Transcriptional Overexpression Research Articles

Transcription Factor PdTP1 Regulates the Biotransformation of Limonene to α-Terpineol and the Growth of Penicillium digitatum.

α-Terpineol, an alcoholic monoterpene with lilac-like aroma, possesses diverse biological activities and has found applications in the food, pharmaceutical, cosmetic, and agricultural industries. Our previous studies indicated that gene PdTP1 was highly expressed in Penicillium digitatum DSM 62840 during the biotransformation of limonene to α-terpineol, while its actual biological functions are not fully understood. Here, PdTP1 was functionally characterized with bioinformatics analysis, subcellular localization, transcriptional activation activity, overexpression, and RNA interference (RNAi) silencing and RNA-seq analysis. Results showed that PdTP1 protein contained a GAL4-like Zn2Cys6 DNA-binding domain and a fungal_trans domain, was located in the nucleus and cell membrane and presented transcriptional activation effect, suggesting that PdTP1 encoded a Zn2Cys6 type transcription factor. Overexpression of PdTP1 in P. digitatum promoted limonene biotransformation and increased α-terpineol production, and opposite results were observed after the silencing of PdTP1. Moreover, transcription factor PdTP1 was found to affect the growth of P. digitatum and participate in ionic stress and oxidative stress responses. RNA-seq data revealed that altering the PdTP1 expression influenced the expression of some genes related to terpene metabolism or biosynthesis, fungal growth, and stress responses. In summary, PdTP1, which encoded a Zn2Cys6 transcription factor, played important roles in improving the production of α-terpineol from limonene and regulating fungal growth and environmental stress responses.

EPEN-26. DRUG SCREENING IN PATIENT-DERIVED MODELS OF POSTERIOR FOSSA A EPENDYMOMA REVEALS NOVEL PHARMACOLOGICAL VULNERABILITIES

Abstract Ependymoma (EPN) is the third most common malignant intracranial pediatric brain tumor. The most common and aggressive EPN subgroup, posterior fossa ependymoma group A (PFA-EPN), occurs predominantly in younger children and has a 5-year progression free survival of only 33%. Despite progress in the molecular characterization and subtyping of EPN, the standard treatment remains surgery with adjuvant radiation therapy. There is currently no approved role for chemotherapy in PFA-EPN. As such, identifying novel therapies for PFA-EPN is an important unmet medical need. To define therapeutic sensitivities for PFA-EPN, we performed a drug screen on three patient-derived PFA-EPN cell lines. We found that PFA-EPN cell lines were sensitive to several clinically relevant drugs, most notably those targeting epigenetic regulators. This is of a particular interest as a common molecular feature of PFA-EPN is the global reduction of the repressive post-translational histone modification H3K27me3 due to transcriptional EZHIP overexpression or H3 K27M mutations. To further characterize our PFA-EPN cell lines, we performed optical genome mapping analysis, revealing previously undescribed structural variants affecting genes encoding proteins responsible for signaling and transcription. One PFA-EPN sample that showed profound sensitivity to EGFR inhibitors, also had a duplication of a genomic region containing the EGFR ligand encoding genes, AREG, BTC, EREG, and EPGN. We are currently working to validate these results in larger cohorts of PFA-EPN models. Collectively, these findings provide new clues on drugs, signaling and putative therapeutic targets in these rare yet aggressive tumors.

Citrus transcription factor CsERF098 cooperates with CsWRKY70 to participate in response of fruit to green mold

As plant-specific transcription factors (TFs), AP2/ERF can participate in regulating disease resistance in plants. Currently, researches on regulation of disease resistance of AP2/ERF TFs were mainly focused on model plants, and the regulatory mechanisms in non-model plants such as citrus fruit, an important economical fruit which is seriously infected by Penicillium digitatum, are still unclear. In this study, transcriptional activator CsERF098 overexpression enhanced the resistance of citrus fruit against green mold. Transcriptome sequencing revealed that CsERF098 was involved in four disease resistance pathways, including salicylic acid (SA) signaling pathway, lignin and waxy biosynthesis pathway, chitinase and jasmonic acid signaling pathway. CsERF098 could bind to promoter of SA pathway CsSAMT and CsSABP2, activate the transcriptional activities of CsSAMT, CsSABP2, CsTLP1b and CsTLP. Furthermore, the interaction of CsERF098 and CsWRKY70 was confirmed, which enhanced the trans-activation of CsERF098 on CsSAMT and CsSABP2. These results indicated that CsERF098 could mediate salicylic acid signaling pathway and enhance the synergistic regulation of CsSAMT and CsSABP2 through interaction with CsWRKY70, thereby in controlling the resistance of citrus fruit to Penicillium digitatum. The present study provides new thoughts on the role and mechanism of AP2/ERF transcription factor involved in regulating citrus disease resistance.

