Dynamic exchange at regulatory elements during chromatin remodeling underlies assisted loading mechanism.

The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.

Replication fork stability confers chemoresistance in BRCA-deficient cells.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

Transcription factor AP1 potentiates chromatin accessibility and glucocorticoid receptor binding.

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.

7T-fMRI: Faster temporal resolution yields optimal BOLD sensitivity for functional network imaging specifically at high spatial resolution.

Recent developments in accelerated imaging methods allow faster acquisition of high spatial resolution images. This could improve the applications of functional magnetic resonance imaging at 7 Tesla (7T-fMRI), such as neurosurgical planning and Brain Computer Interfaces (BCIs). However, increasing the spatial and temporal resolution will both lead to signal-to-noise ratio (SNR) losses due to decreased net magnetization per voxel and T1-relaxation effect, respectively. This could potentially offset the SNR efficiency gains made with increasing temporal resolution. We investigated the effects of varying spatial and temporal resolution on fMRI sensitivity measures and their implications on fMRI-based BCI simulations. We compared temporal signal-to-noise ratio (tSNR), observed percent signal change (%∆S), volumes of significant activation, Z-scores and decoding performance of linear classifiers commonly used in BCIs across a range of spatial and temporal resolution images acquired during an ankle-tapping task. Our results revealed an average increase of 22% in %∆S (p=0.006) and 9% in decoding performance (p=0.015) with temporal resolution only at the highest spatial resolution of 1.5×1.5×1.5mm3, despite a 29% decrease in tSNR (p<0.001) and plateaued Z-scores. Further, the volume of significant activation was indifferent (p>0.05) across spatial resolution specifically at the highest temporal resolution of 500ms. These results demonstrate that the overall BOLD sensitivity can be increased significantly with temporal resolution, granted an adequately high spatial resolution with minimal physiological noise level. This shows the feasibility of diffuse motor-network imaging at high spatial and temporal resolution with robust BOLD sensitivity with 7T-fMRI. Importantly, we show that this sensitivity improvement could be extended to an fMRI application such as BCIs.

Spatially dynamic recurrent information flow across long-range dorsal motor network encodes selective motor goals.

Performing voluntary movements involves many regions of the brain, but it is unknown how they work together to plan and execute specific movements. We recorded high-resolution ultra-high-field blood-oxygen-level-dependent signal during a cued ankle-dorsiflexion task. The spatiotemporal dynamics and the patterns of task-relevant information flow across the dorsal motor network were investigated. We show that task-relevant information appears and decays earlier in the higher order areas of the dorsal motor network then in the primary motor cortex. Furthermore, the results show that task-relevant information is encoded in general initially, and then selective goals are subsequently encoded in specifics subregions across the network. Importantly, the patterns of recurrent information flow across the network vary across different subregions depending on the goal. Recurrent information flow was observed across all higher order areas of the dorsal motor network in the subregions encoding for the current goal. In contrast, only the top-down information flow from the supplementary motor cortex to the frontoparietal regions, with weakened recurrent information flow between the frontoparietal regions and bottom-up information flow from the frontoparietal regions to the supplementary cortex were observed in the subregions encoding for the opposing goal. We conclude that selective motor goal encoding and execution rely on goal-dependent differences in subregional recurrent information flow patterns across the long-range dorsal motor network areas that exhibit graded functional specialization.

Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing.

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ± 50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.

The accessible chromatin landscape of the human genome.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.

An expansive human regulatory lexicon encoded in transcription factor footprints.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

Optimized partial-coverage functional analysis pipeline (OPFAP): a semi-automated pipeline for skull stripping and co-registration of partial-coverage, ultra-high-field functional images.

OBJECTIVE: Ultra-high-field functional MRI (UHF-fMRI) allows for higher spatiotemporal resolution imaging. However, higher-resolution imaging entails coverage limitations. Processing partial-coverage images using standard pipelines leads to sub-optimal results. We aimed to develop a simple, semi-automated pipeline for processing partial-coverage UHF-fMRI data using widely used image processing algorithms. MATERIALS AND METHODS: We developed automated pipelines for optimized skull stripping and co-registration of partial-coverage UHF functional images, using built-in functions of the Centre for Functional Magnetic Resonance Imaging of the Brain's (FMRIB's) Software library (FSL) and advanced normalization tools. We incorporated the pipelines into the FSL's functional analysis pipeline and provide a semi-automated optimized partial-coverage functional analysis pipeline (OPFAP). RESULTS: Compared to the standard pipeline, the OPFAP yielded images with 15 and 30% greater volume of non-zero voxels after skull stripping the functional and anatomical images, respectively (all p = 0.0004), which reflected the conservation of cortical voxels lost when the standard pipeline was used. The OPFAP yielded the greatest Dice and Jaccard coefficients (87 and 80%, respectively; all p < 0.0001) between the co-registered participant gyri maps and the template gyri maps, demonstrating the goodness of the co-registration results. Furthermore, the greatest volume of group-level activation in the most number of functionally relevant regions was observed when the OPFAP was used. Importantly, group-level activations were not observed when using the standard pipeline. CONCLUSION: These results suggest that the OPFAP should be used for processing partial-coverage UHF-fMRI data for detecting high-resolution macroscopic blood oxygenation level-dependent activations.

C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements.

Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocorticoid injection. Upon activation of the glucocorticoid receptor (GR), proximal regions of activated and repressed genes are remodelled, and these remodelling events correlate with RNA polymerase II occupancy of regulated genes. GR is exclusively associated with accessible chromatin and 62% percent of GR-binding sites are occupied by C/EBPß. At the majority of these sites, chromatin is preaccessible suggesting a priming function of C/EBPß for GR recruitment. Disruption of C/EBPß binding to chromatin results in attenuation of pre-programmed chromatin accessibility, GR recruitment and GR-induced chromatin remodelling specifically at sites co-occupied by GR and C/EBPß. Collectively, we demonstrate a highly cooperative mechanism by which C/EBPß regulates selective GR binding to the genome in liver tissue. We suggest that selective targeting of GR in other tissues is likely mediated by the combined action of cell-specific priming proteins and chromatin remodellers.

Coupling transcription factor occupancy to nucleosome architecture with DNase-FLASH.

It is currently not possible to resolve the genome-wide relationship of transcription factors (TFs) and nucleosomes at the level of individual chromatin templates despite rapidly increasing data on TF and nucleosome occupancy in the human genome. Here we describe DNase I-released fragment-length analysis of hypersensitivity (DNase-FLASH), an approach that directly couples mapping of TF occupancy, via quantification of DNA microfragments released from individual TF recognition sites in regulatory DNA, to the surrounding nucleosome architecture, via analysis of larger DNA fragments, in a single assay. DNase-FLASH enables coupling of individual TF footprints to nucleosome occupancy, identifying TFs that precisely demarcate the regulatory DNA-nucleosome interface.

Modulation of RNA polymerase assembly dynamics in transcriptional regulation.

The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcription factors or serves a regulatory role is unknown. Here we have used single-cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA Pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA Pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady-state population of assembling polymerase. As a consequence, polymerase assembly efficiency and, ultimately, the rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly.

Interaction of the glucocorticoid receptor with the chromatin landscape.

The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.

The evolution of endovascular electroencephalography: historical perspective and future applications.

Current standard practice requires an invasive approach to the recording of electroencephalography (EEG) for epilepsy surgery, deep brain stimulation (DBS), and brain-machine interfaces (BMIs). The development of endovascular techniques offers a minimally invasive route to recording EEG from deep brain structures. This historical perspective aims to describe the technical progress in endovascular EEG by reviewing the first endovascular recordings made using a wire electrode, which was followed by the development of nanowire and catheter recordings and, finally, the most recent progress in stent-electrode recordings. The technical progress in device technology over time and the development of the ability to record chronic intravenous EEG from electrode arrays is described. Future applications for the use of endovascular EEG in the preoperative and operative management of epilepsy surgery are then discussed, followed by the possibility of the technique's future application in minimally invasive operative approaches to DBS and BMI.

New Face for Chromatin-Related Mesenchymal Modulator: n-CHD9 Localizes to Nucleoli and Interacts With Ribosomal Genes.

Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes.

Extensive chromatin remodelling and establishment of transcription factor 'hotspots' during early adipogenesis.

Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPß and -δ) to transcription factor 'hotspots'. Our results demonstrate that C/EBPß marks a large number of these transcription factor 'hotspots' before induction of differentiation and chromatin remodelling and is required for their establishment. Furthermore, a subset of early remodelled C/EBP-binding sites persists throughout differentiation and is later occupied by PPARγ, indicating that early C/EBP family members, in addition to their well-established role in activation of PPARγ transcription, may act as pioneering factors for PPARγ binding.

DNA methylation status predicts cell type-specific enhancer activity.

Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR-DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors.

Diverse gene reprogramming events occur in the same spatial clusters of distal regulatory elements.

The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation.

Corrosion inhibition of mild steel in 1 M HCl using water soluble chitosan derivative of vanillin.

The water-soluble chitosan derivative (WSCD) was made by mixing chitosan with sodium hydroxide, treating the mixture with chloroacetic acid, and then forming a Schiff base with vanillin in an acidic medium. In this study, we examined the corrosion-inhibiting ability of a WSCD on mild steel surfaces in acidic environments. Weight loss, EIS, PDP, LPS, and OCP measurements were used to study the corrosion resistance on mild steel surfaces in 1 M HCl solutions with known concentrations of WSCD. The results show that WSCD functions effectively as a mixed-type anodic and cathodic inhibitor, providing 87 % corrosion inhibition efficiency at 75 ppm. Using SEM to investigate the morphology of corroded mild steel with and without varying amounts of WSCD, impedance measurements show the development of a thin film of inhibitor on the metal surface, the extent of which increases as the inhibitor concentration rises. The WSCD molecule first adsorbs on mild steel and follows Langmuir adsorption isotherm. It is found that the (∆Gads0)adsorption's free energy is -17.473 kJ/mol. The contact angle measurements confirm that the hydrophobicity of the metal surface has increased as a result of the inhibitor's thin film development.