Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

Assessment of Feng-Liao-Chang-Wei-Kang as a potential inducer of cytochrome P450 3A4 and pregnane X receptors.

Feng-Liao-Chang-Wei-Kang (FLCWK), a traditional Chinese patent medicine, consists primarily of Polygonum hydropiper and Daphniphyllum calycinum roots. As a complex containing several kinds of flavonoids, FLCWK has the potential to impact the drug metabolism enzyme P450 3A4 (CYP3A4) and nuclear receptors. The purpose of this research was to probe the effects of FLCWK on CYP3A1, the homolog of CYP3A4 in rats, and to confirm whether FLCWK interferes with PXR and CAR-mediated transactivation of CYP3A4. The effects of FLCWK on Cyp3a1 mRNA, catalytic activity levels, and protein expression in Sprague-Dawley (SD) rat liver tissues were examined using real-time PCR, western blotting, and high-performance liquid chromatography (HPLC) assays, respectively. The efficacy of PXR and CAR on CYP3A4 transcriptional activity were detected using luciferase reporter assays and further research of the impact of FLCWK on CYP3A4 gene expression mediated by the PXR pathway was examined by transient transfection of PXR siRNA. FLCWK significantly increased Cyp3a1 mRNA, CYP3A1 activity, and protein expression levels in SD rats. FLCWK highly induced CYP3A4 luciferase activity mediated by PXR in PXRCYP3A4 co-transfected cells. A siRNA-mediated drop-off in PXR expression greatly cut the effect of FLCWK on CYP3A4 mRNA expression in HepG2 cells. These findings show that FLCWK up-regulates CYP3A4 levels via the PXR pathway. This effect should be considered being applied in clinical use as FLCWK has the potential to interact with other drugs.

Transcriptome-wide identification and screening of WRKY factors involved in the regulation of taxol biosynthesis in Taxus chinensis.

WRKY, a plant-specific transcription factor family, plays important roles in pathogen defense, abiotic cues, phytohormone signaling, and regulation of plant secondary metabolism. However, little is known about the roles, functions, and mechanisms of WRKY in taxane biosynthesis in Taxus spp. In this study, 61 transcripts were identified from Taxus chinensis transcriptome datasets by using hidden Markov model search. All of these transcripts encoded proteins containing WRKY domains, which were designated as TcWRKY1-61. After phylogenetic analysis of the WRKY domains of TcWRKYs and AtWRKYs, 16, 8, 10, 14, 5, 7, and 1 TcWRKYs were cladded into Group I, IIa-IIe, and III, respectively. Then, six representative TcWRKYs were selected to classify their effects on taxol biosynthesis. After MeJA (methyl jasmonate acid) and SA (salicylic acid) treatments, all of the six TcWRKYs were upregulated by MeJA treatment. TcWRKY44 (IId) and TcWRKY47 (IIa) were upregulated, whereas TcWRKY8 (IIc), TcWRKY20 (III), TcWRKY26 (I), TcWRKY41 (IIe), and TcWRKY52 (IIb) were downregulated by SA treatment. Overexpression experiments showed that the six selected TcWRKYs exerted different effects on taxol biosynthesis. In specific, TcWRKY8 and TcWRKY47 significantly improved the expression levels of taxol-biosynthesis-related genes. Transcriptome-wide identification of WRKY factors in Taxus not only enhances our understanding of plant WRKY factors but also identifies candidate regulators of taxol biosynthesis.

Functional assessment of genetic variants located in the promoter of SHP1 (NR0B2).

