Exogenous Promoter Research Articles

Evidence for a non-stochastic two-field hypothesis for persistent skin cancer risk

With recurring carcinogen exposures, individual tumors develop in a field of genetic mutations through a stepwise process of clonal expansion and evolution. Once established, this “cancer field” persists in the absence of continued carcinogen exposures, resulting in a sustained risk for cancer development. Using a bioimaging approach, we previously demonstrated that a dermal premalignant field characterized by inflammatory angiogenesis persists following the cessation of ultraviolet light exposures and accurately predicts future overlying epidermal tumor formation. Following ultraviolet light treatments, others have observed that patches of p53 immunopositive cells persist stochastically throughout the epidermal stem cell population. However, these studies were done by random biopsies, introducing sampling bias. We now show that, rather than being randomly distributed, p53+ epidermal cells are enriched only in areas overlying this multi-focal dermal field. Moreover, we also show that the dermal field is characterized by a senescent phenotype. We propose that persistence of the overlying epithelial cancerization field in the absence of exogenous carcinogens or promoters requires a two-field composite consisting of a dermal senescent field driving the persistence of the overlying epidermal cancer field. These observations challenge current models that suggest that persistence of cancer risk in the absence of continued carcinogen exposures is simply a function of stochastically arranged, long-lived but dormant epithelial clonal stem cells mutants. The model proposed here could provide new insights into how cancer risk persists following cessation of carcinogenic exposures.

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.

The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.

Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget.

Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden – as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.

Differential Transgeneration Methylation of Exogenous Promoters in T1 Transgenic Wheat (Triticum aestivum)

Abstract—DNA methylation has become an essential molecular approach to regulate gene expression through the regulation of methyl group addition/removal at the 5th position of Cytosine. The T1 progeny of T0 transgenic wheat plants were used to study the transgeneration methylation of promoter proximal regions of two exogenous promoters using bisulfite sequencing. The progeny of low and high T0 IFS expressers with high and low methylation levels respectively were used. Results of this study revealed that T1 plants that have 35S promoter driving the IFS inherited the methylation status of 35S promoter and IFS expression, especially methylation at the –56 and –88 CpG islands. On the other hand, the high IFS expresser of T0 plants with OL promoter driving the IFS expression passed the same pattern of IFS expression and methylation to their T1 progeny, whereas the low IFS expresser of T0 changed the pattern of expression and methylation to the high T0 expresser in their T1 progeny. This indicates that T1 wheat plants were able to demethylates DNA of the OL promoter proximal region, especially at –106 and –151 and reconstitute the IFS expression from low to high expresser through one generation. This also could indicate that plant promoters are more suitable for driving transgene in plant biotechnology. Results will improve our understanding of regulation of gene expression by DNA methylation and their application in plant biotechnology.

Screening and application of inducible promoters in Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is a promising candidate for producing renewable green chemicals from cellulose due to its cellulolytic and ethanologenic capabilities. It is of significance to screen effective, and convenient-to-use inducible promoters that can be used for regulating the gene expression in R. papyrosolvens. We characterized two endogenous inducible promoters and investigated another two exogenous ones on the adaptability in R. papyrosolvens. Both of the endogenous xylan-inducible promoter Pxyl and exogenous lactose-inducible promoter Plac are found of high specificity and stringency. Pxyl has a short time to be induced while Plac has a low concentration of inducer. With these findings, a mazF-based counter selectable system has been constructed for promoting the efficiency of mutant screening via plasmid curing. The inducible gene expression systems provided novel tools for enhancing the capability of genetic manipulation in engineering R. papyrosolvens. SIGNIFICANCE AND IMPACT OF THE STUDY: Four inducible promoters from Clostridia were characterized in R. papyrosolvens. Xylan-inducible promoter Pxyl was found of a short time while lactose-inducible promoter Plac needs a low concentration of inducer to induce. Employing them, we successfully construct a mazF-based counter selectable system, which would be used to increase the mutant screening efficiency via induction of plasmid curing. The inducible gene expression systems provided novel tools for enhancing the capability of genetic manipulation in engineering R. papyrosolvens.

Methylation of Exogenous Promoters Regulates Soybean Isoflavone Synthase (GmIFS) Transgene in T0 Transgenic Wheat (Triticum aestivum)

DNA methylation is one epigenetic strategy for gene regulation in living organisms. In this study, the expression of soybean isoflavone synthase (GmIFS) transgene in T0 transgenic wheat plants was investigated at the RNA and the final product genestin level. T0 plants showed variations in the GmIFS expression. Methylation status of the exogenous promoters (35S or Oleocin (OL)) proximal sequence was investigated in T0 plants using bisulphite sequencing to disclose their methylation in parallel with the GmIFS level of expression. Results concluded that the high GmIFS expressers of T0 plants exhibited high methylation of exogenous promoter proximal sequences as well as low expression of DNA methyltransferases (Mets). Variation in GmIFS was associated with the level of methylation in the 35S or OL promoters. High expression of GmIFS was negatively correlated with methylation level of 35S and OL promoter proximal regions. In 35S promoter, methylation level of the CpG sites –56 and –88 is strongly linked to GmIFS expression and is involved in the regulation of GmIFS gene. In OL promoter, the CpG site could be involved in the regulation of the GmIFS. Wheat Met3 expression varied among T0 transgenic plants. Its expression profile could explain the regulation of GmIFS transgene by methylation.

