Hypersensitive Research Articles

Pepper immunity against Ralstonia solanacearum is positively regulated by CaWRKY3 through modulation of different WRKY transcription factors

BackgroundSeveral WRKY transcription factors (TFs), including CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 are known to govern the resistance of pepper (Capsicum annuum L.) plants to Ralstonia solanacearum infestation (RSI) and other abiotic stresses. However, the molecular mechanisms underlying these processes remain elusive.MethodsThis study functionally described CaWRKY3 for its role in pepper immunity against RSI. The roles of phytohormones in mediating the expression levels of CaWRKY3 were investigated by subjecting pepper plants to 1 mM salicylic acid (SA), 100 µM methyl jasmonate (MeJA), and 100 µM ethylene (ETH) at 4-leaf stage. A virus-induced gene silencing (VIGS) approach based on the Tobacco Rattle Virus (TRV) was used to silence CaWRKY3 in pepper, and transiently over-expressed to infer its role against RSI.ResultsPhytohormones and RSI increased CaWRKY3 transcription. The transcriptions of defense-associated marker genes, including CaNPR1, CaPR1, CaDEF1, and CaHIR1 were decreased in VIGS experiment, which made pepper less resistant to RSI. Significant hypersensitive (HR)-like cell death, H2O2 buildup, and transcriptional up-regulation of immunological marker genes were noticed in pepper when CaWRKY3 was transiently overexpressed. Transcriptional activity of CaWRKY3 was increased with overexpression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40, and vice versa. In contrast, Pseudomonas syringae pv tomato DC3000 (Pst DC3000) was easily repelled by the innate immune system of transgenic Arabidopsis thaliana that overexpressed CaWRKY3. The transcriptions of defense-related marker genes like AtPR1, AtPR2, and AtNPR1 were increased in CaWRKY3-overexpressing transgenic A. thaliana plants.ConclusionIt is concluded that CaWRKY3 favorably regulates phytohormone-mediated synergistic signaling, which controls cell death in plant and immunity of pepper plant against bacterial infections.

Decoding Human Somatosensory Sensitivity Through Resting EEG and Behavioral Analysis: A Multimodal Fusion Approach.

In precision medicine and clinical pain management, the creation of quantitative, objective indicators to assess somatosensory sensitivity was essential. This study proposed a fusion approach for decoding human somatosensory sensitivity, which combined multimodal (quantitative sensory test and neurophysiology) features to classify the dataset on individual somatosensory sensitivity and reveal distinct types of brain activation patterns. Sixty healthy participants took part in the experiment on somatosensory sensitivity that implemented cold, heat, mechanical punctate, and pressure stimuli, and the resting-state electroencephalography (EEG) was collected using BrainVision. The quantitative sensory testing (QST) scores of the participants were clustered using the unsupervised k-means algorithm into four subgroups: generally hypersensitive (HS), generally non-sensitive (NS), predominantly thermally sensitive (TS), and predominantly mechanically sensitive (MS). Furthermore, two types of power spectral density (PSD), band-based PSD (BB-PSD) and frequency-based PSD (FB-PSD), and two types of inter-electrode connectivity (IEC), band-based connectivity (BBC) and frequency-based connectivity (FBC), derived from resting-state EEG were subjected to feature selection with a proposed prior-compared minimum-redundancy maximum-relevance (PCMRMR) protocol. Their effectiveness was then tested by the supervised classification tasks using support vector machine (SVM), k-nearest neighbor (kNN), random forest (RF), and Gaussian classifier (GC). Brain networks of four somatosensory types were revealed by decoding fused multimodal data, namely type-averaged connectivity. The data from sixty healthy individuals were divided into training (n =59) and validation (n =1) datasets according to leave-one-subject-out (LOSO) criteria. The FBC was identified, which can serve as better brain signatures than BB-PSD, FB-PSD, and BBC to classify subjects as HS, NS, TS, or MS groups. Using the SVM, kNN, RF, and GC models, the best accuracy of 87% was obtained when classifying participants into HS, NS, TS, or MS groups. Moreover, the brain networks were decoded from HS, NS, TS, and MS groups by decoding the type-averaged connectivity fused from somatosensory phenotypes and selected FBC. It indicated that quantified multi-parameter somatosensory sensitivity could be achieved with acceptable accuracy, leading to considerable possibilities for using objective pain perception evaluation in clinical practice.

Site-specific, silicon-induced structural and molecular defence responses against powdery mildew infection in roses.

Silicon (Si) application to miniature potted roses can decrease severity of powdery mildew (Podosphaera pannosa) and this is associated with increased accumulation of callose and hydrogen peroxide (H2 O2 ) as well as hypersensitive (HR) cells. We used microscopy, gene expression and specific inhibitors of callose and H2 O2 to determine how effective these plant responses are in stopping infection. Pathogen arrest in Si-treated (Si+) plants was accompanied by increased accumulation of callose and H2 O2 in papillae and HR cells, respectively. These responses were reduced by application of specific inhibitors (2-deoxy-d-glucose for callose and catalase for H2 O2 ), which increased disease severity in Si+, but not in Si- plants. As markers for HR and callose, expression of the HR-specific gene hsr203J and the wound-related callose synthase GSL5, respectively, was studied. An up-regulation of expression was only seen after isolation of HR cells with laser capture microdissection. The up-regulation was higher in Si+ than in Si- plants and occurred concomitantly with more efficient photosynthesis in Si+ plants at high disease severity as compared to Si- plants. Silicon-mediated activation of callose and H2 O2 are decisive factors in the defence of rose against P. pannosa and these responses were accompanied with more efficient photosynthesis to strengthen the plant. Only by isolation of HR cells using laser capture microdissection as compared to analysis of whole leaf tissues allowed detection of elevated transcript levels of hsr203J and GSL5 at infection sites as markers for HR. © 2021 Society of Chemical Industry.

Strain-Specific Hypersensitive and Extreme Resistance Phenotypes Elicited by Potato virus Y Among 39 Potato Cultivars Released in Three World Regions Over a 117-Year Period.

Strain-specific hypersensitive (HR) and extreme resistance (ER) phenotypes elicited in potato plants by three Potato virus Y (PVY) isolates in strain groups PVYO (BL and DEL3) and PVYD (KIP1) were studied. PVYO and PVYD isolates elicit HR genes Ny or putative Nd, respectively, and all three isolates elicit ER gene Ry. They were inoculated to 39 Australasian, European, or North American potato cultivars released over a 117-year period and harvested tubers were replanted. Both primary and secondary symptoms were recorded. Two European cultivars always developed ER following sap and graft inoculation and, thus, carried comprehensive PVY resistance gene Ry. One Australasian and two European cultivars always developed susceptible phenotypes and, thus, lacked genes Ry, Ny, and putative Nd. Sap inoculation with isolate KIP1 elicited localized HR (LHR) in 31 cultivars and both LHR and systemic HR (SHR) in three others; thus, all carried putative Nd. Isolates BL and DEL3 both elicited susceptible phenotypes in 11 of these 34 cultivars but LHR alone, SHR alone, or both LHR and SHR in the other 23 which, therefore, all carry Ny. With these two isolates, SHR expression ranged from very severe to very weak, with the greatest numbers of isolate-cultivar combinations occurring in the severe category with BL (n = 11) and moderate category (n = 12) with DEL3. Within the same isolate-cultivar combination, overall, SHR symptom expression was weaker with secondary than primary infection. With both primary and secondary infection, SHR expression was most severe with KIP1 and weakest with DEL3. Genes Ny and putative Nd were present in cultivars released between 1939 and 2010 or 1893 and 2010, respectively, occurring in cultivars from all three world regions. These findings have important implications concerning breeding new PVY-resistant potato cultivars, especially for countries lacking healthy seed potato stocks, or where subsistence farmers cannot afford them. An alternative to including gene Ry is incorporating as many strain-specific PVY resistance genes as possible.

The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b–mediated resistance dissociated of their cell-to-cell movement function