Small heterodimer partner 1 (SHP1, NR0B2) is a member of the superfamily of nuclear receptors (NRs). Even if this orphan receptor, unlike other NRs, lacks the DNA-binding domain, it is capable of regulating transcription by repressing the activity of other NRs by direct protein-protein interaction. Accordingly, SHP1 is part of negative feedback loops of the transcriptional regulation of genes involved in drug metabolism and various metabolic pathways including bile acid and glucose homeostasis. Although it is known that several interacting partners of SHP1 also modulate its expression, there is little information about genetic variability of this regulatory mechanism. Our study aimed to identify genetic variants in the NR0B2 promoter region and to determine their impact on NR0B2 transcription. For this, DNA samples originating from 119 participants of the population-based cohort Study of Health in Pomerania were analyzed by Sanger sequencing revealing four genetic variants: NR0B2:c.-594T>C (rs71636795), NR0B2:c.-414G>C (newly identified), NR0B2:c.-423C>T (rs78182695), and NR0B2:c.-224delCTGA (rs145613139) localized in the 5' untranslated region of NR0B2. The impact of these variants on transactivation of the NR0B2 promoter by NRs known to be regulators of SHP1 expression (hepatocyte nuclear factor 4α, liver receptor homolog-1, and farnesoid X receptor) was assessed in a cell-based reporter gene assay, showing that transactivation by hepatocyte nuclear factor 4α and liver receptor homolog-1 was significantly decreased in the presence of the genetic variant NR0B2:c.-594T>C, even though this effect was cell specific. However, SHP1 mRNA expression in a small collection of human kidney samples was not affected by these genetic variants.

HDAC4 mediates IFN-γ induced disruption of energy expenditure-related gene expression by repressing SIRT1 transcription in skeletal muscle cells.

Metabolic homeostasis is achieved through balanced energy storage and output. Impairment of energy expenditure is a hallmark event in patients with obesity and type 2 diabetes. Previously we have shown that the pro-inflammatory cytokine interferon gamma (IFN-γ) disrupts energy expenditure in skeletal muscle cells via hypermethylated in cancer 1 (HIC1)-class II transactivator (CIITA) dependent repression of SIRT1 transcription. Here we report that repression of SIRT1 transcription by IFN-γ paralleled loss of histone acetylation on the SIRT1 promoter region with simultaneous recruitment of histone deacetylase 4 (HDAC4). IFN-γ activated HDAC4 in vitro and in vivo by up-regulating its expression and stimulating its nuclear accumulation. HIC1 and CIITA recruited HDAC4 to the SIRT1 promoter and cooperated with HDAC4 to repress SIRT1 transcription. HDAC4 depletion by small interfering RNA or pharmaceutical inhibition normalized histone acetylation on the SIRT1 promoter and restored SIRT1 expression in the presence of IFN-γ. Over-expression of HDAC4 suppressed the transcription of genes involved in energy expenditure in a SIRT1-dependent manner. In contrast, HDAC4 knockdown/inhibition neutralized the effect of IFN-γ on cellular metabolism by normalizing SIRT1 expression. Therefore, our data reveal a role for HDAC4 in regulating cellular energy output and as such provide insights into rationalized design of novel anti-diabetic therapeutics.

Transcriptional activity assessment of three different promoters for mouse in utero electroporation system.

In utero electroporation (IUE) is a simple and rapid approach to in vivo investigate exogenous gene function in mouse brain, and intensive studies using IUE have greatly contributed to analyze the characterization of specific steps during mouse brain development. Because the efficiency of IUE is highly dependent on the plasmid used and its concentration, and the transcriptional activity of plasmid is not only regulated by the host defense system, but also by the promoter of the expression vectors. Therefore, in the present study, we evaluated the transcriptional activity of three commonly used promoters, CMV, CAG and SV40, in IUE system through measuring the fluorescence intensity of green fluorescent protein which serves as an indicator. Our results demonstrated that the artificially-designed CAG promoter is a potent promoter that effectively drives target gene expression in IUE system.

Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation.

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (-0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.

Assessment of genotoxicity and cytotoxicity of "lixeira" (Curatella americanaL. ) using the prophage λ induction test (SOS inductest) / Avaliação da genotoxicidade e citotoxicidade de "lixeira" (Curatella americana L.) utilizando o teste de indução profago λ (teste de indução SOS)