Conjugation-Mediated Plasmid Transfer Enables Genetic Modification of Diverse Bacillus Species.

Performing genetic manipulations in Bacillus strains is often hindered by difficulty in identifying conditions appropriate for DNA uptake. This shortcoming limits our understanding of the functional diversity within this genus and the practical application of new strains. We have developed a simple method for increasing the genetic tractability of Bacillus spp. through conjugation-mediated plasmid transfer via a diaminopimelic acid (DAP) auxotrophic Escherichia coli donor strain. We observe transfer into representatives of the Bacillus clades subtilis, cereus, galactosidilyticus, and Priestia megaterium and successfully applied this protocol to 9 out of 12 strains attempted. We utilized the BioBrick 2.0 plasmids pECE743 and pECE750, as well as the CRISPR plasmid pJOE9734.1, to generate a xylose-inducible green-fluorescent protein (GFP)-expressing conjugal vector, pEP011. The use of xylose-inducible GFP ensures ease of confirming transconjugants, which enables users to quickly rule out false positives. Additionally, our plasmid backbone offers the flexibility to be used in other contexts, including transcriptional fusions and overexpression, with only a few modifications. IMPORTANCE Bacillus species are widely used to produce proteins and to understand microbial differentiation. Unfortunately, outside a few lab strains, genetic manipulation is difficult and can prevent thorough dissection of useful phenotypes. We developed a protocol that utilizes conjugation (plasmids that initiate their own transfer) to introduce plasmids into a diverse range of Bacillus spp. This will facilitate a deeper study of wild isolates for both industrial and pure research uses.

Cell cycle kinases (AUKA, CDK1, PLK1) are prognostic biomarkers and correlated with tumor-infiltrating leukocytes in HBV related HCC

Hepatocellular carcinoma (HCC) is one of the high incidence cancers and third leading cause of cancer-related mortality. HBV is the top most risk factor accounting for 50–80% of the HCC cases. Kinases: Aurora kinase A (AURKA), cyclin-dependent kinase (CDK1) and Polo-like kinase 1 (PLK1), the key regulators of cell mitosis are overexpressed in varieties of cancers including HCC. However, the exact role of these genes in prognosis of HCC is not fully unveiled. In addition, there is no such an accurate prognostic biomarker for HBV-related HCC. To address this issue, we performed a multidimensional analysis of AURKA, CDK1 and PLK1 with a series of publicly available databases in multiple cancers and with experimental validation in HBV-related HCC tissues. Overexpression of AURKA, CDK1 and PLK1 was found in multiple cancers including HCC. Elevated expression of these genes could result from lowered DNA methylation and genomic alterations. Transcriptional overexpression was significantly correlated with poor prognosis of HCC patients. The expression levels were also significantly positively associated with tumor grades and stages. Furthermore, the expression levels of these genes had a strong correlation with infiltration of immune cells. Our analysis shows that AURKA, CDK1 and PLK1 are correlated with immune infiltration and are the prognostic biomarkers for HBV-induced HCC. Communicated by Ramaswamy H. Sarma

Physiological analysis of the improved ε-polylysine production induced by reactive oxygen species