The cell-to-cell movement protein (NSM) of tomato spotted wilt virus (TSWV) has been recently identified as the effector of the single dominant Sw-5b resistance gene from tomato (Solanum lycopersicum L.). Although most TSWV isolates shows a resistance-inducing (RI) phenotype, regular reports have appeared on the emergence of resistance-breaking (RB) isolates in tomato fields, and suggested a strong association with two point mutations (C118Y and T120N) in the NSM protein. In this study the Sw-5b gene has been demonstrated to confer not only resistance against TSWV but to members of five additional, phylogenetically-related classified within the so-called “American” evolutionary clade, i.e., Alstroemeria necrotic streak virus (ANSV), chrysanthemum stem necrosis virus (CSNV), groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and tomato chlorotic spot virus (TCSV). Remarkably, bean necrotic mosaic virus (BeNMV), a recently discovered tospovirus classified in a distinct American subclade and circulating on the American continent, did not trigger a Sw-5b-mediated hypersensitive (HR) response. Introduction of point mutations C118Y and T120N into the NSM protein of TSWV, TCSV and CSNV abrogated the ability to trigger Sw-5b-mediated HR in both transgenic-N. benthamiana and tomato isolines harboring the Sw-5b gene whereas it had no effect on BeNMV NSM. Truncated versions of TSWV NSM lacking motifs associated with tubule formation, cell-to-cell or systemic viral movement were made and tested for triggering of resistance. HR was still observed with truncated NSM proteins lacking 50 amino acids (out of 301) from either the amino- or carboxy-terminal end. These data altogether indicate the importance of amino acid residues C118 and T120 in Sw-5b-mediated HR only for the NSM proteins from one cluster of tospoviruses within the American clade, and that the ability to support viral cell-to-cell movement is not required for effector functionality.

In Vitro Diagnosis of Hypersensitivity to Nonsteroidal Anti-Inflammatory Drugs (NSAID) Comparison of Two Methods

Objective: Current concept distinguishes between cross intolerance (non-immune) and single or multiple hypersensitivity based (immune) adverse reactions of non-steroidal anti-inflammatory drugs (NSAID) due to their potential to inhibit cyclooxygenase (COX) isoenzymes (COX-1, COX-2). Recently we described a rapid IL-6 release assay using blood mononuclear cells of patients with various clinical forms of drug hypersensitivity. Here we present data of a comprehensive analysis if the IL-6 release test and the classical IgE immuno-assay for their sensitivity in cases with adverse reactions to NSAIDs grouped according to the new clinical classification. Methods: Total and specific serum IgE against 9 different HSA coupled-NSAIDs were determined by manual ELISA tests (55 cases) and compared to drug-specific release from preformed IL-6 pool of PBMCs of patients sensitized to the same NSAIDs after short (20’) incubation of 4 standardized concentrations (51 cases and 9 controls) and IL-6 measurement from their cell free supernatants including positive and negative intraassay controls. Results: The ratio of cross intolerant to specific hypersensitive (HS) cases was higher in the IgE group (and total IgE too,) than in the IL-6 release tested ones. There was no difference, however, in the overall ratio of early and accelerated plus late onset adverse events based on individual histories. Nine NSAIDs were tested in both groups which represented all major COX-1 inhibitors. The positivity of validated test results was double within the IL-6 tested group (65.4% vs. 36.9%). In some cases non-drug components of NSAID formulations were responsible for the observed (mainly) anaphylactic reactions. Positive results in both groups were scattered amongst cross intolerant and single to multiple hypersensitive (HS) subgroups. To our knowledge no comprehensive analysis had been performed before either on clinical phenotypes dependent IgE immunoassays or on NSAID-induced “early” Tcell activation after those specified adverse events. Conclusion: Specific HS and multiple non cross-reactive NSAID sensitizations exceeded non- immune reactions in both in vitro tested groups. Some intolerant patients revealed detectable ASA antibodies of IgE type. Preformed IL-6 release by PBMC was more sensitive than specific IgE immunoassays as an in vitro diagnostic tool. The results indicate that checking of non-drug components should be considered in allergy workups. ASA in vivo provocations need further standardization.

Nine things to know about elicitins.

888 I. 888 II. 889 III. 889 IV. 889 V. 891 VI. 891 VII. 891 VIII. 892 IX. 892 X. 893 XI. 893 893 References 893 SUMMARY: Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium oomycete pathogen species. They were first described in the late 1980s as abundant proteins in Phytophthora culture filtrates that have the capacity to elicit hypersensitive (HR) cell death and disease resistance in tobacco. Later, they became well-established as having features of microbe-associated molecular patterns (MAMPs) and to elicit defences in a variety of plant species. Research on elicitins culminated in the recent cloning of the elicitin response (ELR) cell surface receptor-like protein, from the wild potato Solanum microdontum, which mediates response to a broad range of elicitins. In this review, we provide an overview on elicitins and the plant responses they elicit. We summarize the state of the art by describing what we consider to be the nine most important features of elicitin biology.