Curatella americana L., commonly known as "lixeira" in Brazil, has been used in folk medicine to treat ulcers and inflammations. The purpose of the present work was to evaluate the cytotoxic and genotoxic potential of the ethanolic extract of C. americana stem bark using the prophage λ induction test (SOS inductest). To evaluate the cytotoxicity of this plant, after treatment with different concentrations of the extract, Escherichia coli WP2s(λ) cultures were diluted in M9 buffer, inoculated into LB plates, and incubated for 24 h at 37 ºC. To assess genotoxicity, the lysogenic strain E. coli WP2s(λ) was treated with different concentrations of the extract. Then, the lysogenic strain was added to the indicator strain (RJF013), LB(1/2)(malt/amp), seeded into plates with the matches, and incubated for 24 h at 37 ºC. After this period, the total number of colonies and the number of plaques were counted to evaluate C. americana cytotoxicity and genotoxicity, respectively. Our results showed that although the extract of "lixeira" did not modify the survival of bacteria (p > 0.05), it caused a significant increase in prophage λ induction, especially at the higher concentrations (p<0.05). Therefore, we conclude that the ethanolic extract of C. americana stem bark did not present cytotoxic effect, but some genotoxic potential was observed.

Curatella americana L., comumente conhecida como "lixeira" no Brasil, é utilizada em medicina popular para tratamento de úlceras e inflamações. O presente trabalho teve como objetivo avaliar o potencial citotóxico e genotóxico do extrato etanólico das cascas de C. americana utilizando o Induteste SOS. Para avaliar a citotoxicidade da planta, depois de tratadas com diferentes concentrações do extrato, culturas de E. coli WP2s(λ) foram diluνdas em tampão M9 e semeadas em placas LB. Para avaliar a genotoxicidade da planta, a cepa lisogênica WP2s(λ) de E. coli foi tratada com diferentes concentrações do extrato. Em seguida, esta foi adicionada à cepa indicadora (RJF013) e ambas foram semeadas em placas em meio LB(1/2)(malt)(amp). Todas as culturas foram incubadas por 24 h a 37 ºC. Posteriormente, o número total de colônias e o número de centros infecciosos foram computados para a avaliação da citotoxidade e da genotoxicidade desta planta, respectivamente. Os resultados mostraram que embora o extrato de C. americana não tenha modificado a sobrevivência bacteriana (p > 0,05), provocou aumento significativo (p < 0,05) na indução do profago λ, especialmente nas concentrações mais altas. Assim, concluiu-se que o extrato etanólico das cascas de C. americana não apresentou atividade citotóxica, mas foi observada ação genotóxica direta.

Alternative splicing and caspase-mediated cleavage generate antagonistic variants of the stress oncoprotein LEDGF/p75.

There is increasing evidence that an augmented state of cellular oxidative stress modulates the expression of stress genes implicated in diseases associated with health disparities such as certain cancers and diabetes. Lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, is emerging as a survival oncoprotein that promotes resistance to oxidative stress-induced cell death and chemotherapy. We previously showed that LEDGF/p75 is targeted by autoantibodies in prostate cancer patients and is overexpressed in prostate tumors, and that its stress survival activity is abrogated during apoptosis. LEDGF/p75 has a COOH-terminally truncated splice variant, p52, whose role in stress survival and apoptosis has not been thoroughly investigated. We observed unbalanced expression of these proteins in a panel of tumor cell lines, with LEDGF/p75 generally expressed at higher levels. During apoptosis, caspase-3 cleaved p52 to generate a p38 fragment that lacked the NH(2)-terminal PWWP domain and failed to transactivate the Hsp27 promoter in reporter assays. However, p38 retained chromatin association properties and repressed the transactivation potential of LEDGF/p75. Overexpression of p52 or its variants with truncated PWWP domains in several tumor cell lines induced apoptosis, an activity that was linked to the presence of an intron-derived COOH-terminal sequence. These results implicate the PWWP domain of p52 in transcription function but not in chromatin association and proapoptotic activities. Consistent with their unbalanced expression in tumor cells, LEDGF/p75 and p52 seem to play antagonistic roles in the cellular stress response and could serve as targets for novel antitumor therapies.

In vitro assessment of transcriptional activation of the estrogen and androgen receptors of mosquitofish, Gambusia affinis affinis.

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor-ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERalpha; ESR1), ERbeta1 (ERbeta1) and ERbeta2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (alpha versus beta1), 43% (alpha versus beta2), and 52% (beta1 versus beta2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.

Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness.

We have employed a hidden Markov model (HMM) based on known cAMP responsive elements to search for putative CREB target genes. The best scoring sites were positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments revealed a core promoter requirement for transcriptional induction via CREB; TATA-less promoters were unresponsive to cAMP compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescued cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration.