BZW2 is involved in tumorigenesis in several cancers. However, little is known about the role of BZW2 in hepatocellular carcinoma (HCC). In this study, we first analyzed the gene expression profile of BZW2 in multiple HCC datasets. Next, we explored the biological effects of BZW2 in HCC cell lines. BZW2 was overexpressed in different HCC cohorts. Multivariate analysis confirmed that increased BZW2 expression is an independent prognostic indicator of shorter overall survival. BZW2 coexpressed genes were mainly enriched in the biological processes of ribonucleoprotein complex biogenesis, rRNA metabolism, translational initiation, and negative regulation of metabolic processes. BZW2 depletion reduced proliferation, clonality, and invasion and increased apoptosis in MHCC97-H cells. Furthermore, BZW2 overexpression or knockdown enhanced or impaired c-Myc expression, respectively. Overall, these findings identified BZW2 as a biomarker of HCC and provided novel insight that the effect of BZW2 on the translatome is a potential mechanism that promotes HCC progression via the c-Myc pathway.

Synchronization of proline, ascorbate and oxidative stress pathways under the combination of salinity and heat in tomato plants

Adverse environmental conditions have a devastating impact on plant productivity. In nature, multiple abiotic stresses occur simultaneously, and plants have evolved unique responses to cope against this combination of stresses. Here, we coupled genome-wide transcriptional profiling and untargeted metabolomics with physiological and biochemical analyses to characterize the effect of salinity and heat applied in combination on the metabolism of tomato plants. Our results demonstrate that this combination of stresses causes a unique reprogramming of metabolic pathways, including changes in the expression of 1388 genes and the accumulation of 568 molecular features. Pathway enrichment analysis of transcript and metabolite data indicated that the proline and ascorbate pathways act synchronously to maintain cellular redox homeostasis, which was supported by measurements of enzymatic activity and oxidative stress markers. We also identified key transcription factors from the basic Leucine Zipper Domain (bZIP), Zinc Finger Cysteine-2/Histidine-2 (C2H2) and Trihelix families that are likely regulators of the identified up-regulated genes under salinity + heat combination. Our results expand the current understanding of how plants acclimate to environmental stresses in combination and unveil the synergy between key cellular metabolic pathways for effective ROS detoxification. Our study opens the door to elucidating the different signaling mechanisms for stress tolerance.

Cloning and functional analysis of c/ebpα as negative regulator of dmrt1 in Chinese tongue sole (Cynoglossus semilaevis).

c/ebpα is a member of the C/EBP family of transcription factors, which are involved in cell growth and differentiation and have a conserved basic leucine zipper (bZIP) domain. However, little is known about its function in sex determination and differentiation. In the present study, c/ebpα was cloned from the gonads of Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of c/ebpα was 1583bp, with a 198-bp 5' UTR, a 446-bp 3' UTR, and a 939-bp open reading frame encoding a 312-amino acid peptide. qRT-PCR revealed that c/ebpα was predominantly expressed in undifferentiated gonads of male C. semilaevis at 30 dpf and 60 dpf and peaked at 60 dpf. Expression levels of c/ebpα in the testis were constantly higher than those in ovaries at all developmental stages. Moreover, a dual-luciferase assay revealed that c/ebpα could negatively regulate the male-determining gene dmrt1 in vitro. These results provide fundamental information indicating that C. semilaevis c/ebpa might be involved in early gonadal differentiation and functions as a negative regulator of dmrt1 by repressing its transcription.

IPO11 Regulates the Nuclear Import of BZW1/2 and Is Necessary for AML Cells and Stem Cells

While most patients with acute myeloid leukemia (AML) achieve remission with initial therapy, the majority relapse leading to poor overall survival. Relapse is frequently driven by a rare subset of leukemic stem cells (LSC). Understanding biological mechanisms that maintain LSCs will help identify new therapeutic strategies for this disease. To identify such vulnerabilities, we overlaid the results of a genome-wide CRISPR screen with the expression of genes enriched in functionally defined LSCs. Through our CRISPR screen, we identified 570 genes whose knockout reduced the growth and viability of OCI-AML2 cells. Essential genes for growth and viability by our CRIPSR screen were enriched in the LSC+ population. By overlaying the hits from our CRISPR screen with genes upregulated in LSCs, we identified IPO11, as a top hit, with 7.5-fold increase in the LSC+ fraction compared to the LSC- fraction. IPO11 is a member of the importin-β family of proteins and facilitates the import of protein cargo into the nucleus. Further analysis showed that IPO11 was upregulated in LSC+ (engrafting) vs. LSC- (non-engrafting) primary AML samples, CD34+ vs CD34- AML samples, undifferentiated progenitor vs. myeloid cluster AML samples, and relapse vs de novo AML. IPO11 was increased in AML cells compared to normal hematopoietic cells and increased IPO11 expression was associated with decreased overall survival in AML. By immunoblotting, IPO11 protein was increased in primary AML (n=4) compared to normal hematopoietic cells (n=4). To determine whether IPO11 is necessary for AML growth and viability, we knocked down IPO11 in OCI-AML2, TEX and NB4 leukemia cells with shRNA in lentiviral vectors. Knockdown of IPO11 reduced AML growth and viability by 80-90%. In contrast, knockdown of another importin-β family member, IPO5, that was not a hit in our CRIPSR screen, did not reduce AML growth and viability. Knockdown of IPO11 increased differentiation of AML cells as evidenced by the changes in gene expression, decreased chromatin accessibility, increased CD11b expression and increased non-specific esterase staining. Finally, knockdown of IPO11 reduced the engraftment of TEX cells and the low passage primary AML 8227 cells into immune deficient mice by over 90%. Importantly, IPO11 knockdown reduced engraftment of primary AML cells into mouse marrow. To identify novel cargos of IPO11, we performed proximity-dependent biotin labeling (BioID) coupled with mass spectrometry and identified proteins that interacted with IPO11. Among the top hits were BZW1 and BZW 2 (Basic leucine zipper and W2 domains 1 and 2). BZW1 and BZW2 are members of the bZIP super family of transcription factors. Knockdown of IPO11 reduced levels of BZW1 in the nucleus detected by immunoblotting and confocal microscopy. Commercial antibodies could not detect BZW2. To determine if the nuclear import of BZW1 and 2 were functionally important for the effects of IPO11 on AML stem cell function and differentiation, we knocked down BZW1 and BZW2. Dual knockdown of BZW1 and BZW2 (but not individual) mimicked the effects of IPO11 inhibition and decreased the growth and viability of AML cells. Changes in gene expression after BZW1/2 knockdown were similar to IPO11 knockdown with enrichment in myeloid-differentiated genes. By pathway analysis, we identified that IPO11 knockdown, as well as BZW1/2 knockdown decreased expression of MYC target genes, suggesting a mechanism by which these proteins regulate AML stem cell function. Thus, in summary, we identified IPO11 as an essential gene for the viability of AML cells and stem cells. This work highlights a previously unappreciated role of the protein import pathway in regulating AML stem cell function and highlights a potential new therapeutic target for AML. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .