DNaseI Sensitivity Research Articles

Mechanical stretch facilitates tenomodulin expression to induce tenocyte migration via MAPK signaling pathway

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that regulates tendon development and maturation. Our previous study indicated that mechanical stretch could induce Tnmd expression to promote tenocyte migration, associated with reinforcement of fibrous actin (F-actin) stress fibers and chromatin decondensation. However, the detailed molecular mechanisms of this processes are far from clear. Activation of mitogen-activated protein kinase (MAPK) signaling occurs in response to various extracellular stimuli and controls a large number of fundamental cellular processes. The present study we investigated the influence of MAPK signaling on mechanical stretch-induced Tnmd expression and its action way. Expression and activities of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK (p38) were determined by Western blot. Cell migration was detected by Transwell assay. Immunofluorescence staining was used to detect F-actin stress fibers. Nuclear chromatin decondensation was detected by in situ DNaseI sensitivity assay. It was found that mechanical stretch promoted Tnmd expression by activating ERK1/2, JNK and p38 signaling. The inhibition of the ERK1/2, JNK or p38 repressed mechanical stretch-promoted tenocyte migration and mechanical stretch-induced reinforcement of F-actin stress fibers. However, only ERK1/2 and p38 inhibitor could repress mechanical stretch-induced chromatin decondensation, and the JNK inhibitor had no significant effect. Moreover, latrunculin (Lat A), the most widely used reagent to depolymerize actin filaments, could inhibit the stretch-induced chromatin decondensation. Taken together, our findings elucidated a molecular pathway by which a mechanical signal is transduced via activation of MAPK signaling to influence reinforcement of F-actin stress fibers and chromatin decondensation, which could further lead Tnmd expression to promote tenocyte migration.

EPISPOT: An epigenome-driven approach for detecting and interpreting hotspots in molecular QTL studies

SummaryWe present EPISPOT, a fully joint framework which exploits large panels of epigenetic annotations as variant-level information to enhance molecular quantitative trait locus (QTL) mapping. Thanks to a purpose-built Bayesian inferential algorithm, EPISPOT accommodates functional information for both cis and trans actions, including QTL hotspot effects. It effectively couples simultaneous QTL analysis of thousands of genetic variants and molecular traits with hypothesis-free selection of biologically interpretable annotations which directly contribute to the QTL effects. This unified, epigenome-aided learning boosts statistical power and sheds light on the regulatory basis of the uncovered hits; EPISPOT therefore marks an essential step toward improving the challenging detection and functional interpretation of trans-acting genetic variants and hotspots. We illustrate the advantages of EPISPOT in simulations emulating real-data conditions and in a monocyte expression QTL study, which confirms known hotspots and finds other signals, as well as plausible mechanisms of action. In particular, by highlighting the role of monocyte DNase-I sensitivity sites from >150 epigenetic annotations, we clarify the mediation effects and cell-type specificity of major hotspots close to the lysozyme gene. Our approach forgoes the daunting and underpowered task of one-annotation-at-a-time enrichment analyses for prioritizing cis and trans QTL hits and is tailored to any transcriptomic, proteomic, or metabolomic QTL problem. By enabling principled epigenome-driven QTL mapping transcriptome-wide, EPISPOT helps progress toward a better functional understanding of genetic regulation.

Multiple SCN5A variant enhancers modulate its cardiac gene expression and the QT interval

The rationale for genome-wide association study (GWAS) results is sequence variation in cis-regulatory elements (CREs) modulating a target gene's expression as the major cause of trait variation. To understand the complete molecular landscape of one of these GWAS loci, we performed in vitro reporter screens in cardiomyocyte cell lines for CREs overlapping nearly all common variants associated with any of five independent QT interval (QTi)-associated GWAS hits at the SCN5A-SCN10A locus. We identified 13 causal CRE variants using allelic reporter activity, cardiomyocyte nuclear extract-based binding assays, overlap with human cardiac tissue DNaseI hypersensitive regions, and predicted impact of sequence variants on DNaseI sensitivity. Our analyses identified at least one high-confidence causal CRE variant for each of the five sentinel hits that could collectively predict SCN5A cardiac gene expression and QTi association. Although all 13 variants could explain SCN5A gene expression, the highest statistical significance was obtained with seven variants (inclusive of the five above). Thus, multiple, causal, mutually associated CRE variants can underlie GWAS signals.

Commentary: The beginning of the end of the 'modern synthesis'? Commentary on Jablonka E, Lamb MJ. The inheritance of acquired epigenetic variations.

The 1989 paper entitled ‘The inheritance of acquired epigenetic variations’ was one of a couple that Eva Jablonka and Marion Lamb published in the Journal of Theoretical Biology in the late 1980s to initiate their now famous collaboration which led to their books Epigenetic Inheritance and Evolution: the Lamarckian Dimension in 1995 and the ‘catch-all’ exploration, Evolution in Four Dimensions, in 2005. Marion Lamb brought to that 1989 collaboration 15 years of experimental Drosophila genetic work on ageing in relation to radiation and temperature. Eva Jablonka had done rodent studies using the DNA-demethylating agent, 5-azacytidine, to increase DNase-I sensitivity (and therefore potential gene expression) on the inactive chromosome X, and to change the timing of X replication during cell division. The 1980s was a remarkable decade for medical and human molecular genetics and I was glad to be in the thick of it in London. That decade saw the robustness of Mendelian genetics and classic genetic linkage approaches confirmed, as well as some big surprises that by 1992 would challenge a commonly accepted view of the genome as remarkably stable with the DNA sequence and its variants honed over aeons of evolutionary time. Genomic imprinting, i.e. parent of origin-specific gene expression, was demonstrated in humans, as was the molecular basis of the fragile X syndrome, the first of a class of ‘dynamic’ mutations. There had been a long history of experimental research on (induced) phenotype transmission before 1989—principally in an attempt to demonstrate Lamarckian inheritance of acquired characteristics in inbred animals—but without much success. Experiments had shown transgenerational effects of drug and hormone treatments in mammals but, as a review in 1988 demonstrates, the authors were struggling to propose a plausible mechanism. However, the advances in DNA analysis and understanding of epigenetic regulation began to reveal potential mediating mechanisms for transgenerational effects. Modification of DNA bases (without changing the DNA sequence) by methylation of cytosines was shown in 1975 and it soon became clear that methylation patterns influenced some gene expression and were stably inherited in cell lines. But it was in the 1980s that the study of DNA methylation and its role in regulating gene expression during embryonic development and beyond really took off. DNA methylation started to be incorporated into existing models of chromatin modification. Jablonka and Lamb seized the moment, and their 1989 paper was probably the first to discuss the role of the inheritance of acquired epigenetic variations in evolution itself. They conclude: ‘Therefore, inherited changes in the structure of chromatin can influence neo-Darwinian evolution as well as cause a type of ‘Lamarckian’ inheritance,’ a very big claim in the

The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia

Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor – decitabine (DAC) – in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.

The accessible chromatin landscape of the human genome.

DNaseI 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 choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. 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.

Ultrastructural Probes of Clusters of Open Regulatory Elements (Core) Within Chromatin

Introduction: Mammalian cells during interphase display clustering of DNase I-sensitive sites within the 10 nm micro-fibrils of active euchromatin (Song L, et al, Genome Research, August 19, 2011). Over 870,000 distinct CORE sites were identified as poised or active in gene transcription across an analysis of seven human cell lines, (ibid). Methods: We have developed a high-resolution electron microscopic technique for detecting DNase I-sensitive sites within intact single human cells. All such sites are confined to the euchromatin portion of the cell nucleus, and can be analyzed for location, number, shape, and size within each single cell analyzed within native biopsied tissue. Such ultrastructural probes can detect sites greater than 10 nm in diameter, and offer a global 3D view in 10 nm thick serial sections of the probed tissue after instant fixation during biopsy. Results: DNase I-sensitive probes were visualized within tissue biopsies of normal human bone marrow or Hodgkin lymphoma lymph nodes before therapy. Single cell analysis revealed all probe sites are confined to the euchromatin portion of the cell nucleus, and are visualized as ellipsoid clusters ranging in size from 25 - 100 nm (small), and 100 - 350 nm (large), to over 350 nm in overall diameter (giant). Within probed clusters, fine granulation is apparent of approximately 10 nm size, and the clusters are found near nuclear pores. Normal bone marrow cells display a reduction in size and number of probed clusters during cell differentiation and maturation, while Hodgkin lymphoma apposed lymphocytes display an increase in size and number of probed giant clusters during in-vivo immune activation. Conclusions: Cluster of open regulatory elements (CORE) ultrastructural probes based on the DNase I-sensitivity of active transcription sites, can be useful in single-cell analysis within biopsied intact tissues.

Dynamic reprogramming of chromatin accessibility during Drosophilaembryo development

BackgroundThe development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology.ResultsWe used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm.ConclusionsOur results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features.

Efficient cell migration requires global chromatin condensation

Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes.

Efficient cell migration requires global chromatin condensation

Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes.

Sequencing newly replicated DNA reveals widespread plasticity in human replication timing

Faithful transmission of genetic material to daughter cells involves a characteristic temporal order of DNA replication, which may play a significant role in the inheritance of epigenetic states. We developed a genome-scale approach--Repli Seq--to map temporally ordered replicating DNA using massively parallel sequencing and applied it to study regional variation in human DNA replication time across multiple human cell types. The method requires as few as 8,000 cytometry-fractionated cells for a single analysis, and provides high-resolution DNA replication patterns with respect to both cell-cycle time and genomic position. We find that different cell types exhibit characteristic replication signatures that reveal striking plasticity in regional replication time patterns covering at least 50% of the human genome. We also identified autosomal regions with marked biphasic replication timing that include known regions of monoallelic expression as well as many previously uncharacterized domains. Comparison with high-resolution genome-wide profiles of DNaseI sensitivity revealed that DNA replication typically initiates within foci of accessible chromatin comprising clustered DNaseI hypersensitive sites, and that replication time is better correlated with chromatin accessibility than with gene expression. The data collectively provide a unique, genome-wide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.

Context-Specific KLF-1 Mediated Transcriptional Activation at the Alpha Hemoglobin-Stabilizing Protein and Dematin Promoters in Erythroid Cells.

Abstract 461Krüppel-like factor 1 (KLF1) is essential for erythroid gene expression. Key molecular mechanisms modulated by this transacting factor have been elucidated at the b-globin locus. KLF1 has been associated with recruitment of SWI/SNF and RNA polymerase (PolII) complexes necessary for chromatin remodeling and gene transcription respectively, and for facilitating the apposition of the promoter with the far-upstream locus control region. More recently, KLF1 has been implicated in the regulation of an erythroid-specific gene program unlinked to the b-globin locus. Coordinated expression of these genes, including Alpha Hemoglobin-Stabilizing Protein (AHSP), a factor required for globin tetramer stability, and the red cell membrane protein Dematin, are critical for erythroid ontogeny. To compare the role(s) of KLF1 at these loci, we have used a unique 4-OH-Tamoxifen (4-OHT) inducible erythroid cell line, which facilitates the characterization of the temporal kinetics of KLF1-dependent erythroid gene activation. In preliminary experiments, we observed that KLF1 binding was maximal at the three loci within 60 minutes of 4-OHT induction. AHSP and dematin primary RNA transcripts followed similar kinetics, being maximal at 60-90 minutes post-induction. In contrast, b-globin gene transcription reached a plateau 4-6 hours post-induction. From these observations, we hypothesized that transcriptional activation at AHSP and dematin differs from that observed at the b-globin cluster. Consistent with this hypothesis, we observed significant differences in chromatin remodeling at the three loci. At the b-globin promoter, we observed a small but statistically significant increase in DNaseI sensitivity, a measure of chromatin remodeling, with KLF1 binding. In contrast, we observed a complete loss of DNaseI resistance after KLF1 binding at the AHSP and dematin promoters. Consistent with these findings, we observed a five-fold reduction in histone H3 occupancy at the AHSP and dematin promoters, contrasting with no significant change in occupancy at the b-promoter. Importantly, these differences were not observed in regions 1-5 kb upstream of the promoters. These observations, coupled with similar differences in DNaseI hypersensitivity and histone occupancy in fetal liver erythroblasts from wild type and KLF1-null mice, suggest a profound difference in the mechanisms of chromatin remodeling at KLF1-dependent erythroid gene loci. To explore the potential mechanisms underlying these differences in chromatin accessibility, we examined the kinetics of recruitment of other transacting factors and co-activators to the three loci. We observed similar increases in binding of serine-5 phosphorylated PolII, GATA-1, and p45NF-E2 at the promoters. In contrast, binding of BRG1, the core ATPase component of the SWI/SNF complex differed between the b–promoter and the other erythroid genes. Although BRG1 binding was co-incident with KLF1 binding to the b-gene, we observed significant albeit weak binding of this complex to the AHSP and Dematin promoters only after maximal gene transcription had occurred. Our results suggest that different KLF1 multiprotein complexes are recruited to remodel target gene promoters in vivo. Furthermore, we propose that KLF1's chromatin remodeling capabilities are not limited to the recruitment of the SWI/SNF complexes Disclosures:No relevant conflicts of interest to declare.