Cryptococcus adeliensis inciting branch canker on stone fruit trees

Stem and branch cankers of stone fruits are among the most destructive diseases throughout stone fruit growing areas of the world, including Iran. Symptoms similar to those produced by Pseudomonas syringae pv. syringae were observed on stone fruit trees in several fruit growing areas in the Khorasan province in North Eastern Iran in 2011 and 2012. The isolates (48) recovered from almost all affected tissues had cream to pinkish, circular, glistening, pasty and convex colonies with entire margins on sucrose nutrient agar (SNA). A number of isolates induced a hypersensitive (HR)-like reaction in tobacco (Nicotiana tabacum) 11 days after infiltration of leaves with a turbid cell suspensions thereof. Pathogenicity of representative isolates (seven of 48) was confirmed by inoculation of peach (Prunus persica) twigs with a lightly turbid cell suspension of each. Blast analysis of the ITS-sequences of representative isolates revealed their 100 % homology with those of Cryptococcus adeliensis isolates present in GenBank. Analysis of fatty acid methyl esters, partial sequences of 26SrRNA and RPB2 as well as their phenotypic characteristics verified the identity of the yeast isolates as C. adeliensis. The stem canker caused by C. adeliensis appears to be a newly emerged disease of stone fruit trees.

NADPH oxidase RBOHD contributes to autophagy and hypersensitive cell death during the plant defense response in Arabidopsis thaliana

Autophagy has been implicated as a cellular protein degradation process that is used to recycle cytoplasmic components under biotic and abiotic stresses and so restrict programmed cell death (PCD). In this study, we report a novel regulatory mechanism by which NADPH oxidase respiratory burst oxidase homolog D (RBOHD) regulated pathogen-induced autophagy and hypersensitive (HR) cell death. We found that the Pseudomonas syringae pv tomato bacteria DC3000 expressing avrRps4 (Pst-avrRps4) induction of RBOHD-dependent reactive oxygen species (ROS) production promoted the onset of autophagy, whereas a pretreatment with an NADPH oxidase RBOHD inhibitor reversed this trend. The inhibitor significantly blocked pathogen-induced autophagosome formation and ROS increase. Moreover, we also show that in the wild-type and atrbohF mutant, Pst-avrRps4-induced cell death was limited, whereas in the case of the atrbohD mutant, the infection triggered a spreading-type necrosis. Our results demonstrate that the RBOHD-dependent ROS accumulation stimulated autophagosome formation and limited HR cell death.

Expression of an Engineered Heterologous Antimicrobial Peptide in Potato Alters Plant Development and Mitigates Normal Abiotic and Biotic Responses

Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

Effect of Silicon Absorption on Soybean Resistance to Phakopsora pachyrhizi in Different Cultivars.

Silicon (Si) is recognized for its prophylactic role in alleviating diseases when absorbed by plants and has been proposed as a possible solution against soybean rust, caused by Phakopsora pachyrhizi. However, little is known about its potential effects on soybean (Glycine max) because the plant's ability to absorb Si is poorly defined. In this work, our objectives were to evaluate and quantify the absorption of Si in leaves of different soybean cultivars and to determine if such absorption was able to enhance resistance to soybean rust. In a first set of experiments with cv. Williams 82, hydroponic plants were supplied or not with Si and inoculated with urediniospores of P. pachyrhizi. Chemical analyses revealed no significant differences in the plants' Si content regardless of the treatment, which translated into no effect on rust incidence. However, in a second set of experiments with different cultivars, plants of Korean cultivar Hikmok sorip absorbed nearly four times more Si than those of Williams 82. At the same time, plants from this cultivar exhibited a near absence of disease symptoms when supplied with Si. This resistance appeared to be the result of hypersensitive (HR) reactions that were triggered when plants were fed with Si. These results support the concept that a plant's innate ability to absorb Si will dictate the benefits conferred by a treatment with Si and provide evidence that Si can protect soybean plants against soybean rust through mediated resistance.

