Sub-nucleosomal Genome Structure Reveals Distinct Nucleosome Folding Motifs.

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and ß-rhombus analogous to α helix and ß sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.

iNucs: inter-nucleosome interactions.

MOTIVATION: Deciphering nucleosome-nucleosome interactions is an important step toward mesoscale description of chromatin organization but computational tools to perform such analyses are not publicly available. RESULTS: We developed iNucs, a user-friendly and efficient Python-based bioinformatics tool to compute and visualize nucleosome-resolved interactions using standard pairs format input generated from pairtools. AVAILABILITYAND IMPLEMENTATION: https://github.com/Karimi-Lab/inucs/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nucleosome-level 3D organization of the genome.

Nucleosomes are the unitary structures of chromosome folding, and their arrangements are intimately coupled to the regulation of genome activities. Conventionally, structural analyses using electron microscopy and X-ray crystallography have been used to study such spatial nucleosome arrangements. In contrast, recent improvements in the resolution of sequencing-based methods allowed investigation of nucleosome arrangements separately at each genomic locus, enabling exploration of gene-dependent regulation mechanisms. Here, we review recent studies on nucleosome folding in chromosomes from these two methodological perspectives: conventional structural analyses and DNA sequencing, and discuss their implications for future research.

Protein expression analyses at the single cell level.

The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

Effects of chronic vardenafil treatment persist after end of treatment in rats with acute arteriogenic erectile dysfunction.

INTRODUCTION: In our previous study, chronic vardenafil treatment improved erectile function soon after the end of the treatment in rats with acute arteriogenic erectile dysfunction (ED). AIM: The aim of this study is to evaluate whether the effects of chronic vardenafil treatment persist after the end of treatment using rats with acute arteriogenic ED. METHODS: Rats were randomly divided into three groups: (i) control; (ii) ligation; and (iii) vardenafil + no treatment. Rats in the ligation and vardenafil + no treatment groups underwent ligature of the bilateral internal iliac arteries to induce acute arteriogenic ED and were subsequently treated with vehicle or vardenafil (4.0 mg/kg/day), respectively, for 3 weeks. Subsequently, all rats were kept for a further 2 weeks with no treatment. Rats in the control group underwent sham surgery. MAIN OUTCOME MEASURES: Erectile function was assessed by changes in intracavernous pressure (ICP). Smooth muscle (SM)/collagen ratios in corpus cavernosum were analyzed by Masson trichrome staining. Transforming growth factor-ß1 (TGF-ß1 ) mRNA and protein levels in corpus cavernosum (CC) were, respectively, evaluated by real-time polymerase chain reaction (PCR) analysis and Western blotting analysis. RESULTS: ICP/mean arterial pressure (MAP) in the ligation group remained significantly lower than that in control group (P < 0.01). Despite no treatment for 2 weeks, ICP/MAP in the var + no treatment group remained significantly higher than that in ligation group (P < 0.05). SM/collagen ratio in the ligation group remained significantly lower when compared with the control group (P < 0.01). The ratio in the var + no treatment group remained significantly higher when compared with the ligation group at 2 weeks after the end of treatment (P < 0.05). TGF-ß(1) mRNA and protein levels did not differ among the groups. CONCLUSIONS: The effects of chronic vardenafil treatment on erectile function and penile structure persist, even after the end of treatment, in acute arteriogenic ED rats.

Interactions of thyroid hormone receptor with Ku proteins and interleukin enhancer binding factor 3 modulate the promoter activity of thyroid-stimulating hormone alpha.

We previously identified Ku proteins and interleukin enhancer binding factor 3 (ILF3) as cofactors for the nuclear receptor farnesoid X receptor and liver receptor homolog-1, respectively. Here we provide further evidence that these cofactors modulate the promoter activity of the nuclear receptor thyroid hormone receptor (TR) target gene, thyroid-stimulating hormone alpha (TSHα), which is negatively regulated by the TR ligand triiodothyronine (T(3)). Ku proteins suppressed TSHα promoter activity independent of T(3), whereas ILF3 enhanced TSHα activity, especially in the presence of T(3). Taken together, our results suggest that Ku proteins and ILF3 function as co-regulators for TR-mediated TSHα expression.

Interleukin enhancer-binding factor 3 functions as a liver receptor homologue-1 co-activator in synergy with the nuclear receptor co-activators PRMT1 and PGC-1α.

LRH-1 (liver receptor homologue-1), a transcription factor and member of the nuclear receptor superfamily, regulates the expression of its target genes, which are involved in bile acid and cholesterol homoeostasis. However, the molecular mechanisms of transcriptional control by LRH-1 are not completely understood. Previously, we identified Ku80 and Ku70 as LRH-1-binding proteins and reported that they function as co-repressors. In the present study, we identified an additional LRH-1-binding protein, ILF3 (interleukin enhancer-binding factor 3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor co-activators PRMT1 (protein arginine methyltransferase 1) and PGC-1α (peroxisome proliferator-activated receptor γ co-activator-1α). We demonstrated that ILF3, PRMT1 and PGC-1α were recruited to the promoter region of the LRH-1-regulated SHP (small heterodimer partner) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in co-operation with PRMT1 and PGC-1α through the C-terminal region of ILF3. In addition, we found that the small interfering RNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1α at the SHP promoter and SHP expression. Taken together, our results suggest that ILF3 functions as a novel LRH-1 co-activator by acting synergistically with PRMT1 and PGC-1α, thereby promoting LRH-1-dependent gene expression.

Hi-CO: 3D genome structure analysis with nucleosome resolution.

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1-10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2-3 weeks for sequencing library preparation and 2 weeks for simulation.

Repression of the promoter activity mediated by liver receptor homolog-1 through interaction with ku proteins.

Nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a crucial role in the homeostasis of bile acids and cholesterol by controlling the expression of genes central to bile acid synthesis and efflux, reverse cholesterol transport, and high density lipoprotein-remodeling. However, the molecular mechanisms that modulate the transactivation activity of LRH-1 remain unclear. It is proposed that LRH-1's activity is regulated by post-modifications, the binding of small heterodimer partner (SHP), or the binding of coregulators. To search for cofactors that regulate the transactivation activity of LRH-1, we performed a pull-down assay using glutathione S-transferase (GST) fused to the N-terminal portion of LRH-1 and nuclear extracts from HeLa cells, and identified Ku proteins as interacting proteins with LRH-1. We also found that Ku proteins associate with LRH-1 through its DNA-binding domain and hinge region. Luciferase reporter assays revealed that Ku proteins repressed the SHP promoter activity mediated by LRH-1. Furthermore, Ku proteins suppressed the coactivating effect of peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha), an LRH-1 coactivator, on the LRH-1-mediated SHP promoter activity. Previously, we showed that Ku proteins interacted with nuclear receptor farnesoid X receptor (FXR; NR1H4) and decreased the expression of its target gene. In this study, we demonstrated that Ku proteins also interacted with not only LRH-1 but various nuclear receptors, such as the estrogen receptor, PPAR, and Rev-erb. Ku proteins may function as corepressors for various nuclear receptors including LRH-1.

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression.

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

Efficacy of continuous ingestion of dewaxed brown rice on the cognitive functions of the residents of elderly welfare facilities: A pilot test using crossover trial.

Compared with regular brown rice, dewaxed brown rice (DBR), prepared by excluding only the wax layer in the outermost layer of brown rice using a new rice milling technique, has improved water absorbency, digestibility, and taste. Dewaxed brown rice has a nutritional value close to that of brown rice and contains a large amount of lipopolysaccharides (LPS), which are known to improve the cognitive function of mice. In this study, we examined the effect of continuous DBR ingestion on the cognitive function of elderly people. A crossover comparison test was performed, in which elderly people who moved into an elderly welfare facility were divided into two groups and ingested DBR or polished white rice for three meals a day for 6 months, followed by a change in test meals for the next 6 months. Cognitive function was assessed using Revised Hasegawa's Dementia Scale (HDS-R) before starting the test and 6 months after ingesting each test meal. No subjects withdrew or discontinued DBR intake during the study period, and all subjects continued the test for 6 months. In subjects with low cognitive function (defined as subjects with HDS-R total score of ≥1 but <10 at the start of the study), there was a significant association between continuous DBR ingestion and cognitive function improvement (increase in total HDS-R score). Our findings suggest that the long-term DBR ingestion as a staple food could be useful for preventing cognitive decline in elderly; it offers an easily implemented option as a daily diet for preventing cognitive decline.

Effect of Continuous Dewaxed Brown Rice Ingestion on the Cognitive Function of Elderly Individuals.

Dewaxed brown rice (DBR), which is prepared by removing only the outermost wax layer of brown rice using a new rice milling technique, has improved water absorbency, digestibility, and taste compared to regular brown rice. DBR has a nutritional value close to that of brown rice, including a rich amount of lipopolysaccharides that are known to improve cognitive function in mice. This study thus aimed to verify the influence of continuous DBR ingestion on cognitive function among elderly individuals. The present study employed a crossover comparison design using the Revised Hasegawa Dementia Scale to assess cognitive function. Our findings confirmed that long-term DBR ingestion contributed to the prevention and reduction of overall cognitive decline, especially among elderly individuals with low cognitive function. Thus, DBR has the potential to be a useful staple food that maintains brain homeostasis among elderly individuals.

[Functional analysis of nuclear receptor FXR controlling metabolism of cholesterol].

Nuclear receptors function as ligand-inducible transcription factors that regulate various physiological functions such as development, reproduction, and metabolism. Dysregulation of the metabolism of cholesterol, triglyceride, and glucose leads to the metabolic syndrome including type 2 diabetes mellitus, obesity, dyslipidemia, and atherosclerosis. Studies of nuclear receptors promise to provide discoveries of therapeutic agents against the metabolic syndrome. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily and is activated by bile acids. FXR regulates the metabolism of not only bile acid but also cholesterol, lipoprotein, triglyceride, and glucose, and is considered a potential therapeutic target for the metabolic syndrome because of these functions. Nuclear receptors have two regions for transactivation, a constitutive activation function (AF-1) and a ligand-dependent activation function (AF-2). AF-1 and AF-2 seem to require interactions with coactivators for the activation function and both work synergistically to give full transactivation of nuclear receptors. However, coactivators for AF-1 activity are poorly understood, whereas coactivators required for AF-2 activity have been well studied. To understand the molecular mechanism of AF-1 in FXR, we isolated proteins associated with AF-1 by GST pull-down assay using the N-terminal region of FXR and nuclear extracts from HeLa cells. This review focuses on the roles of FXR and our new findings regarding FXR-associated factors.

Dynamic behavior of giant liposomes at desired osmotic pressures.

To apply accurate and uniform osmotic pressures to liposomes, they can be formed using the spontaneous transfer method in solutions with different osmolarities. The majority of liposomes unexpectedly opened large holes (several micrometers in diameter) in response to the osmotic pressure regardless of its strength, that is, the difference between the outside and inside solute (sucrose or KCl) concentrations. However, the lag time for any response, including the opening of a hole, after the formation of the liposome decreased with increasing osmotic pressure.