Fusion of Spectroscopy and Cobalt Electrochemistry Data for Estimating Phosphate Concentration in Hydroponic Solution.

Phosphate is a key element affecting plant growth. Therefore, the accurate determination of phosphate concentration in hydroponic nutrient solutions is essential for providing a balanced set of nutrients to plants within a suitable range. This study aimed to develop a data fusion approach for determining phosphate concentrations in a paprika nutrient solution. As a conventional multivariate analysis approach using spectral data, partial least squares regression (PLSR) and principal components regression (PCR) models were developed using 56 samples for calibration and 24 samples for evaluation. The R2 values of estimation models using PCR and PLSR ranged from 0.44 to 0.64. Furthermore, an estimation model using raw electromotive force (EMF) data from cobalt electrodes gave R2 values of 0.58-0.71. To improve the model performance, a data fusion method was developed to estimate phosphate concentration using near infrared (NIR) spectral and cobalt electrochemical data. Raw EMF data from cobalt electrodes and principle component values from the spectral data were combined. Results of calibration and evaluation tests using an artificial neural network estimation model showed that R2 = 0.90 and 0.89 and root mean square error (RMSE) = 96.70 and 119.50 mg/L, respectively. These values are sufficiently high for application to measuring phosphate concentration in hydroponic solutions.

Crystal structure of heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding.

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed-back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117-124. © 2016 Wiley Periodicals, Inc.

Osteogenic Effects of the Diospyros lotus L. Leaf Extract on MC3T3-E1 Pre-Osteoblasts and Ovariectomized Mice via BMP2/4 and TGF ß Pathways.

Osteoporosis, a disease defined by the primary bone strength due to a low bone mineral density, is a bone disorder associated with increased mortality in the older adult population. Osteoporosis is mainly treated via hormone replacement therapy, bisphosphates, and anti-bone resorption agents. However, these agents exert severe side effects, necessitating the development of novel therapeutic agents. Many studies are focusing on osteogenic agents as they increase the bone density, which is essential for osteoporosis treatment. Here, we aimed to investigate the effects of Diospyros lotus L. leaf extract (DLE) and its components on osteoporosis in MC3T3-E1 pre-osteoblasts and ovariectomized mice and to elucidate the underlying related pathways. DLE enhanced the differentiation of MC3T3-E1 pre-osteoblasts, with a 1.5-fold elevation in ALP activity, and increased the levels of osteogenic molecules, RUNX family transcription factor 2, and osterix. This alteration resulted from the activation of bone morphogenic protein 2/4 (BMP2/4) and transformation of growth factor ß (TGF ß) pathways. In ovariectomized mice, DLE suppressed the decrease in bone mineral density by 50% and improved the expression of other bone markers, which was confirmed by the 3~40-fold increase in osteogenic proteins and mRNA expression levels in bone marrow cells. The three major compounds identified in DLE exhibited osteogenic and estrogenic activities with their aglycones, as previously reported. Among the major compounds, myricitrin alone was not as strong as whole DLE with all its constituents. The osteogenic activity of DLE was partially suppressed by the inhibitor of estrogen signaling, indicating that the estrogenic activity of DLE participated in its osteogenic activity. Overall, DLE suppresses osteoporosis by inducing osteoblast differentiation.

Rex1/Zfp42 as an epigenetic regulator for genomic imprinting.

Zfp42/Rex1 (reduced expression gene 1) is a well-known stem-cell marker that has been duplicated from YY1 in the eutherian lineage. In the current study, we characterized the in vivo roles of Rex1 using a mutant mouse line disrupting its transcription. In contrast to the ubiquitous expression of YY1, Rex1 is expressed only during spermatogenesis and early embryogenesis and also in a very limited area of the placenta. Yet, the gene dosage of Rex1 is very critical for the survival of the late-stage embryos and neonates. This delayed phenotypic consequence suggests potential roles for Rex1 in establishing and maintaining unknown epigenetic modifications. Consistently, Rex1-null blastocysts display hypermethylation in the differentially methylated regions (DMRs) of Peg3 and Gnas imprinted domains, which are known to contain YY1 binding sites. Further analyses confirmed in vivo binding of Rex1 only to the unmethylated allele of these two regions. Thus, Rex1 may function as a protector for these DMRs against DNA methylation. Overall, the functional connection of Rex1 to genomic imprinting represents another case where newly made genes have co-evolved with lineage-specific phenomena.