Epsilon-poly-L-lysine (ε-PL) is produced by Streptomyces species in acidic and aerobic conditions, which inevitably induces rapid generation of reactive oxygen species (ROS). The devastating effects of ROS on biomolecules and cell vitality have been well-studied, while the positive effects of ROS are rarely reported. In this study, we found that a proper dose of intracellular ROS (about 3.3 μmol H2O2 /g DCW) could induce a physiological modification to promote the ε-PL production (from 1.2 to 1.5 g/L). It resulted in larger sizes of colony and mycelial pellets as well as vibrant, aggregated, and more robust mycelia, which were of high capability of ROS detoxication. Physiological studies showed that appropriate doses of ROS activated the metabolism of the pentose phosphate pathway at both transcriptional and enzymatic levels, which was beneficial for biomass accumulation. The biosynthesis of lysine was also promoted in terms of transcriptional regulatory overexpression, increased transcription and enzymatic activity of key genes, larger pools of metabolites in the TCA cycle, replenishment pathway, and diaminoheptanedioic acid pathway. In addition, energy provision was ensured by activated metabolism of the TCA cycle, a larger pool of NADH, and higher activity of the electron transport system. Increased transcription of HrdD and pls further accelerated the ε-PL biosynthesis. These results indicated that ROS at proper intracellular dose could act as an inducing signal to activate the ε-PL biosynthesis, which laid a foundation for further process regulation to maintain optimal ROS dose in industrial ε-PL production and was of theoretical and practical significance. • A proper dose of intracellular ROS positively influences the ε-PL production. • Proper dose of ROS enhanced the mycelial activity and antioxidative capability. • ROS increased lysine synthesis metabolism, energy provision and pls expression.

Short-term effects of estradiol and bisphenol A on gene expression associated with early head mineralization in the seabass Dicentrarchus labrax

IntroductionNatural and synthetic estrogens are pollutants found in aquatic ecosystems at low concentrations reaching ng.L-1 to μg.L-1. At these concentrations, they are able to interfere with the fish endocrine system. When waterborne exposure occurs at early life stages, when blood estrogens concentrations are low, this may have significant consequences for estrogen-sensitive functions such as skeletal development.MethodsTo better understand how (xeno)estrogens may affect early head mineralization, 12 days post-hatch larvae of the European seabass Dicentrarchus labrax were experimentally exposed for 4 days to the natural estrogen estradiol E2 and to the xenoestrogen bisphenol A (BPA), both used at either regulatory concentration of water quality or a 100 times higher concentration. Head mineralization level was assessed using Alizarin red staining, together with the relative quantification of mRNA expression levels of several genes playing key roles in skeletogenesis and estrogen signaling pathways.ResultsWe showed that (xeno)estrogen exposure at early larval stage increases the expression of skeleton-associated genes: matrix proteins encoding genes (col1a2, col2a1a, col2a1b, bgp1a, bgp1b, sparc), proteolytic enzyme encoding genes (ctsk) and transcription and signaling factors (sox9a, sox9b, ihha, runx2, rankl). Although transcriptional overexpression of these genes was significant in larvae exposed to 40 ng.L-1 E2 and to 1.6 and 160 μg.L-1 BPA, increased mineralization was detected only in E2-exposed larvae, suggesting a difference in head skeleton development and remodeling in BPA-treated larvae.DiscussionOur results suggest that these phenotypic differences could be due to the implication of other estrogenic signaling pathways involving both nuclear and membrane-bound estrogen receptors (ERs and GPERs), but also estrogen-related receptors (ERRs). This study brings new insights into the regulatory mechanisms of skeletogenesis by E2 and BPA and into the effects of waterborne exposure to (xeno)estrogens on the early skeletal development of teleost fishes.

Glutathione S-transferase (GST) genes and their function associated with pyrethroid resistance in the malaria vector Anopheles sinensis.

Glutathione S-transferases (GSTs), a multifunctional protein family, are involved in insecticide resistance. However, a systematic analysis of GSTs in Anopheles sinensis, an important vector for malaria transmission, is lacking. In this study, we investigated the diversity and characteristics of GST genes, and analyzed their expression patterns and functions associated with insecticide resistance in this species. We identified 32 putative cytosolic and three putative microsomal GST genes in the An.sinensis genome. Transcriptome analysis showed that GSTs were highly expressed in larvae, and mainly expressed in the antennae, midgut and Malpighian tubules of adults. In addition, we found that GSTd2 and GSTe2 were significantly upregulated in four An.sinensis pyrethroid-resistant field populations. Furthermore, silencing of GSTd2 and GSTe2 significantly increased the susceptibility of An.sinensis to deltamethrin, and recombinant GSTd2 and GSTe2 exhibited high enzymatic activity in the metabolism of 1-chloro-2,4-dinitrobenzene and dichlorodiphenyltrichloroethane (DDT). These results showed that GSTs are involved in the development of insecticide resistance in An.sinensis through transcriptional overexpression and enzymatic metabolization, facilitating our understanding of insecticide resistance in insects. © 2022 Society of Chemical Industry.

A potato intragene overexpressing GSL1 confers resistance to Pectobacterium atrosepticum

ABSTRACT An intragene involves the rearrangement of gene configurations based on switching coding sequences and regulatory regions within a species. The overexpression of specific GSL (Gibberellin Stimulated-Like) genes is known to confer enhanced disease resistance. A potato intragenic expression cassette, with the potato GSL1 gene placed under the regulatory control of the light-inducible Lhca3 gene from potato, was designed and constructed. The resulting intragene was transformed using Agrobacterium-mediated gene transfer into potato cultivar Iwa to investigate whether overexpression of the potato GSL1 intragene can confer disease resistance. The transgenic status of the resulting plants was confirmed by PCR. The majority of the transgenic potato lines were determined to overexpress the GSL1 gene at the mRNA level using quantitative RT–PCR analysis. From these results, seven lines were selected for further characterisation and thoroughly evaluated in bioassays for resistance to Pectobacterium atrosepticum which causes blackleg disease in potato. These pathogenicity bioassays demonstrated that high transcriptional overexpression of GSL1 confers high resistance to blackleg disease. This confirms a functional role of the GSL1 gene in plant defence against pathogens and provides an intragenic approach for disease resistance in potatoes.

The histone demethylase Lsd1 regulates multiple repressive gene programs during T cell development.

Analysis of the transcriptional profiles of developing thymocytes has shown that T lineage commitment is associated with loss of stem cell and early progenitor gene signatures and the acquisition of T cell gene signatures. Less well understood are the epigenetic alterations that accompany or enable these transcriptional changes. Here, we show that the histone demethylase Lsd1 (Kdm1a) performs a key role in extinguishing stem/progenitor transcriptional programs in addition to key repressive gene programs during thymocyte maturation. Deletion of Lsd1 caused a block in late T cell development and resulted in overexpression of interferon response genes as well as genes regulated by the Gfi1, Bcl6, and, most prominently, Bcl11b transcriptional repressors in CD4+CD8+ thymocytes. Transcriptional overexpression in Lsd1-deficient thymocytes was not always associated with increased H3K4 trimethylation at gene promoters, indicating that Lsd1 indirectly affects the expression of many genes. Together, these results identify a critical function for Lsd1 in the epigenetic regulation of multiple repressive gene signatures during T cell development.

Fibroblast Growth Factor Receptor (FGFR) Signaling in GIST and Soft Tissue Sarcomas.

Sarcomas are a heterogeneous group of rare malignancies originating from mesenchymal tissues with limited therapeutic options. Recently, alterations in components of the fibroblast growth factor receptor (FGFR) signaling pathway have been identified in a range of different sarcoma subtypes, most notably gastrointestinal stromal tumors, rhabdomyosarcomas, and liposarcomas. These alterations include genetic events such as translocations, mutations, and amplifications as well as transcriptional overexpression. Targeting FGFR has therefore been proposed as a novel potential therapeutic approach, also in light of the clinical activity shown by multi-target tyrosine kinase inhibitors in specific subtypes of sarcomas. Despite promising preclinical evidence, thus far, clinical trials have enrolled very few sarcoma patients and the efficacy of selective FGFR inhibitors appears relatively low. Here, we review the known alterations of the FGFR pathway in sarcoma patients as well as the preclinical and clinical evidence for the use of FGFR inhibitors in these diseases. Finally, we discuss the possible reasons behind the current clinical data and highlight the need for biomarker stratification to select patients more likely to benefit from FGFR targeted therapies.

Molecular and functional characterization of three novel carboxylesterases in the detoxification of permethrin in the mosquito, Culex quinquefasciatus.