Cooperation of genes in HPV16 E6/E7-dependent cervicovaginal carcinogenesis trackable by endoscopy and independent of exogenous estrogens or carcinogens.

Human papillomavirus (HPV) infection is necessary but insufficient for progression of epithelial cells from dysplasia to carcinoma-in situ (CIS) to invasive cancer. The combination of mutant cellular and viral oncogenes that regulate progression of cervical cancer (CC) remains unclear. Using combinations of HPV16 E6/E7 (E+), mutant Kras (mKras) (K+) and/or loss of Pten (P-/-), we generated autochthonous models of CC without exogenous estrogen, carcinogen or promoters. Furthermore, intravaginal instillation of adenoCre virus enabled focal activation of the oncogenes/inactivation of the tumor suppressor gene. In P+/+ mice, E6/E7 alone (P+/+E+K-) failed to cause premalignant changes, while mKras alone (P+/+E-K+) caused persistent mucosal abnormalities in about one-third of mice, but no cancers. To develop cancer, P+/+ mice needed both E6/E7 and mKras expression. Longitudinal endoscopies of P+/+E+K+ mice predicted carcinoma development by detection of mucosal lesions, found on an average of 23 weeks prior to death, unlike longitudinal quantitative PCRs of vaginal lavage samples from the same mice. Endoscopy revealed that individual mice differed widely in the time required for mucosal lesions to appear after adenoCre and in the time required for these lesions to progress to cancer. These cancers developed in the transition zone that extends, unlike in women, from the murine cervix to the distal vagina. The P-/-E+K+ genotype led to precipitous cancer development within a few weeks and E6/E7-independent cancer development occurred in the P-/-E-K+ genotype. In the P-/-E+K- genotype, mice only developed CIS. Thus, distinct combinations of viral and cellular oncogenes are involved in distinct steps in cervical carcinogenesis.

Early fate of exogenous promoters in E.coli.

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.

Agrobiological evaluation of new apple clonal rootstocks of the Michurinsk State Agrarian University selection using different breeding methods

The results of many years of research on propagation of promising forms of clonal stocks of apple trees of the Michurinsk State Agrarian University selection in the mother plantation by stooling, by herbaceous cuttings and by clonal micropropagation are presented. According to the complex of economic and biological features, promising clonal rootstocks of an apple tree with high shoot-forming capacity in the mother plant were identified: dwarf apple trees 2-3-49, 2-15-2, 2-15-15, 3-4-7, 5-21-27; semi dwarf apple trees 2-3-14, 2-9-49, 2-9-102, 2-12-10, 5-24-1, 5-27-1. The rootstocks that are promising for propagation by herbaceous cuttings – 2-3-14, 2-3-49, 2-12-10, 2-15-15, 3-4-7, 5-21-93; at the same time, treatment with exogenous promoters such as IBA and NAA at a concentration of 50 mg/l increases the yield and quality of stock material. A modified MSO nutrient medium with the addition of 0.5-1.0 mg/l of 6-BAP and 0.1-0.2 mg/l of IBA is selected for the clonal micropropagation of apple stocks.

Thy1 promoter activity in the Rosa26 locus in mice: lessons from Dre-rox conditional expression system.

The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.

Exogenous Promoter Triggers APETALA3 Silencing through RNA-Directed DNA Methylation Pathway in Arabidopsis.

The development of floral organs plays a vital role in plant reproduction. In our research, the APETALA3 (AP3) promoter-transgenic lines showed abnormal developmental phenotypes in stamens and petals. The aim of this study is to understand the molecular mechanisms of the morphological defects in transgenic plants. By performing transgenic analysis, it was found that the AP3-promoted genes and the vector had no relation to the morphological defects. Then, we performed the expression analysis of the class A, B, and C genes. A dramatic reduction of transcript levels of class B genes (AP3 and PISTILLATA) was observed. Additionally, we also analyzed the methylation of the promoters of class B genes and found that the promoter of AP3 was hypermethylated. Furthermore, combining mutations in rdr2-2, drm1/2, and nrpd1b-11 with the AP3-silencing lines rescued the abnormal development of stamens and petals. The expression of AP3 was reactivated and the methylation level of AP3 promoter was also reduced in RdDM-defective AP3-silencing lines. Our results showed that the RdDM pathway contributed to the transcriptional silencing in the transgenic AP3-silencing lines. Moreover, the results revealed that fact that the exogenous fragment of a promoter could trigger the methylation of homologous endogenous sequences, which may be ubiquitous in transgenic plants.

Ester Formation via Symbiotic Activation Utilizing Trichloroacetimidate Electrophiles.