Identification of antifungal niphimycin from Streptomyces sp. KP6107 by screening based on adenylate kinase assay.

Microbial culture extracts are used for natural product screening to find antifungal lead compounds. A microbial culture extract library was constructed using 343 actinomycete isolates to examine the value of the adenylate kinase (AK) assay for screening to identify antifungal metabolites that disrupt cell integrity in plant pathogenic fungi. A culture extract of Streptomyces sp. strain KP6107 lysed cells of Fusarium oxysporum f.sp. lycopersici which resulted in high AK activity. The active ingredient N-1 was purified from the culture extract using various chromatographic procedures and identified to be the guanidyl-polyol macrolide antibiotic, niphimycin, which is a potent fungal cell membrane disruptor. Niphimycin showed broad-spectrum antifungal activity against Alternaria mali, Aspergillus oryzae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Cercospora canescens, Cylindrocarpon destructans, F. oxysporum f.sp. cucumerinum, F. oxysporum f.sp. lycopersici, and Rhizoctonia solani at concentrations of 8-64 µg ml(-1). Anthracnose development in pepper plants was completely inhibited by treatment with 50 µg ml(-1) niphimycin, which was as effective as chlorothalonil. These results show that the AK assay is an efficient and selective tool in screening for cell membrane/wall disruptors of plant pathogenic fungi.

A peptide encoded by a highly conserved gene belonging to the genus Streptomyces shows antimicrobial activity against plant pathogens.

The genus Streptomyces has been unceasingly highlighted for the versatility and diversity of the antimicrobial agents they produce. Moreover, it is a heavily sequenced taxon in the phylum Actinobacteria. In this study, 47 sequence profiles were identified as proteins highly conserved within the genus Streptomyces. Significant hits to the 38 profiles were found in more than 2000 Streptomyces genomes, 11 of which were further conserved in more than 90% of Actinobacterial genomes analyzed. Only a few genes corresponding to these sequence profiles were functionally characterized, which play regulatory roles in the morphology and biosynthesis of antibiotics. Here a highly conserved sequence, namely, SHC-AMP (Streptomyces highly conserved antimicrobial peptide), which exhibited antimicrobial activity against bacterial and fungal plant pathogens, was reported. In particular, Arabidopsis thaliana was effectively protected against infection with Pseudomonas syringae pv. tomato DC3000 by treatment with this peptide. Results indicated the potential application of this peptide as an antimicrobial agent for control of plant diseases. Our results suggest putative target genes for controlling Streptomyces spp., including the one exhibiting antimicrobial activity against a wide range of phytopathogens.

Identification and biocontrol efficacy of Streptomyces miharaensis producing filipin III against Fusarium wilt.

A number of bacterial strains were isolated from the internal tissue of Trapa japonica. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of Streptomyces miharaensis showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP-1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH-20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP-1 inhibited the mycelial growth of plant pathogenic fungi such as Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum at 1-10 µg ml(-1) and also markedly inhibited the development of Fusarium wilt caused by F. oxysporum f.sp. lycopersici in tomato plants by treatment with 10 µg ml(-1) under greenhouse conditions. The efficacy of FP-1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An egfp -tagged strain of KPE62302H confirmed its ability to colonize tomato plants.

A Hyperspectral Data 3D Convolutional Neural Network Classification Model for Diagnosis of Gray Mold Disease in Strawberry Leaves.

Gray mold disease is one of the most frequently occurring diseases in strawberries. Given that it spreads rapidly, rapid countermeasures are necessary through the development of early diagnosis technology. In this study, hyperspectral images of strawberry leaves that were inoculated with gray mold fungus to cause disease were taken; these images were classified into healthy and infected areas as seen by the naked eye. The areas where the infection spread after time elapsed were classified as the asymptomatic class. Square regions of interest (ROIs) with a dimensionality of 16 × 16 × 150 were acquired as training data, including infected, asymptomatic, and healthy areas. Then, 2D and 3D data were used in the development of a convolutional neural network (CNN) classification model. An effective wavelength analysis was performed before the development of the CNN model. Further, the classification model that was developed with 2D training data showed a classification accuracy of 0.74, while the model that used 3D data acquired an accuracy of 0.84; this indicated that the 3D data produced slightly better performance. When performing classification between healthy and asymptomatic areas for developing early diagnosis technology, the two CNN models showed a classification accuracy of 0.73 with regards to the asymptomatic ones. To increase accuracy in classifying asymptomatic areas, a model was developed by smoothing the spectrum data and expanding the first and second derivatives; the results showed that it was possible to increase the asymptomatic classification accuracy to 0.77 and reduce the misclassification of asymptomatic areas as healthy areas. Based on these results, it is concluded that the proposed 3D CNN classification model can be used as an early diagnosis sensor of gray mold diseases since it produces immediate on-site analysis results of hyperspectral images of leaves.

Investigating combinatorial approaches in virtual screening on human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): a case study for small molecule kinases.

Efforts toward improving the predictiveness in tier-based approaches to virtual screening (VS) have mainly focused on protein kinases. Despite their significance as drug targets, small molecule kinases have been rarely tested with these approaches. In this paper, we investigate the efficacy of a pharmacophore screening-combined structure-based docking approach on the human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, an emerging target for cancer chemotherapy. Six out of a total 1364 compounds from NCI's Diversity Set II were selected as true actives via throughput screening. Using a database constructed from these compounds, five programs were tested for structure-based docking (SBD) performance, the MOE of which showed the highest enrichments and second highest screening rates. Separately, using the same database, pharmacophore screening was performed, reducing 1364 compounds to 287 with no loss in true actives, yielding an enrichment of 4.75. When SBD was retested with the pharmacophore filtered database, 4 of the 5 SBD programs showed significant improvements to enrichment rates at only 2.5% of the database, with a 7-fold decrease in an average VS time. Our results altogether suggest that combinatorial approaches of VS technologies are easily applicable to small molecule kinases and, moreover, that such methods can decrease the variability associated with single-method SBD approaches.

Root treatment with rhizobacteria antagonistic to Phytophthora blight affects anthracnose occurrence, ripening, and yield of pepper fruit in the plastic house and field.

We previously selected rhizobacterial strains CCR04, CCR80, GSE09, ISE13, and ISE14, which were antagonistic to Phytophthora blight of pepper. In this study, we investigated the effects of root treatment of rhizobacteria on anthracnose occurrence, ripening, and yield of pepper fruit in the plastic house and field in 2008 and 2009. We also examined the effects of volatiles produced by the strains on fruit ripening and on mycelial growth and spore development of Colletotrichum acutatum and Phytophthora capsici in the laboratory, identifying the volatile compounds by gas chromatography-mass spectrometry (GC-MS). In the house tests, all strains significantly (P < 0.05) reduced anthracnose incidence on pepper fruit; strains GSE09 and ISE14 consistently produced higher numbers of pepper fruit or increased the fresh weight of red fruit more than the controls in both years. In the field tests, all strains significantly (P < 0.05) reduced anthracnose occurrence on either green or red pepper fruit; strain ISE14 consistently produced higher numbers or increased fresh weights of red fruit more than the controls in both years. In the laboratory tests, volatiles produced by strains GSE09 and ISE13 only stimulated maturation of pepper fruit from green (unripe) to red (ripe) fruit; the volatiles of certain strains inhibited the growth and development of C. acutatum and P. capsici. On the other hand, GC-MS analysis of volatiles of strains GSE09 and ISE13 revealed 17 distinct compounds in both strains, including decane, dodecane, 1,3-di-tert-butylbenzene, tetradecane, 2,4-di-tert-butylphenol, and hexadecane. Among these compounds, 2,4-di-tert-butylphenol only stimulated fruit ripening and inhibited growth and development of the pathogens. Taken together, strains GSE09 and ISE14 effectively reduced anthracnose occurrence and stimulated pepper fruit ripening and yield, possibly via bacterial volatiles. Therefore, these two strains could be potential agents for controlling Phytophthora blight and anthracnose, and for increasing fruit ripening and yield. To our knowledge, this is the first report of volatiles such as 2,4-di-tert-butylphenol produced by rhizobacteria being related to both fruit ripening and pathogen inhibition.

YY1's role in DNA methylation of Peg3 and Xist.

Unusual clusters of YY1 binding sites are located within several differentially methylated regions (DMRs), including Xist, Nespas and Peg3, which all become methylated during oogenesis. In this study, we performed conditional YY1 knockdown (KD) to investigate YY1's roles in DNA methylation of these DMRs. Reduced levels of YY1 during spermatogenesis did not cause any major change in these DMRs although the same YY1 KD caused hypermethylation in these DMRs among a subset of aged mice. However, YY1 KD during oogenesis resulted in the loss of DNA methylation on Peg3 and Xist, but there were no changes on Nespas and H19. Continued YY1 KD from oogenesis to the blastocyst stage caused further loss in DNA methylation on Peg3. Consequently, high incidents of lethality were observed among embryos that had experienced the reduced levels of YY1 protein. Overall, the current study suggests that YY1 likely plays a role in the de novo DNA methylation of the DMRs of Peg3 and Xist during oogenesis and also in the maintenance of unmethylation status of these DMRs during spermatogenesis.

In vivo YY1 knockdown effects on genomic imprinting.

The YY1 transcription factor is predicted to control several imprinted domains, including the Peg3, Gnas and Xist/Tsix regions. To test this possibility, we have used RNA interference strategies to generate transgenic mouse lines that express reduced levels of the cellular YY1 protein. As predicted, lowering YY1 levels resulted in global expression changes in these three imprinted domains. In neonatal brains, most imprinted genes of the Peg3 domain were up-regulated. In the Gnas domain, Nespas was down-regulated, whereas three other imprinted transcripts were up-regulated, including Nesp, Gnasxl and Exon1A. In the Xist/Tsix domain, no obvious change was detected in the expression levels of the two genes in female mice. However, male mice showed low-level coordinated, up- and down-regulation of Xist and Tsix, respectively, suggesting potential de-repression of Xist in a subset of male cell populations. YY1 knockdown also changed the methylation levels at the imprinting control regions (ICRs) of these domains in a target-specific manner. In addition, breeding experiments indicated that the birth weights of 20% of the transgenic females were much lower than those of normal female littermates. We surmise that this gender-specific outcome is caused by the YY1 knockdown effect on the Xist locus of females. In sum, these results demonstrate that YY1 indeed functions as a trans factor for transcriptional regulation and DNA methylation of these imprinted domains in vivo.

Characterization of a multifunctional feather-degrading Bacillus subtilis isolated from forest soil.

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO(3), 0.1% (w/v) K(2)HPO(4), 0.06% (w/v) KH(2)PO(4) and 0.04% (w/v) MgCl(2)·6H(2)O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free -SH group, soluble protein and amino acids production. The concentration of free -SH group in the culture medium was 15.5 ± 0.2 µM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 µM. Proline (2809.9 µM), histidine (371.3 µM) and phenylalanine (172.0 µM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.

YY1's longer DNA-binding motifs.

