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This study aimed to measure the fracture strengths and hardness of final restorative milled and 3D-printed materials and evaluate the appropriate crown thickness for their clinical use for permanent prosthesis. One type of milled material (group M) and two types of 3D-printed materials (groups P1 and P2) were used. Their crown thickness was set to 0.5, 1.0, and 1.5 mm for each group, and the fracture strength was measured. Vickers hardness was measured and analyzed to confirm the hardness of each material. Scanning electron microscopy was taken to observe the surface changes of the 3D-printed materials under loads of 900 and 1500 N. With increased thickness, the fracture strength significantly increased for group M but significantly decreased for group P1. For group P2, the fracture strengths for the thicknesses of 0.5 mm and 1.5 mm significantly differed, but that for 1.0 mm did not differ from those for other thicknesses. The hardness of group M was significantly higher than that of groups P1 and P2. For all thicknesses, the fracture strength was higher than the average occlusal force for all materials; however, an appropriate crown thickness is required depending on the material and component.

In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Vision is vital for daily activities, and yet the most common eye diseases-cataracts, DR, ARMD, and glaucoma-lead to blindness in aging eyes. Cataract surgery is one of the most frequently performed surgeries, and the outcome is typically excellent if there is no concomitant pathology present in the visual pathway. In contrast, patients with DR, ARMD and glaucoma often develop significant visual impairment. These often-multifactorial eye problems can have genetic and hereditary components, with recent data supporting the role of DNA damage and repair as significant pathogenic factors. In this article, we discuss the role of DNA damage and the repair deficit in the development of DR, ARMD and glaucoma.

We visited 1519 villages across 19 Indian states in 2009 to (a) count all health care providers and (b) elicit their quality as measured through tests of medical knowledge. We document three main findings. First, 75% of villages have at least one health care provider and 64% of care is sought in villages with 3 or more providers. Most providers are in the private sector (86%) and, within the private sector, the majority are 'informal providers' without any formal medical training. Our estimates suggest that such informal providers account for 68% of the total provider population in rural India. Second, there is considerable variation in quality across states and formal qualifications are a poor predictor of quality. For instance, the medical knowledge of informal providers in Tamil Nadu and Karnataka is higher than that of fully trained doctors in Bihar and Uttar Pradesh. Surprisingly, the share of informal providers does not decline with socioeconomic status. Instead, their quality, along with the quality of doctors in the private and public sector, increases sharply. Third, India is divided into two nations not just by quality of health care providers, but also by costs: Better performing states provide higher quality at lower per-visit costs, suggesting that they are on a different production possibility frontier. These patterns are consistent with significant variation across states in the availability and quality of medical education. Our results highlight the complex structure of health care markets, the large share of private informal providers, and the substantial variation in the quality and cost of care across and within markets in rural India. Measuring and accounting for this complexity is essential for health care policy in India., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

A DNA dosimeter (DNAd) was previously developed that uses double-strand breaks (DSB) to measure dose. This dosimeter has been tested to measure dose in scenarios where transient-charged particle equilibrium (TCPE) has been established. The probability of double strand break (PDSB o ), which is the ratio of broken double-stranded DNA (dsDNA) to the initial unbroken dsDNA in the dosimeter, was used to quantify DSBs and related to dose. The goal of this work is to produce a new technique to process and analyze the DNAd and quantify DNA-DSBs. This technique included simultaneously processing multiple DNAds and also establishing a new form to the probability of double strand break (PDSB n ), which was then used to test the DNAd in a non-TCPE condition by taking beam penumbra measurements. The technique utilized a 384-well plate, and the measurements were made at the edge of a 10 × 10 cm field and compared to film measurements. During these penumbra measurements, while observing the positional differences in the higher gradient region at 4.1 and 4.55 cm from the center of the radiation field, the distance to agreement of PDSB o to film were 0.38 cm and 0.26 cm while the distance to agreement of PDSB n to film were 0.11 cm and 0.06 cm, respectively. Finally, the developed new separation technique reduced the time needed for the analysis of 25 samples from 200 min to 30 min., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Fibroblast dysfunction is the main pathogenic mechanism of idiopathic pulmonary fibrosis (IPF). S100 calcium-binding protein A4 (S100A4) plays critical roles in the proliferation of fibroblasts and in the development of pulmonary, hepatic, and renal fibrosis. However, the clinical implications of S100A4 in IPF have not been evaluated., Methods and Materials: The S100A4 mRNA and protein levels were measured by real-time PCR and immunoblotting in fibroblasts from IPF patients and controls. The S100A4 level was measured by enzyme-linked immunosorbent assay in bronchoalveolar lavage fluid (BALF) from the normal controls (NCs; n = 33) and from patients with IPF (n = 87), non-specific interstitial pneumonia (NSIP; n = 22), hypersensitivity pneumonitis (HP; n = 19), and sarcoidosis (n = 9). S100A4 localization was evaluated by immunofluorescence staining., Results: The S100A4 mRNA and protein levels were significantly higher in fibroblasts from IPF patients (n = 14) than in those from controls (n = 10, p < 0.001). The S100A4 protein level in BALF was significantly higher in the IPF (89.25 [49.92-203.02 pg/mL]), NSIP (74.53 [41.88-131.45 pg/mL]), HP (222.36 [104.92-436.92 pg/mL]) and sarcoidosis (101.62 [59.36-300.62 pg/mL]) patients than in the NCs (7.57 [1.31-14.04 pg/mL], p < 0.01, respectively). Cutoff S100A4 levels of 18.85 and 28.88 pg/mL had 87.4% and 87.8% accuracy, respectively, for discriminating IPF and other lung diseases from NCs., Conclusions: S100A4 is expressed by α-SMA-positive cells in the interstitium of the IPF patients. S100A4 may participate in the development of IPF, and its protein level may be a candidate diagnostic and therapeutic marker for IPF., (Copyright © 2020. Published by Elsevier Ltd.)

In this work, we developed a DNA dosimeter, consisting of 4-kb DNA strands attached to magnetic streptavidin beads and labeled with fluorescein, to detect double-strand breaks (DSBs). The purpose here was to evaluate whether the DNA dosimeter readings reflect the relative biological effects of 160 kVp and 6 MV X rays. AVarian 600 C/D linac (6 MV) and a Faxitron cabinet X-ray system (160 kVp), both calibrated using traceable methods, were used to deliver high- and low-energy photons, respectively, to DNA dosimeters and multiple cell lines (mNs-5, HT-22 and Daoy). The responses were fit versus dose, and were used to quantify the dose of low-energy photons that produced the same response as that of the high-energy photons, at doses of 3, 6 and 9 Gy. The equivalent doses were utilized to calculate the relative biological effectiveness (RBEDSB and RBEcell survival). Additionally, a neutral comet assay was performed to measure the amount of intracellular DNA DSB, and ultimately the RBEcomet assay. The results of this work showed 160-kVp photon RBE values and 95% confidence intervals of 1.12 ± 0.04 (mNS-5), 1.16 ± 0.06 (HT-22), 1.25 ± 0.09 (Daoy) and 1.21 ± 0.24 (DNA dosimeter) at 9 Gy and 1.32 ± 0.16 (comet assay) at 3 Gy. Within the current error, the DNA dosimeter measured RBEDSB values in agreement with the RBEcell survival and assay from the cell survival and comet assay RBEcomet measurements. These results suggest that the DNA dosimeter can measure the changes in the radiobiological effects from different energy photons., (©2020 by Radiation Research Society. All rights of reproduction in any form reserved.)

Our previous transcriptome study of cultured fibroblasts identified 178 genes that were differentially expressed by 8 idiopathic pulmonary fibrosis (IPF) fibroblasts compared with 4 controls. Here, we performed genome-wide DNA methylation analysis to evaluate the relationship of CpG methylation to differential gene expression. Among 485,577 loci, 5850 loci on 2282 genes showed significant differences between the 2 groups (delta-beta >10.21 and p -value <0.05). Among these, beta values of 80 CpGs (30 hypermethylated and 50 hypomethylated) were significantly correlated with mRNA expression of 34 genes (19.1%) of the 178 differentially expressed genes between the 2 groups (13 downregulated and 21 upregulated). Gene ontology enrichment of these genes included cell adhesion, molecule binding, chemical homeostasis, surfactant homeostasis, and receptor binding. One-third of them are involved in the known process of fibrosis; the others are novel genes with respect to pulmonary fibrosis. We identified relationships between the altered DNA methylation levels and about one-fifth of the corresponding changes in gene expression by lung tissue fibroblasts. Findings from this study provide new information on novel genes responsible for the pathogenesis of IPF under the control of CpG methylation changes in IPF lungs.

We developed a dosimeter that measures biological damage following delivery of therapeutic beams in the form of double-strand breaks (DSBs) to DNA. The dosimeter contains DNA strands that are labeled on one end with biotin and on the other with fluorescein and attached to magnetic microbeads. Following irradiation, a magnet is used to separate broken from unbroken DNA strands. Then, fluorescence is utilized to measure the relative amount of broken DNA and determine the probability for DSB. The long-term goal for this research is to evaluate whether this type of biologically based dosimeter holds any advantages over the conventional techniques. The purpose of this work was to optimize the dosimeter fabrication and usage to enable higher precision for the long-term research goal. More specifically, the goal was to optimize the DNA dosimeter using three metrics: the response, precision, and cost per dosimeter. Six aspects of the dosimeter fabrication and usage were varied and evaluated for their effect on the metrics: (1) the type of magnetic microbeads, (2) the microbead to DNA mass ratio at attachment, (3) the type of suspension buffer used during irradiation, (4) the concentration of the DNA dosimeter during irradiation, (5) the time waited between fabrication and irradiation of the dosimeter, and (6) the time waited between irradiation and read out of the response. In brief, the best results were achieved with the dosimeter when attaching 4.2 µg of DNA with 1 mg of MyOne T1 microbeads and by suspending the microbead-connected DNA strands with 200 µl of phosphate-buffered saline for irradiation. Also, better results were achieved when waiting a day after fabrication before irradiating the dosimeter and also waiting an hour after irradiation to measure the response. This manuscript is meant to serve as guide for others who would like to replicate this DNA dose measurement technique.

Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52 -independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast., Competing Interests: The authors declare no conflict of interest.

Ribonucleoside monophosphates (rNMPs) mis-incorporated during DNA replication are removed by RNase H2-dependent excision repair or by topoisomerase I (Top1)-catalyzed cleavage. The cleavage of rNMPs by Top1 produces 3' ends harboring terminal adducts, such as 2',3'-cyclic phosphate or Top1 cleavage complex (Top1cc), and leads to frequent mutagenesis and DNA damage checkpoint induction. We surveyed a range of candidate enzymes from Saccharomyces cerevisiae for potential roles in Top1-dependent genomic rNMP removal. Genetic and biochemical analyses reveal that Apn2 resolves phosphotyrosine-DNA conjugates, terminal 2',3'-cyclic phosphates, and their hydrolyzed products. APN2 also suppresses 2-base pair (bp) slippage mutagenesis in RNH201-deficient cells. Our results define additional activities of Apn2 in resolving a wide range of 3' end blocks and identify a role for Apn2 in maintaining genome integrity during rNMP repair.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort ( n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions in vitro . Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer., (©2018 American Association for Cancer Research.)

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes., (© 2018 Wang et al.)

Purpose: Many types of dosimeters are used to measure radiation dose and calibrate radiotherapy equipment, but none directly measure the biological effect of this dose. The purpose here is to create a dosimeter that can measure the probability of double-strand breaks (DSB) for DNA, which is directly related to the biological effect of radiation., Methods: A DNA dosimeter, consisting of magnetic streptavidin beads attached to four kilobase pair DNA strands labeled with biotin and fluorescein amidite (FAM) on opposing ends, was suspended in phosphate-buffered saline (PBS). Fifty microliter samples were placed in plastic tubes inside a water tank setup and irradiated at the dose levels of 25, 50, 100, 150, and 200 Gy. After irradiation, the dosimeters were mechanically separated into beads (intact DNA) and supernatant (broken DNA/FAM) using a magnet. The fluorescence was read and the probability of DSB was calculated. This DNA dosimeter response was benchmarked against a Southern blot analysis technique for the measurement of DSB probability., Results: For the DNA dosimeter, the probabilities of DSB at the dose levels of 25, 50, 100, 150, and 200 Gy were 0.043, 0.081, 0.149, 0.196, and 0.242, respectively, and the standard errors of the mean were 0.002, 0.003, 0.006, 0.005, and 0.011, respectively. For the Southern blot method, the probabilities of DSB at the dose levels of 25, 50, 100, 150, and 200 Gy were 0.053, 0.105, 0.198, 0.235, and 0.264, respectively, and the standard errors of the mean were 0.013, 0.024, 0.040, 0.044, and 0.063, respectively., Conclusions: A DNA dosimeter can accurately determine the probability of DNA double-strand break (DSB), one of the most toxic effects of radiotherapy, for absorbed radiation doses from 25 to 200 Gy. This is an important step in demonstrating the viability of DNA dosimeters as a measurement technique for radiation., (© 2018 American Association of Physicists in Medicine.)

Yeast Rad1-Rad10 (XPF-ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1-Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1-Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1-Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1-Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1-Rad10 removes ICL via NER, whereas in S/G2, Rad1-Rad10 facilitates NER-independent replication-coupled ICLR.

DNA double-strand breaks (DSBs) are induced by a variety of genotoxic agents, including ionizing radiation and chemotherapy drugs for treating cancers. The elimination of DSBs proceeds via distinctive error-free and error-prone pathways. Repair by homologous recombination (HR) is largely error-free and mediated by RAD51/BRCA2 gene products. Classical non-homologous end joining (C-NHEJ) requires the Ku heterodimer and can efficiently rejoin breaks, with occasional loss or gain of DNA information. Recently, evidence has unveiled another DNA end-joining mechanism that is independent of recombination factors and Ku proteins, termed alternative non-homologous end joining (A-NHEJ). While A-NHEJ-mediated repair does not require homology, in a subtype of A-NHEJ, DSB breaks are sealed by microhomology (MH)-mediated base-pairing of DNA single strands, followed by nucleolytic trimming of DNA flaps, DNA gap filling, and DNA ligation, yielding products that are always associated with DNA deletion. This highly error-prone DSB repair pathway is termed microhomology-mediated end joining (MMEJ). Dissecting the mechanisms of MMEJ is of great interest because of its potential to destabilize the genome through gene deletions and chromosomal rearrangements in cells deficient in canonical repair pathways, including HR and C-NHEJ. In addition, evidence now suggests that MMEJ plays a physiological role in normal cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden.

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by the complex interaction of cells involved in chronic inflammation and fibrosis. Global gene expression of a homogenous cell population will identify novel candidate genes., Methods: Gene expression of fibroblasts derived from lung tissues (8 IPF and 4 controls) was profiled, and ontology and functional pathway were analyzed in the genes exhibiting >2 absolute fold changes with p-values < 0.05. CCL8 mRNA and protein levels were quantified using real-time PCR and ELISA. CCL8 localization was evaluated by immunofluorescence staining., Results: One hundred seventy eight genes differentially expressed and 15 genes exhibited >10-fold change. Among them, 13 were novel in relation with IPF. CCL8 expression was 22.8-fold higher in IPF fibroblasts. The levels of CCL8 mRNA and protein were 3 and 9-fold higher in 14 IPF fibroblasts than those in 10 control fibroblasts by real-time PCR and ELISA (p = 0.022 and p = 0.026, respectively). The CCL8 concentrations in BAL fluid was significantly higher in 86 patients with IPF than those in 41 controls, and other interstitial lung diseases including non-specific interstitial pneumonia (n = 22), hypersensitivity pneumonitis (n = 20) and sarcoidosis (n = 19) (p < 0.005, respectively). Cut-off values of 2.29 pg/mL and 0.43 pg/mL possessed 80.2 and 70.7% accuracy for the discrimination of IPF from NC and the other lung diseases, respectively. IPF subjects with CCL8 levels >28.61 pg/mL showed shorter survival compared to those with lower levels (p = 0.012). CCL8 was expressed by α-SMA-positive cells in the interstitium of IPF., Conclusions: Transcriptome analysis identified several novel IPF-related genes. Among them, CCL8 is a candidate molecule for the differential diagnosis and prediction of survival.

Prevalence of microhomology (MH) at the breakpoint junctions in somatic and germ-line chromosomal rearrangements and in the programmed immune receptor rearrangements from cells deficient in classical end joining reveals an enigmatic process called MH-mediated end joining (MMEJ). MMEJ repairs DNA double strand breaks (DSBs) by annealing flanking MH and deleting genetic information at the repair junctions from yeast to humans. Being genetically distinct from canonical DNA DSB pathways, MMEJ is involved with the fusions of eroded/uncapped telomeres as well as with the assembly of chromosome fragments in chromothripsis. In this review article, we will discuss an up-to-date model representing the MMEJ process and the mechanism by which cells regulate MMEJ to limit repair-associated mutagenesis. We will also describe the possible therapeutic gains resulting from the inhibition of MMEJ in recombination deficient cancers. Lastly, we will embark on two contentious issues associated with MMEJ such as the significance of MH at the repair junction to be the hallmark of MMEJ and the relationship of MMEJ to other mechanistically related DSB repair pathways., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Plant lectins have attracted much attention for biomedical applications including targeted drug delivery system and therapy against tumors and microbial infections. The main problem of using lectins as a biomedical tool is a batch-to-batch variation in isoforms content. The production of lectins using recombination tools has the advantage of obtaining high amounts of proteins with more precise properties, but there are only a handful of functional recombinant lectins presently available. A fetuin/asialo-fetuin specific lectin, Rhodobindin, has unique tandem repeats structure which makes it useful in exploiting for recombinant lectin. We developed three functional recombinant lectins using E. coli expression system: one from full cDNA sequence and two from fragmentary sequences of Rhodobindin. Hemagglutinating activity and solubility of the recombinant lectins were highest at OD 0.7 cell concentration at 20 °C. The optimized process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.

Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive dyspnea and worsening lung function. ACE is increased in the bronchoalveolar lavage fluid from patients with IPF, suggesting the role of ACE in the pathogenesis of IPF. We evaluated the role of single-nucleotide polymorphisms (SNPs) in the development risk of IPF., Methods: Two-hundred twenty patients with IPF and 456 healthy subjects were included in this study. Eleven polymorphisms were selected among those reported previously. Genotype was performed by single base extension., Results: Although absolute LD (|D'|= 1 and r(2 )= 1) was not present, 11 SNPs showed tight LDs. The logistic analysis of the all of 11 SNPs on the ACE genes between patients with IPF and healthy subjects were found to be related with the risk of IPF in recessive type. However, in patients with IPF diagnosed by surgical lung biopsy, only two SNP of -5538T>C and +21288&#95;insdel SNPs were related with the risk of IPF in co-dominant type, and there were no SNPs related with the risk of IPF in dominant type. In patients with IPF diagnosed by clinical criteria or surgical lung biopsy, four SNPs on promoter (-5538T>C, -5508A>C, -3927T>C, -115T>C), one on intron (+15276A>G), one on exon (+21181G>A), and one in three prime region (+21288&#95;insdel) were related with the risk of IPF., Conclusions: This study showed a newly discovered SNP of ACE associated with the risk of development of IPF. ACE -5538T>C and -5508A>C significantly associated with risk of IPF in Korea.

Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s) responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR) events after a DNA double-strand break (DSB) in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements., Competing Interests: The authors have declared that no competing interests exist.

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5' strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11-Rad50-Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase-topoisomerase complex Sgs1-Top3-Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways.

Background: Frailty is considered to be a clinical syndrome characterized by decreased physiological reserves associated with a greater risk of health-related problems, hospitalization, and death. The current study examined hospitalization, falls, cognitive decline and disability between robust, prefrail and frail elderly in one year., Methods: 110 participants aged 65 or more who visited two senior welfare centers in Seoul from February 2008 to June 2008 were surveyed again from March 2009 to June 2009 with demographic characteristics, number of chronic diseases and medication, study of osteoporotic fractures (SOF) frailty index, instrumental activity of daily living (IADL), depression, mini-mental state examination-Korean version (MMSE-K), falling history and admission history within one year. These results were compared with participants' previous survey done one year ago., Results: Among total 110 subjects, 48 (44%) robust, 30 (27%) prefrail, and 32 (29%) frail subjects changed to 26 (24%), 54 (49%), and 30 (27%) respectively over the year. There were statistical significances in age, number of chronic disease, depressive mood, MMSE, falls, hospitalization, IADL disability contributing to frailty (P < 0.05). Frailty defined by SOF frailty index was associated with greater risk of adverse outcomes. Frail subjects had a higher age-adjusted risk of cognitive function decline (odds ratio [OR], 3.57), disability (OR, 9.64), fall (OR, 5.42), and hospitalization (OR, 4.45; P < 0.005)., Conclusion: The frailty index like SOF frailty index might predict risk of falls, disability, hospitalization, and cognitive decline in the elderly, emphasizing special attention to the individuals showing frailty in outpatient examination.

Single-stranded DNA constitutes an important early intermediate for homologous recombination and damage-induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double-strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5'-strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2-dependent initiation to the Exo1- and Dna2-dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.

Islet transplantation (ITx) has potential as a therapy for patients with type 1 diabetes. For successful engraftment and insulin independence, the transplanted islets must establish an adequate, stable blood supply. Angiopoientin-1 (Ang1) is a specific growth factor that induces vascularization via the Tie2 or Tie1 receptor. In this study, we used an in vitro angiogenesis assay to evaluate islet function following transplantation and the effect of the Ang1 variant cartilage oligomeric matrix protein (COMP) Ang1 on isolated islets. The enhanced function of islets transduced with COMP-Ang1 was also confirmed in a streptozotocin (STZ)-induced diabetic mice model. In a three-dimensional collagen-based culture system, the transduction of COMP-Ang1 into islets significantly increased angiogenesis compared with the bacterial-β-galactosidase (LacZ)-transduced controls and an intact, nontransduced islet negative control group. COMP-Ang1 transduced islets also attenuated hyperglycemia in syngeneic diabetic C57BL/6 mice and enhanced glucose tolerance by areas under the curves of intraperitoneal glucose tolerance tests. These findings demonstrated the capacity of COMP-Ang1 to promote revascularization in cultured islets, which may contribute to successful transplantation in vivo., (2010 Elsevier Inc. All rights reserved.)

Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G(1) cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5' to 3' resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G(1), and markedly repressed at G(2). Repression of NHEJ at G(2) is achieved by efficient end resection and by the reduced association of core NHEJ proteins with DNA breaks, both of which depend on the CDK1 activity. Importantly, repression of 5' end resection by CDK1 inhibition at G(2) alone did not fully restore either physical association of Ku/Dnl4-Lif1 with DSBs or NHEJ proficiency to the level at G(1). Expression of excess Ku can partially offset the inhibition of end joining at G(2). The results suggest that regulation of Ku/Dnl4-Lif1 affinity for DNA ends may contribute to the cell cycle-dependent modulation of NHEJ efficiency.

Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.

Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5'-to-3' degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.

Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Delta maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.

The Mediator-related transcription co-factors integrate positive and negative inputs and recruit and activate the RNA polymerase II complex. To understand the role of Mediator during transcription, it is important to identify Mediator subunits that are essential for its functions. In the yeast Mediator, the conserved component Rgr1 is associated with multiple subunits that are required for specific activation or repression events. Yeast rgr1 is essential for viability, for certain repression mechanisms, and for activation of heat shock genes, but it is not known whether rgr1 is generally important for transcription. Here we have performed the first analysis of rgr-1 function in a metazoan. We found that in the developing Caenorhabditis elegans embryo rgr-1 is broadly required for transcription and for phosphorylation of both Ser-2 and Ser-5 of the RNA polymerase II C-terminal domain repeat. We conclude that RGR-1 fulfills a critical Mediator function that is broadly essential for metazoan mRNA transcription and that RGR-1 may be required at an early recruitment or initiation step.

