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Progesterone receptor (PR)-interacting compounds in the environment are associated with serious health hazards. However, methods for their detection in environmental samples are cumbersome. We report a sensitive activity-based biosensor for rapid and reliable screening of progesterone receptor (PR)-interacting endocrine disrupting chemicals (EDCs). The biosensor is a cell line which expresses nuclear mCherry-NF1 and a green fluorescent protein (GFP)-tagged chimera of glucocorticoid receptor (GR) N terminus fused to the ligand binding domain (LBD) of PR (GFP-GR-PR). As this LBD is shared by the PRA and PRB, the biosensor reports on the activation of both PR isoforms. This GFP-GR-PR chimera is cytoplasmic in the absence of hormone and translocates rapidly to the nucleus in response to PR agonists or antagonists in concentration- and time-dependent manner. In live cells, presence of nuclear NF1 label eliminates cell fixation and nuclear staining resulting in efficient screening. The assay can be used in screens for novel PR ligands and PR-interacting contaminants in environmental samples. A limited screen of river water samples indicated a widespread, low-level contamination with PR-interacting contaminants in all tested samples. [ABSTRACT FROM AUTHOR]

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis. The involvement of nuclear pore proteins in cellular mechanosensing and metastasis is unclear. Here the authors identify that nuclear pore protein NUP210 promotes metastasis through the interaction with mechanotransducer LINC complex protein and chromatin to regulate mechanosensitive genes. [ABSTRACT FROM AUTHOR]

A widely regarded model for glucocorticoid receptor (GR) action postulates that dimeric binding to DNA regulates unfavorable metabolic pathways while monomeric receptor binding promotes repressive gene responses related to its anti-inflammatory effects. This model has been built upon the characterization of the GRdim mutant, reported to be incapable of DNA binding and dimerization. Although quantitative live-cell imaging data shows GRdim as mostly dimeric, genomic studies based on recovery of enriched half-site response elements suggest monomeric engagement on DNA. Here, we perform genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding even to open chromatin. We also find that GRdim does not exclusively bind half-response elements. Our results do not support a physiological role for monomeric GR and are consistent with a common mode of receptor binding via higher order structures that drives both the activating and repressive actions of glucocorticoids. Glucocorticoid receptors (GR) are thought to bind DNA as dimers or monomers, to regulate different transcription pathways. Here, the authors perform genome-wide studies on GRs with mutations that impair dimerization and provide evidence that monomeric GRs do not play a significant physiologic role. [ABSTRACT FROM AUTHOR]

The cytokines interleukin 1β and 6 (IL-1β, IL-6) mediate the acute phase response (APR). In liver, they regulate the secretion of acute phase proteins. Using RNA-seq in primary hepatocytes, we show that these cytokines regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression. By mapping changes in enhancer landscape and transcription factor occupancy (using ChIP-seq), we show that synergistic gene induction is achieved by assisted loading of STAT3 on chromatin by NF-κB. With IL-6 treatment alone, STAT3 does not efficiently bind 20% of its coordinated binding sites. In the presence of IL-1β, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac. This facilitates STAT3 binding and synergistic gene expression. Our findings reveal an enhancer-specific crosstalk whereby NF-κB enables STAT3 binding at some enhancers while perturbing it at others. This model reconciles seemingly contradictory reports of NF-κB-STAT3 crosstalk. [ABSTRACT FROM AUTHOR]

The interaction of regulatory proteins with the complex nucleoprotein structures that are found in mammalian cells involves chromatin reorganization at multiple levels. Mechanisms that support these transitions are complex on many timescales, which range from milliseconds to minutes or hours. In this Review, we discuss emerging concepts regarding the function of regulatory elements in living cells. We also explore the involvement of these dynamic and stochastic processes in the evolution of fluctuating transcriptional activity states that are now commonly reported in eukaryotic systems. [ABSTRACT FROM AUTHOR]

ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. [ABSTRACT FROM AUTHOR]

Dynamic transitions in the epigenome have been associated with regulated patterns of nuclear organization. The accumulating evidence that chromatin remodeling is implicated in circadian function prompted us to explore whether the clock may control nuclear architecture. We applied the chromosome conformation capture on chip technology in mouse embryonic fibroblasts (MEFs) to demonstrate the presence of circadian long-range interactions using the clock-controlled Dbp gene as bait. The circadian genomic interactions with Dbp were highly specific and were absent in MEFs whose clock was disrupted by ablation of the Bmal1 gene (also called Arntl). We establish that the Dbp circadian interactome contains a wide variety of genes and clock-related DNA elements. These findings reveal a previously unappreciated circadian and clock-dependent shaping of the nuclear landscape. [ABSTRACT FROM AUTHOR]

The effects of pump laser spectral bandwidth on the performance of longitudinally pumped diode-pumped alkali lasers is explored by extending the analytic, three-level model using longitudinally averaged number densities. By assuming a statistical distribution between the upper two levels, the limiting solution for the quasi-two level system is achieved. A second limiting solution is identified for strongly bleached conditions where the atom recycle rate, limited by spin-orbit relaxation, fully specifies the output power. Performance in the intermediate regime depends significantly on the pump bandwidth relative to the atomic absorption line width and requires numerical simulation. The ratio of populations for the two excited, P states completes an analytic solution and depends primarily on pump laser bandwidth, threshold, and alkali concentration. Absorption well into the wings on the atomic profile can be utilized by increasing alkali concentration, but imposes increased pump intensity threshold. [ABSTRACT FROM AUTHOR]