The DNA-binding sites of YY1 located within two newly identified downstream genes of YY1, Peg3 (GGCGCCATnTT) and Xist (CCGCCATnTT), are longer than the known motif of YY1 (CGCCATnTT). Gel shift assays indicated that these DNA-binding sites are previously unnoticed, longer motifs of YY1. Independent DNA-binding motif studies further confirmed that YY1 recognizes a longer sequence (GCCGCCATTTTG) as its consensus motif. This longer motif exhibited higher affinity to the YY1 protein than the known motif. Another DNA-binding motif study was also performed using a protein containing three amino acid substitutions in the first zinc finger unit of YY1, mimicking the zinc finger domain of pho (a drosophila homologue of YY1). The substitutions cause the weakening of DNA-binding specificity at both 5'- and 3'-sides of the longer motif, yielding a much shorter sequence (GCCAT) as a consensus motif. This indicates that the intact first finger unit is required for recognition of the longer motif of YY1. Also, this shortening suggests that the DNA recognition by YY1 is mediated through the concerted, but not modular, contribution by its four zinc finger units.

YY1 is autoregulated through its own DNA-binding sites.

BACKGROUND: The transcription factor Yin Yang 1 (YY1) is a ubiquitously expressed, multifunctional protein that controls a large number of genes and biological processes in vertebrates. As a general transcription factor, the proper levels of YY1 protein need to be maintained for the normal function of cells and organisms. However, the mechanism for the YY1 homeostasis is currently unknown. RESULTS: The current study reports that the YY1 gene locus of all vertebrates contains a cluster of its own DNA-binding sites within the 1st intron. The intact structure of these DNA-binding sites is absolutely necessary for transcriptional activity of the YY1 promoter. In an inducible cell line system that over-expresses an exogenous YY1 gene, the overall increased levels of YY1 protein caused a reduction in transcription levels of the endogenous YY1 gene. Reversion to the normal levels of YY1 protein restored the transcriptional levels of the endogenous YY1 to normal levels. This homeostatic response was also mediated through its cluster of YY1 binding sites. CONCLUSION: Taken together, the transcriptional level of YY1 is self-regulated through its internal DNA-binding sites. This study identifies YY1 as the first known autoregulating transcription factor in mammalian genomes.

Retroposition and evolution of the DNA-binding motifs of YY1, YY2 and REX1.

YY1 is a DNA-binding transcription factor found in both vertebrates and invertebrates. Database searches identified 62 YY1 related sequences from all the available genome sequences ranging from flying insects to human. These sequences are characterized by high levels of sequence conservation, ranging from 66% to 100% similarity, in the zinc finger DNA-binding domain of the predicted proteins. Phylogenetic analyses uncovered duplication events of YY1 in several different lineages, including flies, fish and mammals. Retroposition is responsible for generating one duplicate in flies, PHOL from PHO, and two duplicates in placental mammals, YY2 and Reduced Expression 1 (REX1) from YY1. DNA-binding motif studies have demonstrated that YY2 still binds to the same consensus sequence as YY1 but with much lower affinity. In contrast, REX1 binds to DNA motifs divergent from YY1, but the binding motifs of REX1 and YY1 share some similarity at their core regions (5'-CCAT-3'). This suggests that the two duplicates, YY2 and REX1, although generated through similar retroposition events have undergone different selection schemes to adapt to new roles in placental mammals. Overall, the conservation of YY2 and REX1 in all placental mammals predicts that each duplicate has co-evolved with some unique features of eutherian mammals.

Disease control efficacy of 32,33-didehydroroflamycoin produced by Streptomyces rectiviolaceus strain DY46 against gray mold of tomato fruit.

Despite the efficacy of synthetic fungicides in controlling postharvest diseases, public concerns regarding chemical residues in food and an increase in drug-resistant strains of pathogens have led to a need for new agents to control postharvest diseases. The current study was performed to find control agents of microbial origin that are effective on gray mold of tomato fruits. We recently isolated Streptomyces rectiviolaceus DY46, which has antagonistic activity against various plant pathogenic fungi. The incidence of gray mold of tomato fruits was markedly reduced by 80.0% in tomatoes treated with the cell extract of Streptomyces rectiviolaceus DY46 compared with the control tomatoes. The active ingredient was purified from the cell extract of DY46 and identified to be 32,33-didehydroroflamycoin (DDHR). DDHR displayed MICs (minimal inhibitory concentrations) against the mycelial growth of various plant pathogenic fungi at concentrations of 8-64 mg L-1. The incidence of gray mold in tomato fruits inoculated with conidial suspension (104 conidia mL-1) of Botrytis cinerea was markedly reduced by 88.9% in tomatoes treated with DDHR (100 mg L-1) compared with the control. The DDHR residue in tomato fruit was significantly diminished 2 d after treatment. These results show that DDHR would be relatively safe for use as a postharvest fungicide.