Carboxylesterases (CarEs) belong to a super family of multifunctional enzymes associated with the degradation of endogenous and exogenous compounds. Many insect CarEs are known to play important roles in catalyzing the hydrolysis of organophosphates (OPs), carbamates, and synthetic pyrethroids (SPs). The elevation of esterase activity through gene amplification and overexpression of estα2 and estβ2 genes contributes to the development of resistance to OP insecticides in the mosquito Culex quinquefasciatus. Three additional CarE genes are upregulated in permethrin-resistant Cx. quinquefasciatus according to an RNA-seq analysis, but their function remains unknown. In this study, we, for the first time, characterized the function of these three novel genes using in vitro protein expression, an insecticide metabolism study and molecular docking analysis. All three CarE genes were significantly overexpressed in resistant mosquito larvae, but not adults, compared to susceptible strain. No gene copy differences in these three genes were found in the mosquitoes tested. In vitro high-performance liquid chromatography (HPLC) revealed that CPIJ018231, CPIJ018232, and CPIJ018233 metabolized 30.4% ± 2.9%, 34.7% ± 6.8%, and 23.2% ± 2.2% of the permethrin, respectively. No mutations in resistant strains might significantly affect their CarE hydrolysis ability. A docking analysis further confirmed that these three CarEs from resistant strain all potentially metabolize permethrin. Taken together, these three carboxylesterase genes could play important roles in the development of permethrin resistance in Cx. quinquefasciatus larvae through transcriptional overexpression, metabolism, and detoxification.

GLI1 reduces drug sensitivity by regulating cell cycle through PI3K/AKT/GSK3/CDK pathway in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma‐associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

Identification, Biological Characteristics, and Active Site Residues of 3-Ketosteroid Δ1-Dehydrogenase Homologues from Arthrobacter simplex.

3-Ketosteroid Δ1-dehydrogenase (KsdD) is the key enzyme responsible for Δ1-dehydrogenation, which is one of the most valuable reactions for steroid catabolism. Arthrobacter simplex has been widely used in the industry due to its superior bioconversion efficiency, but KsdD information is not yet fully clear. Here, five KsdD homologues were identified in A. simplex CGMCC 14539. Bioinformatic analysis indicated their distinct properties and structures. Each KsdD was functionally confirmed by transcriptional response, overexpression, and heterologous expression. The substantial difference in substrate profiles might be related to the enzyme loop structure. Two promising enzymes (KsdD3 and KsdD5) were purified and characterized, exhibiting strong organic solvent tolerance and clear preference for 4-ene-3-oxosteroids. KsdD5 seemed to be more versatile due to good activity on substrates with or without a substituent at C11 and high optimal temperature and also possessed unique residues. It is the first time that KsdDs have been comprehensively disclosed in the A. simplex industrial strain.

Leonurus sibiricus root extracts decrease airway remodeling markers expression in fibroblasts.

Bronchial asthma is believed to be provoked by the interaction between airway inflammation and remodeling. Airway remodeling is a complex and poorly understood process, and controlling it appears key for halting the progression of asthma and other obstructive lung diseases. Plants synthesize a number of valuable compounds as constitutive products and as secondary metabolites, many of which have curative properties. The aim of this study was to evaluate the anti-remodeling properties of extracts from transformed and transgenic Leonurus sibiricus roots with transformed L. sibiricus roots extract with transcriptional factor AtPAP1 overexpression (AtPAP1). Two fibroblast cell lines, Wistar Institute-38 (WI-38) and human fetal lung fibroblast (HFL1), were incubated with extracts from transformed L. sibiricus roots (TR) and roots with transcriptional factor AtPAP1 over-expression (AtPAP1 TR). Additionally, remodeling conditions were induced in the cultures with rhinovirus 16 (HRV16). The expressions of metalloproteinase 9 (MMP)-9, tissue inhibitor of metalloproteinases 1 (TIMP-1), arginase I and transforming growth factor (TGF)-β were determined by quantitative polymerase chain reaction (qPCR) and immunoblotting methods. AtPAP1 TR decreased arginase I and MMP-9 expression with no effect on TIMP-1 or TGF-β mRNA expression. This extract also inhibited HRV16-induced expression of arginase I, MMP-9 and TGF-β in both cell lines (P<0·05) Our study shows for the first time to our knowledge, that transformed AtPAP1 TR extract from L. sibiricus root may affect the remodeling process. Its effect can be attributed an increased amount of phenolic acids such as:chlorogenic acid, caffeic acid or ferulic acid anddemonstrates the value of biotechnology in medicinal research.

Eravacycline susceptibility was impacted by genetic mutation of 30S ribosome subunits, and branched-chain amino acid transport system II carrier protein, Na/Pi cotransporter family protein in Staphylococcus aureus

BackgroundOur previous research indicated the excellent in vitro antibacterial activity of Eravacycline (Erava) and its heteroresistance frequency against clinical Staphylococcus aureus isolates. In this study, we further aimed to investigate the mechanisms of Erava resistance and heteroresistance in S. aureus. Eight parental S. aureus isolates were induced under Erava pressure in vitro and the Erava-resistant isolates were selected and identified. Then, the genetic mutations of 30S ribosomal subunits were analyzed by PCR and sequence alignment. RT-qPCR analysis were performed to compare the relative expression of eight candidate genes impacting the susceptibility of tetracycline (Tet) between the resistant or heteroresistant and parental isolates. Furthermore, the in vitro overexpression vectors of three selected candidate genes were constructed to test their impact on the heteroresistance and resistance of Erava in S. aureus.ResultsThe MICs elevation in Erava-induced resistant S. aureus isolates were identified and the increasing MICs values of another two Tet class antibiotics, including both omadacycline (Omada) and tigecycline (Tige) were also tested. Genetic mutations in 30S ribosomal protein S10 were found frequently in Erava-derived resistant isolates. RT-qPCR analysis and the in vitro overexpression experiments indicated that USA300HOU_RS00550 (an Na/Pi cotransporter family protein) and USA300HOU_RS01625 (a branched-chain amino acid transport system II carrier protein) contributed to Erava heteroresistance in S. aureus.ConclusionGenetic mutation of 30S ribosome subunits contributed to Erava resistance, and the transcriptional overexpression of USA300HOU_RS01625 and USA300HOU_RS00550 also participated in the occurrence of Erava heteroresistance in S. aureus.

DUF3669, a \u201cdomain of unknown function\u201d within ZNF746 and ZNF777, oligomerizes and contributes to transcriptional repression

BackgroundZNF746 and ZNF777 belong to a subset of the large Krüppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the “domain of unknown function 3669” (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson’s disease.ResultsHere we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669’s impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved “MLE” sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein.ConclusionDUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes

To discover epigenetic changes that may underly neuroblastoma pathogenesis, we identified differentially methylated genes in neuroblastoma cells compared to neural crest cells, the presumptive precursors cells for neuroblastoma, by using genome-wide DNA methylation analysis. We previously described genes that were hypermethylated in neuroblastoma; in this paper we report on 67 hypomethylated genes, which were filtered to select genes that showed transcriptional over-expression and an association with poor prognosis in neuroblastoma, highlighting GATA3 for detailed studies. Specific methylation assays confirmed the hypomethylation of GATA3 in neuroblastoma, which correlated with high expression at both the RNA and protein level. Demethylation with azacytidine in cultured sympathetic ganglia cells led to increased GATA3 expression, suggesting a mechanistic link between GATA3 expression and DNA methylation. Neuroblastomas that had completely absent GATA3 methylation and/or very high levels of protein expression, were associated with poor prognosis. Knock-down of GATA3 in neuroblastoma cells lines inhibited cell proliferation and increased apoptosis but had no effect on cellular differentiation. These results identify GATA3 as an epigenetically regulated component of the neuroblastoma transcriptional control network, that is essential for neuroblastoma proliferation. This suggests that the GATA3 transcriptional network is a promising target for novel neuroblastoma therapies.

Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking

BackgroundHerbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC–MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development.ResultsThe genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum.ConclusionsA herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the “cryptic” herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

Loss of thymidine kinase 1 inhibits lung cancer growth and metastatic attributes by reducing GDF15 expression.

Metabolic alterations that are critical for cancer cell growth and metastasis are one of the key hallmarks of cancer. Here, we show that thymidine kinase 1 (TK1) is significantly overexpressed in tumor samples from lung adenocarcinoma (LUAD) patients relative to normal controls, and this TK1 overexpression is associated with significantly reduced overall survival and cancer recurrence. Genetic knockdown of TK1 with short hairpin RNAs (shRNAs) inhibits both the growth and metastatic attributes of LUAD cells in culture and in mice. We further show that transcriptional overexpression of TK1 in LUAD cells is driven, in part, by MAP kinase pathway in a transcription factor MAZ dependent manner. Using targeted and gene expression profiling-based approaches, we then show that loss of TK1 in LUAD cells results in reduced Rho GTPase activity and reduced expression of growth and differentiation factor 15 (GDF15). Furthermore, ectopic expression of GDF15 can partially rescue TK1 knockdown-induced LUAD growth and metastasis inhibition, confirming its important role as a downstream mediator of TK1 function in LUAD. Collectively, our findings demonstrate that TK1 facilitates LUAD tumor and metastatic growth and represents a target for LUAD therapy.