Trichloroacetimidates are useful reagents for the synthesis of esters under mild conditions that do not require an exogenous promoter. These conditions avoid the undesired decomposition of substrates with sensitive functional groups that are often observed with the use of strong Lewis or Brønsted acids. With heating, these reactions have been extended to benzyl esters without electron-donating groups. These inexpensive and convenient methods should find application in the formation of esters in complex substrates.

TaBT1, affecting starch synthesis and thousand kernel weight, underwent strong selection during wheat improvement.

BRITTLE1 (BT1), responsible for unidirectional transmembrane transport of ADP-glucose, plays a pivotal role in starch synthesis of cereal grain. In this study, we isolated three TaBT1 homoeologous genes located on chromosomes 6A, 6B, and 6D in common wheat. TaBT1 was mainly expressed in developing grains, and knockdown of TaBT1 in common wheat produced a decrease in grain size, thousand kernel weight (TKW), and grain total starch content. High diversity was detected at the TaBT1-6B locus, with 24 polymorphic sites forming three haplotypes (Hap1, Hap2, and Hap3). Association analysis revealed that Hap1 and Hap2 were preferred haplotypes in modern breeding, for their significant correlations with higher TKW. Furthermore, β-glucuronidase (GUS) staining and enzyme activity assays in developing grains of transgenic rice with exogenous promoters indicated that the promoters of Hap1 and Hap2 showed stronger driving activity than that of Hap3. Evolutionary analysis revealed that BT1 underwent strong selection during wheat polyploidization. In addition, the frequency distribution of TaBT1-6B haplotypes revealed that Hap1 and Hap2 were preferred in global modern wheat cultivars. Our findings suggest that TaBT1 has an important effect on starch synthesis and TKW, and provide two valuable molecular markers for marker assisted selection (MAS) in wheat high-yield breeding.

Next Generation Transgenic Rat Model Production.

The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.

Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene (neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Burden-driven feedback control of gene expression.

Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress.

Kaiso is a member of the BTB/POZ zinc finger family, which is involved in cancer progression, cell cycle control, apoptosis, and WNT signaling. Depending on promoter context, it may function as either a transcriptional repressor or activator. Previous studies found that Kaiso might be SUMOylated due to heat shock, but the biological significance of Kaiso SUMOylation is unclear. Here, we find that K42 is the only amino acid within Kaiso that is modified with SUMO. Kaiso is monoSUMOylated at lysine 42 in cell lines of kidney origin under normal physiological conditions. SUMOylated Kaiso can activate transcription from exogenous methylated promoters, wherein the deSUMOylated form of the protein kept the ability to be a repressor. Rapid Kaiso deSUMOylation occurs in response to hyperosmotic stress and is reversible upon return to an isotonic environment. DeSUMOylation occurs within minutes in HEK293 cells treated with 100 mM NaCl and relaxes in 3 h even in a salt-containing medium. Genomic editing of Kaiso by conversion of K42 into R42 (K42R) in HEK293 cells that resulted in fully deSUMOylated endogenous protein led to misregulation of genes associated with ion transport, blood pressure, and the immune response. TRIM25 was significantly repressed in two K42R HEK293 clones. By a series of rescue experiments with K42R and KO HEK293 cells, we show that TRIM25 is a direct transcriptional target for Kaiso. In the absence of Kaiso, the level of TRIM25 is insensitive to hyperosmotic stress. Extending our observations to animal models, we show that in response to a high salt diet, Kaiso knockout mice are characterized by significantly higher blood pressure increases when compared to wild-type animals. Thus, we propose a novel biological role for Kaiso in the regulation of homeostasis.

Establishment of a high-throughput detection system for DNA demethylating agents

ABSTRACTEpigenetic alterations underlie various human disorders, including cancer, and this has resulted in the development of drugs targeting epigenetic alterations. Although DNA demethylating agents are one of the major epigenetic drugs, only two compounds—5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2'-deoxycytidine (5-aza-dC, decitabine)—have obtained clinical approval. Here, we aimed to establish a detection system for DNA demethylating agents suitable for a high-throughput screening (HTS) in mammalian cells. We inserted luciferase and EGFP reporter genes under the UCHL1 promoter, which is methylation-silenced in human colon cancers and can be readily demethylated to drive strong expression. Methylated UCHL1 promoter was introduced into HCT116 colon cancer cells, and transfectants with methylated exogenous UCHL1 promoter were obtained. By screening subclones from each of the epigenetically heterogeneous transfectant clones, we finally obtained three optimal subclones that expressed luciferase and EGFP after 5-aza-dC treatment with high signal-to-noise ratios. Nucleosomes with H3K9me2 were present around the exogenous UCHL1 promoter in all three subclones. Using one of the subclones (HML58-3), HTS was conducted using 19,840 small molecules. Two hit compounds were obtained, and these turned out to be 5-aza-dC and 5-aza-CR. The assay system constructed here demonstrates a robust response to DNA demethylating agents, along with high specificity, and will be useful for screening and biological assays in epigenetics.