Two evolutionarily conserved sequence elements for Peg3/Usp29 transcription.

BACKGROUND: Two evolutionarily Conserved Sequence Elements, CSE1 and CSE2 (YY1 binding sites), are found within the 3.8-kb CpG island surrounding the bidirectional promoter of two imprinted genes, Peg3 (Paternally expressed gene 3) and Usp29 (Ubiquitin-specific protease 29). This CpG island is a likely ICR (Imprinting Control Region) that controls transcription of the 500-kb genomic region of the Peg3 imprinted domain. RESULTS: The current study investigated the functional roles of CSE1 and CSE2 in the transcriptional control of the two genes, Peg3 and Usp29, using cell line-based promoter assays. The mutation of 6 YY1 binding sites (CSE2) reduced the transcriptional activity of the bidirectional promoter in the Peg3 direction in an orientation-dependent manner, suggesting an activator role for CSE2 (YY1 binding sites). However, the activity in the Usp29 direction was not detectable regardless of the presence/absence of YY1 binding sites. In contrast, mutation of CSE1 increased the transcriptional activity of the promoter in both the Peg3 and Usp29 directions, suggesting a potential repressor role for CSE1. The observed repression by CSE1 was also orientation-dependent. Serial mutational analyses further narrowed down two separate 6-bp-long regions within the 42-bp-long CSE1 which are individually responsible for the repression of Peg3 and Usp29. CONCLUSION: CSE2 (YY1 binding sites) functions as an activator for Peg3 transcription, while CSE1 acts as a repressor for the transcription of both Peg3 and Usp29.

Imprinting of an evolutionarily conserved antisense transcript gene APeg3.

APeg3 is an antisense transcript gene of Peg3, which has been recently identified from rat brain. Careful analyses of EST databases indicated that a homologous transcript also exists in other mammalian species, including mouse, cow and human. 5'-and 3'-RACE experiments have subsequently identified a 900-bp cDNA sequence of APeg3 from mouse brain. Mouse APeg3 is localized in the 3'UTR of Peg3 with an intronless genomic structure. The expression of mouse APeg3 is derived mainly from the paternal allele, indicating the imprinting of this antisense transcript gene in brain. Strand-specific RNA analyses also revealed the expression of both human and cow APEG3 in adult brains. In sum, our study confirms that the mammalian PEG3 locus harbors an antisense transcript gene displaying paternal allele-specific expression, and the evolutionary conservation further suggests potential roles of this transcript gene for the function of this imprinted domain.

MacroH2A1 knockdown effects on the Peg3 imprinted domain.

BACKGROUND: MacroH2A1 is a histone variant that is closely associated with the repressed regions of chromosomes. A recent study revealed that this histone variant is highly enriched in the inactive alleles of Imprinting Control Regions (ICRs). RESULTS: The current study investigates the potential roles of macroH2A1 in genomic imprinting by lowering the cellular levels of the macroH2A1 protein. RNAi-based macroH2A1 knockdown experiments in Neuro2A cells changed the expression levels of a subset of genes, including Peg3 and Usp29 of the Peg3 domain. The expression of these genes was down-regulated, rather than up-regulated, in response to reduced protein levels of the potential repressor macroH2A1. This down-regulation was not accompanied with changes in the DNA methylation status of the Peg3 domain. CONCLUSION: MacroH2A1 may not function as a transcriptional repressor for this domain, but that macroH2A1 may participate in the heterochromatin formation with functions yet to be discovered.