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies. [ABSTRACT FROM AUTHOR]

Development, differentiation and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from a myriad of potential genomic binding sites is undetermined. Here we show that up to 95% of de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligand-activated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cell-selective glucocorticoid receptor occupancy patterns appear to be comprehensively predetermined by cell-specific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a framework for understanding regulatory factor-genome interactions and provide a molecular basis for the tissue selectivity of steroid pharmaceuticals and other agents that intersect the living genome. [ABSTRACT FROM AUTHOR]

Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation. [ABSTRACT FROM AUTHOR]

We have identified a novel gene, six transmembrane protein of prostate 2 (STAMP2), named for its high sequence similarity to the recently identified STAMP1 gene. STAMP2 displays a tissue-restricted expression with highest expression levels in placenta, lung, heart, and prostate and is predicted to code for a 459-amino acid six transmembrane protein. Using a form of STAMP2 labeled with green flourescent protein (GFP) in quantitative time-lapse and immunofluorescence confocal microscopy, we show that STAMP2 is primarily localized to the Golgi complex, trans-Golgi network, and the plasma membrane. STAMP2 also localizes to vesicular-tubular structures in the cytosol and colocalizes with the Early Endosome Antigen1 (EEA1) suggesting that it may be involved in the secretory/endocytic pathways. STAMP2 expression is exquisitely androgen regulated in the androgen-sensitive, androgen receptor-positive prostate cancer cell line LNCaP, but not in androgen receptor-negative prostate cancer cell lines PC-3 and DU145. Analysis of STAMP2 expression in matched normal and tumor samples microdissected from prostate cancer specimens indicates that STAMP2 is overexpressed in prostate cancer cells compared with normal prostate epithelial cells. Furthermore, ectopic expression of STAMP2 in prostate cancer cells significantly increases cell growth and colony formation suggesting that STAMP2 may have a role in cell proliferation. Taken together, these data suggest that STAMP2 may contribute to the normal biology of the prostate cell, as well as prostate cancer progression.Oncogene (2005) 24, 4934–4945. doi:10.1038/sj.onc.1208677; published online 9 May 2005 [ABSTRACT FROM AUTHOR]

Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR-/- and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27KIP1. Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.Oncogene (2004) 23, 4400-4412. doi:10.1038/sj.onc.1207553 Published online 12 April 2004 [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor protein plays a critical role in mediating cellular response to stress. Upon DNA damage, post-translational modifications stabilize and activate this nuclear phosphoprotein. To determine the effect of phosphorylation site mutants in the context of the whole p53 protein, we performed reporter assays in p53 and MDM2 knockout mouse embryonic fibroblasts transfected with full-length p53 constructs. We show that mutation of S37 causes a decrease in p53 transcriptional activity compared to wild-type p53. Our data further suggest that the dephosphorylation of p53 at S37 is a regulated event involving protein phosphatase 2A (PP2A). Coimmunoprecipitation and immunofluorescence microscopy studies demonstrate that PP2A and p53 associate with one another in vivo following ?-irradiation. Consistent with these observations, phosphorylated S37 accumulates in cell extracts prepared from ?-irradiated Molt-4 cells in the presence of okadaic acid. Furthermore, in vitro phosphatase assays show that PP2A dephosphorylates p53 at S37. These results suggest that dephosphorylation of p53 at S37 plays a role in the transcriptional regulation of the p53 protein in response to DNA damage.Oncogene (2004) 23, 49-57. doi:10.1038/sj.onc.1207005 [ABSTRACT FROM AUTHOR]

DNA accessibility to transcription regulators varies between cells and modulates gene expression patterns. Several "open" chromatin profiling methods that provide valuable insight into the activity of these regulatory regions have been developed. However, their application to clinical samples has been limited despite the discovery that the Analysis of Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) method can be performed using fewer cells than other techniques. Obtaining fresh rather than stored samples and a lack of adequate optimization and quality controls are major barriers to ATAC's clinical implementation. Here, we describe an optimized ATAC protocol in which we varied nuclear preparation conditions and transposase concentrations and applied rigorous quality control measures before testing fresh, flash frozen, and cryopreserved breast cells and tissue. We obtained high quality data from small cell number. Furthermore, the genomic distribution of sequencing reads, their enrichment at transcription start sites, and transcription factor footprint analyses were similar between cryopreserved and fresh samples. This updated method is applicable to clinical samples, including cells from fine needle aspiration and tissues obtained via core needle biopsy or surgery. Chromatin accessibility analysis using patient samples will greatly expand the range of translational research and personalized medicine by identification of clinically-relevant epigenetic features. [ABSTRACT FROM AUTHOR]

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other's dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level. [ABSTRACT FROM AUTHOR]

Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome. [ABSTRACT FROM AUTHOR]

Diversification and specialization of high-throughput technologies demand assay-specific treatment of data for reliable interpretation. A new study shows that data generated using the Hi-C approach contain hidden features of interchromosomal DNA interactions, which are revealed through analysis with an integrated probabilistic model that corrects for multiple sources of bias in the data. [ABSTRACT FROM AUTHOR]

The article discusses the process of deep sequencing of DNase I method in the U.S. It details how the researchers have used this method to the genome-wide localization of disrupted nucleosomes and the mapping of transcription factor binding cites. It also demonstrates how this method can lead to the revelation of both regions of chromatin transition and specific binding events that occur within those regions.

A correction to the article "Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions" that was published in the 2014 issue, volume 21, pages 73-81 is presented.