Dam mutants provide improved sensitivity and spatial resolution for profiling transcription factor binding.
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ABSTRACT:
SUBMITTER: Szczesnik T
PROVIDER: S-EPMC6567924 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DamID, in which a protein of interest is fused to Dam methylase, enables mapping of protein-DNA binding through readout of adenine methylation in genomic DNA. DamID offers a compelling alternative to chromatin immunoprecipitation sequencing (ChIP-Seq), particularly in cases where cell number or antibody availability are limiting. This comes at a cost, however, of high non-specific signal and a lowered spatial resolution of several kb, limiting its application to transcription factor-DNA binding. Here we show that mutations in Dam, when fused to the transcription factor Tcf7l2, greatly reduce non-specific methylation. Combined with a simplified DamID sequencing protocol, we find that these Dam mutants allow for accurate detection of transcription factor binding at a sensitivity and spatial resolution closely matching that seen in ChIP-seq.
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Project description:Dam mutants provide improved sensitivity and spatial resolution for profiling transcription factor binding
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Project description:An essential component of genome function is the syntax of genomic regulatory elements that determine how diverse transcription factors interact to orchestrate a program of regulatory control. A precise characterization of in vivo spacing constraints between key transcription factors would reveal key aspects of this genomic regulatory language. To discover novel transcription factor spatial binding constraints in vivo, we developed a new integrative computational method, genome wide event finding and motif discovery (GEM). GEM resolves ChIP data into explanatory motifs and binding events at high spatial resolution by linking binding event discovery and motif discovery with positional priors in the context of a generative probabilistic model of ChIP data and genome sequence. GEM analysis of 63 transcription factors in 214 ENCODE human ChIP-Seq experiments recovers more known factor motifs than other contemporary methods, and discovers six new motifs for factors with unknown binding specificity. GEM's adaptive learning of binding-event read distributions allows it to further improve upon previous methods for processing ChIP-Seq and ChIP-exo data to yield unsurpassed spatial resolution and discovery of closely spaced binding events of the same factor. In a systematic analysis of in vivo sequence-specific transcription factor binding using GEM, we have found hundreds of spatial binding constraints between factors. GEM found 37 examples of factor binding constraints in mouse ES cells, including strong distance-specific constraints between Klf4 and other key regulatory factors. In human ENCODE data, GEM found 390 examples of spatially constrained pair-wise binding, including such novel pairs as c-Fos:c-Jun/USF1, CTCF/Egr1, and HNF4A/FOXA1. The discovery of new factor-factor spatial constraints in ChIP data is significant because it proposes testable models for regulatory factor interactions that will help elucidate genome function and the implementation of combinatorial control.
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Project description:The quality of visual information that is available to an animal is limited by the size of its eyes. Differences in eye size can be observed even between closely related individuals, yet we understand little about how this affects vision. Insects are good models for exploring the effects of size on visual systems because many insect species exhibit size polymorphism. Previous work has been limited by difficulties in determining the 3D structure of eyes. We have developed a novel method based on x-ray microtomography to measure the 3D structure of insect eyes and to calculate predictions of their visual capabilities. We used our method to investigate visual allometry in the bumblebee Bombus terrestris and found that size affects specific aspects of vision, including binocular overlap, optical sensitivity, and dorsofrontal visual resolution. This reveals that differential scaling between eye areas provides flexibility that improves the visual capabilities of larger bumblebees.
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Project description:We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Experiment Overall Design: Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further. Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin) in a parallel study (Robbins et al., unpublished results). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturerâs protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.
2007-07-20 | E-GEOD-2928 | biostudies-arrayexpress
Project description:MotivationClusters of protein-DNA interaction events involving the same transcription factor are known to act as key components of invertebrate and mammalian promoters and enhancers. However, detecting closely spaced homotypic events from ChIP-Seq data is challenging because random variation in the ChIP fragmentation process obscures event locations.ResultsThe Genome Positioning System (GPS) can predict protein-DNA interaction events at high spatial resolution from ChIP-Seq data, while retaining the ability to resolve closely spaced events that appear as a single cluster of reads. GPS models observed reads using a complexity penalized mixture model and efficiently predicts event locations with a segmented EM algorithm. An optional mode permits GPS to align common events across distinct experiments. GPS detects more joint events in synthetic and actual ChIP-Seq data and has superior spatial resolution when compared with other methods. In addition, the specificity and sensitivity of GPS are superior to or comparable with other methods.Availabilityhttp://cgs.csail.mit.edu/gps.
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Project description:To understand the impact of genome sequence variation (the genotype) responsible for biological diversity and human health (the phenotype) including cystic fibrosis and Alzheimer's disease, we developed a Gaussian-process-based machine learning (ML) approach, variation spatial profiling (VSP). VSP uses a sparse collection of known variants found in the population that perturb the protein fold to define unknown variant function based on the emergent general principle of spatial covariance (SCV). SCV quantitatively captures the role of proximity in genotype-to-phenotype spatial-temporal relationships. Phenotype landscapes generated through SCV provide a platform that can be used to describe the functional properties that drive sequence-to-function-to-structure design of the polypeptide fold at atomic resolution. We provide proof of principle that SCV can enable the use of population-based genomic platforms to define the origins and mechanism of action of genotype-to-phenotype transformations contributing to the health and disease of an individual.
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Project description:Multishot multiplexed sensitivity-encoding diffusion-weighted imaging is a feasible and easily implementable routine breast MRI protocol that yields high-quality diffusion-weighted breast images.Purpose: To compare multiplexed sensitivity-encoding (MUSE) diffusion-weighted imaging (DWI) and single-shot DWI for lesion visibility and differentiation of malignant and benign lesions within the breast.Materials and Methods: In this prospective institutional review board-approved study, both MUSE DWI and single-shot DWI sequences were first optimized in breast phantoms and then performed in a group of patients. Thirty women (mean age, 51.1 years ± 10.1 [standard deviation]; age range, 27-70 years) with 37 lesions were included in this study and underwent scanning using both techniques. Visual qualitative analysis of diffusion-weighted images was accomplished by two independent readers; images were assessed for lesion visibility, adequate fat suppression, and the presence of artifacts. Quantitative analysis was performed by calculating apparent diffusion coefficient (ADC) values and image quality parameters (signal-to-noise ratio [SNR] for lesions and fibroglandular tissue; contrast-to-noise ratio) by manually drawing regions of interest within the phantoms and breast tumor tissue. Interreader variability was determined using the Cohen κ coefficient, and quantitative differences between MUSE DWI and single-shot DWI were assessed using the Mann-Whitney U test; significance was defined at P < .05.Results: MUSE DWI yielded significantly improved image quality compared with single-shot DWI in phantoms (SNR, P = .001) and participants (lesion SNR, P = .009; fibroglandular tissue SNR, P = .05; contrast-to-noise ratio, P = .008). MUSE DWI ADC values showed a significant difference between malignant and benign lesions (P < .001). No significant differences were found between MUSE DWI and single-shot DWI in the mean, maximum, and minimum ADC values (P = .96, P = .28, and P = .49, respectively). Visual qualitative analysis resulted in better lesion visibility for MUSE DWI over single-shot DWI (κ = 0.70).Conclusion: MUSE DWI is a promising high-spatial-resolution technique that may enhance breast MRI protocols without the need for contrast material administration in breast screening.Keywords: Breast, MR-Diffusion Weighted Imaging, OncologySupplemental material is available for this article.© RSNA, 2020.
| S-EPMC7983772 | biostudies-literature
Project description:Induced morphology changes of cells and organelles are by far the easiest way to determine precise protein sub-locations and organelle quantities in light microscopy. By using hypotonic solutions to swell mammalian cell organelles we demonstrate that precise membrane, lumen or matrix protein locations within the endoplasmic reticulum, Golgi and mitochondria can reliably be established. We also show the benefit of this approach for organelle quantifications, especially for clumped or intertwined organelles like peroxisomes and mitochondria. Since cell and organelle swelling is reversible, it can be applied to live cells for successive high-resolution analyses. Our approach outperforms many existing imaging modalities with respect to resolution, ease-of-use and cost-effectiveness without excluding any co-utilization with existing optical (super)resolution techniques.
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Project description:PurposeThis work aims to investigate the utility of velocity selective inversion pulses for perfusion weighted functional MRI.MethodsTracer kinetic properties of velocity selective inversion (VSI) pulses as an input function for an arterial spin labeling (ASL) experiment were characterized in a group of healthy participants. Numerical simulations were conducted to search for a robust set of timing parameters for FMRI time series acquisition with maximal signal to noise ratio efficiency. The performance of three VSI pulse sequences with different timing parameters was compared with a pseudocontinuous ASL sequence in a simple FMRI experiment conducted on healthy participants.ResultsThe fit to the tracer kinetic model yielded arterial CBV of 1.24% ± 0.52% and 0.45 ± 0.11% and perfusion rates of 60.8 ± 32.3 and 34.4 ± 5.4 mL/min/100 g for gray and white matter, respectively. Bolus arrival times were estimated as 75.7 ± 21 ms and 349 ± 78 ms for gray and white matter, respectively. The FMRI experiments showed that VSI pulses yield comparable sensitivity to PCASL with similar timing parameters (TR = 4 s). However, VSI pulses could be used at a faster acquisition speed (TR = 3 s) and were more sensitive to neuronal activity than PCASL pulses, as evidenced by the 31% higher Z scores obtained on average in the active regions.ConclusionVSI pulses can be very beneficial for perfusion weighted functional MRI because of their tracer kinetic characteristics, which allow a faster acquisition rate while maintaining an efficient labeling input function.
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