The metazoan transcription elongation factor P-TEFb (CDK-9/cyclin T) is essential for HIV transcription, and is recruited by some cellular activators. P-TEFb promotes elongation in vitro by overcoming pausing that requires the SPT-4/SPT-5 complex, but considerable evidence indicates that SPT-4/SPT-5 facilitates elongation in vivo. Here we used RNA interference to investigate P-TEFb functions in vivo, in the Caenorhabditis elegans embryo. We found that P-TEFb is broadly essential for expression of early embryonic genes. P-TEFb is required for phosphorylation of Ser 2 of the RNA Polymerase II C-terminal domain (CTD) repeat, but not for most CTD Ser 5 phosphorylation, supporting the model that P-TEFb phosphorylates CTD Ser 2 during elongation. Remarkably, although heat shock genes are cdk-9-dependent, they can be activated when spt-4 and spt-5 expression is inhibited along with cdk-9. This observation suggests that SPT-4/SPT-5 has an inhibitory function in vivo, and that mutually opposing influences of P-TEFb and SPT-4/SPT-5 may combine to facilitate elongation, or insure fidelity of mRNA production. Other genes are not expressed when cdk-9, spt-4, and spt-5 are inhibited simultaneously, suggesting that these genes require P-TEFb in an additional mechanism, and that they and heat shock genes are regulated through different P-TEFb-dependent elongation pathways.

A high frequency of apoptosis is a conserved hallmark of oocyte development. In C. elegans, about half of all developing oocytes are normally killed by a physiological germline-specific apoptosis pathway, apparently so that they donate cytoplasm to the survivors. We have investigated the functions of CGH-1, the C. elegans ortholog of the predicted RNA helicase ste13/ME31B/RCK/p54, which is germline-associated in metazoans and required for sexual reproduction in yeast. We show that CGH-1 is expressed specifically in the germline and early embryo, and is localized to P granules and other possible mRNA-protein particles. cgh-1 is required for oocyte and sperm function. It is also needed to prevent the physiological germline apoptosis mechanism killing essentially all developing oocytes, making lack of cgh-1 function the first stimulus identified that can trigger this mechanism. We conclude that cgh-1 and its orthologs may perform conserved functions during gametogenesis, that in C. elegans certain aspects of oocyte development are monitored by the physiological germline apoptosis pathway, and that similar surveillance mechanisms may contribute to germline apoptosis in other species.

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.

A DNA polymerase alpha-associated multienzyme complex isolated from mouse LP1-1 cells transfected with the thymidine kinase gene of herpes simplex virus type I (1) showed activities of DNA polymerase alpha, topoisomerase II, and thymidine kinase (TK) in the complex. TK antiserum recognized a 43 kDa polypeptide only in the fraction of the multienzyme complex prepared from the LP1-1 cells but not that from L-M(TK-) cells. In permeabilized cells, hydroxyurea did not show any inhibitory effect on either DNA polymerase or TK, whereas aphidicolin, novobiocin, and TK antiserum inhibited both enzymes. These results provide evidence for the functional association and an allosteric interaction between the viral TK and host DNA polymerase alpha.

Nucleosomes positioned over promoters are usually inhibitory to protein binding and activity. We analyzed at the nucleotide level of resolution the nucleosomal organization of a distal, liver-specific enhancer in various mouse tissues and found that the enhancer exists in an array of three precisely positioned nucleosomes only in liver chromatin, where the enhancer is active. In vivo footprinting reveals that essential transcription factor-binding sites are occupied on apparent nucleosome surfaces, in one case leading to a perturbed nucleosomal structure. A similar nucleosomal array is generated with an in vitro chromatin assembly system in which nucleosome positioning is dependent upon binding to the enhancer of proteins related to hepatocyte nuclear factor 3. We suggest that certain transcription factors can organize nucleosomal structures that define an active enhancer element.