Identification of the Cluster Control Region for the Protocadherin-β Genes Located beyond the Protocadherin-γ Cluster

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

A TWO-STAGE EVOLUTIONARY APPROACH FOR EFFECTIVE CLASSIFICATION OF HYPERSENSITIVE DNA SEQUENCES

Hypersensitive (HS) sites in genomic sequences are reliable markers of DNA regulatory regions that control gene expression. Annotation of regulatory regions is important in understanding phenotypical differences among cells and diseases linked to pathologies in protein expression. Several computational techniques are devoted to mapping out regulatory regions in DNA by initially identifying HS sequences. Statistical learning techniques like Support Vector Machines (SVM), for instance, are employed to classify DNA sequences as HS or non-HS. This paper proposes a method to automate the basic steps in designing an SVM that improves the accuracy of such classification. The method proceeds in two stages and makes use of evolutionary algorithms. An evolutionary algorithm first designs optimal sequence motifs to associate explicit discriminating feature vectors with input DNA sequences. A second evolutionary algorithm then designs SVM kernel functions and parameters that optimally separate the HS and non-HS classes. Results show that this two-stage method significantly improves SVM classification accuracy. The method promises to be generally useful in automating the analysis of biological sequences, and we post its source code on our website.

Induction and Maintenance of IL-4 Expression Are Regulated Differently by the 3′ Enhancer in CD4 T Cells

IL-4 expression is known to be activated in CD4 T cells when they are differentiated to Th2 but not Th1 cells. However, CD4 T cells selected by MH class II-expressing thymocytes, named thymocyte-selected CD4 T cells (T-CD4 T cells), express IL-4 under both Th1 and Th2 conditions. In this study, we investigated molecular mechanisms by which IL-4 gene expression is regulated in T-CD4 T cells. We found that T-CD4 T cells express IL-4 soon after selection in the thymus. Deficiency of DNase I hypersensitive (HS) sites HS5a and HS5 at the 3'-enhancer region in the IL-4 gene decreased IL-4 production, but T-CD4 T cells were able to make IL-4 under the Th1-inducing condition. Consistent with this, IL-4 was expressed in Th1 differentiated T-CD4 T cells in the absence of recombination signal binding protein-J that interacts with HS5. When HS5 was examined separately from other endogenous regulatory elements using a reporter system, CD4 T cells that are selected by thymic epithelial cells cannot transcribe the IL-4 reporter gene with HS5 alone. However, HS5 was able to induce the expression of the IL-4 reporter gene in T-CD4 T cells. Interestingly, the Th1 differentiating signal led to deacetylation at HS5 of the IL-4 endogenous gene, whereas the Th2-inducing environment had no effect. Therefore, in T-CD4 T cells, HS5 plays an essential role during the induction phase of IL-4 expression, but the maintenance of IL-4 expression in Th1 cells requires additional regulatory elements.

Genomic Profiling of HMGN1 Reveals an Association with Chromatin at Regulatory Regions

The interaction of architectural proteins such as the linker histone H1 and high-mobility-group (HMG) proteins with nucleosomes leads to changes in chromatin structure and histone modifications and alters the cellular transcription profile. The interaction of HMG proteins with chromatin is dynamic. However, it is not clear whether the proteins are constantly and randomly redistributed among all the nucleosomes or whether they preferentially associate with, and turn over at, specific regions in chromatin. To address this question, we examined the genome-wide distribution of the nucleosome binding protein HMGN1 and compared it to that of regulatory chromatin marks. We find that HMGN1 is not randomly distributed throughout the genome. Instead, the protein preferentially localizes to DNase I hypersensitive (HS) sites, promoters, functional enhancers, and transcription factor binding sites. Our results suggest that HMGN1 is part of the cellular machinery that modulates transcriptional fidelity by generating, maintaining, or preferentially interacting with specific sites in chromatin.

Sp1 and Sp3 regulate transcription of the chicken GAS41 gene

The 5′-flanking region of the chicken glioma-amplified sequence (GAS) 41 gene is close to the 3′ end of the lysozyme gene and contains no typical TATA box, but several GC boxes. In this study, we have localized the GAS 41 promoter to this narrow region. Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses revealed that Sp1 and Sp3 bind to this promoter. Mapping by a technique of indirect end labeling demonstrated that the Sp1-binding sites contained in this region exactly co-map with two previously identified DNase I hypersensitive (HS) sites, which suggests the important role of Sp1 binding in maintaining an open chromatin structure of the GAS41 promoter. We further found that Sp1 and Sp3 strongly activate CAT expression controlled by the putative GAS41 promoter in Drosophila Schneider S2 cells and that deletion of the Sp1 sites resulted in a loss of promoter activity in chicken HD11 cells. The results indicate that transcription factors of the Sp family play an important role in the transcriptional regulation of the chicken GAS41 gene.

DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements across the Genome from Mammalian Cells

INTRODUCTIONIdentification of active gene regulatory elements is a key to understanding transcriptional control governing biological processes such as cell-type specificity, differentiation, development, proliferation, and response to the environment. Mapping DNase I hypersensitive (HS) sites has historically been a valuable tool for identifying all different types of regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. This method utilizes DNase I to selectively digest nucleosome-depleted DNA (presumably by transcription factors), whereas DNA regions tightly wrapped in nucleosome and higher-order structures are more resistant. The traditional low-throughput method for identifying DNase I HS sites uses Southern blots. Here, we describe the complete and improved protocol for DNase-seq, a high-throughput method that identifies DNase I HS sites across the whole genome by capturing DNase-digested fragments and sequencing them by high-throughput, next-generation sequencing. In a single experiment, DNase-seq can identify most active regulatory regions from potentially any cell type, from any species with a sequenced genome.

NF90 and NF45 Regulate Interleukin‐13 (IL13) Gene Transcription in Human T Cells

The Th2 cytokine IL13 is a critical effector of allergic inflammation. We previously showed human IL13 gene expression is paralleled by chromatin remodeling, leading to the formation of multiple DNase I hypersensitive (HS) sites throughout the locus. The distal IL13 promoter harbors two HS sites, HS4 and HS5, detected in both naïve and polarized CD4+ T helper cells. We now show HS4, which is located ~1.5 kb upstream of the IL13 ATG, acts as a cis‐regulatory element that enhances transcription driven by the IL13 promoter in transiently transfected, activated Jurkat T cells. The enhancing activity mapped to the 3’half of HS4 (HS4‐3’), a region which binds Nuclear Factor‐90 (NF90) and NF45 as demonstrated by DNA‐affinity chromatography and tandem mass spectrometry. The NF90/45 binding motif in HS4‐3’ was further mapped and dissected by gel shift analysis. Chromatin immunoprecipitation confirmed recruitment of NF90 and NF45 to the HS4‐containing region in the endogenous IL13 locus upon T cell stimulation. Moreover, stable overexpression of NF90 and NF45 increased HS4‐mediated enhancement of IL13 transcription by 3‐fold and 4‐fold, respectively. Collectively, our results identify NF90 and NF45 as important regulators of human IL13 transcription in response to T cell activation.

High-Resolution Mapping and Characterization of Open Chromatin across the Genome

Mapping DNase I hypersensitive (HS) sites is an accurate method of identifying the location of genetic regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. We employed high-throughput sequencing and whole-genome tiled array strategies to identify DNase I HS sites within human primary CD4+ T cells. Combining these two technologies, we have created a comprehensive and accurate genome-wide open chromatin map. Surprisingly, only 16%-21% of the identified 94,925 DNase I HS sites are found in promoters or first exons of known genes, but nearly half of the most open sites are in these regions. In conjunction with expression, motif, and chromatin immunoprecipitation data, we find evidence of cell-type-specific characteristics, including the ability to identify transcription start sites and locations of different chromatin marks utilized in these cells. In addition, and unexpectedly, our analyses have uncovered detailed features of nucleosome structure.

Identification and Characterization of Cell Type–Specific and Ubiquitous Chromatin Regulatory Structures in the Human Genome

The identification of regulatory elements from different cell types is necessary for understanding the mechanisms controlling cell type–specific and housekeeping gene expression. Mapping DNaseI hypersensitive (HS) sites is an accurate method for identifying the location of functional regulatory elements. We used a high throughput method called DNase-chip to identify 3,904 DNaseI HS sites from six cell types across 1% of the human genome. A significant number (22%) of DNaseI HS sites from each cell type are ubiquitously present among all cell types studied. Surprisingly, nearly all of these ubiquitous DNaseI HS sites correspond to either promoters or insulator elements: 86% of them are located near annotated transcription start sites and 10% are bound by CTCF, a protein with known enhancer-blocking insulator activity. We also identified a large number of DNaseI HS sites that are cell type specific (only present in one cell type); these regions are enriched for enhancer elements and correlate with cell type–specific gene expression as well as cell type–specific histone modifications. Finally, we found that approximately 8% of the genome overlaps a DNaseI HS site in at least one the six cell lines studied, indicating that a significant percentage of the genome is potentially functional.