Chromatin and DNA Modifications in theOpaque2-Mediated Regulation of Gene Transcription during Maize Endosperm Development

The maize (Zea mays) Opaque2 (O2) gene encodes an endosperm-specific bZIP-type transcription activator. In this study, we analyzed O2 targets for chromatin and DNA modifications and transcription factors binding during endosperm development and in leaves. In leaves, O2 targets exhibit high cytosine methylation levels and transcriptionally silent chromatin, enriched with histones H3 dimethylated at Lys-9 (H3K9me2) and Lys-27 (H3K27me2). Transcriptional activation in the endosperm occurs through a two-step process, with an early potentiated state and a later activated state. The potentiated state has cytosine demethylation at symmetric sites, substitution of H3K9me2 and H3K27me2 with histones H3 acetylated at Lys-14 (H3K14ac) and dimethylated at Lys-4 (H3K4me2), and increased DNaseI sensitivity. During the activated state, the mRNA of O2 targets accumulates in correspondence to RNPII, O2, and Ada2/Gcn5 coactivator binding. The active state also exhibits further increases of H3K14ac/H3K4me2 and DNaseI accessibility levels and deposition of histone H3 acetylated at Lys-9 and trimethylated at Lys-4. Analysis of o2 mutants revealed that O2 targets differ in their dependence on O2 activity for coactivator recruitment and for formation of specific chromatin modification profiles. These results indicate gene-specific involvement of mechanisms that modify chromatin states in the O2-mediated regulation of transcription.

Automated mapping of large-scale chromatin structure in ENCODE

Motivation: A recently developed DNaseI assay has given us our first genome-wide view of chromatin structure. In addition to cataloging DNaseI hypersensitive sites, these data allows us to more completely characterize overall features of chromatin accessibility. We employed a Bayesian hierarchical change-point model (CPM), a generalization of a hidden Markov Model (HMM), to characterize tiled microarray DNaseI sensitivity data available from the ENCODE project.Results: Our analysis shows that the accessibility of chromatin to cleavage by DNaseI is well described by a four state model of local segments with each state described by a continuous mixture of Gaussian variables. The CPM produces a better fit to the observed data than the HMM. The large posterior probability for the four-state CPM suggests that the data falls naturally into four classes of regions, which we call major and minor DNaseI hypersensitive sites (DHSs), regions of intermediate sensitivity, and insensitive regions. These classes agree well with a model of chromatin in which local disruptions (DHSs) are concentrated within larger domains of intermediate sensitivity, the accessibility islands. The CPM assigns 92% of the bases within the ENCODE regions to the insensitive regions. The 5.8% of the bases that are in regions of intermediate sensitivity are clearly enriched in functional elements, including genes and activating histone modifications, while the remaining 2.2% of the bases in hypersensitive regions are very strongly enriched in these elements.Availability: The CPM software is available upon request from the authors.Contact: jstam@stamlab.org; noble@gs.washington.edu; Charles_Lawrence@brown.eduSupplementary information: Supplementary data are available at Bioinformatics online. Source code is available at http://noble.gs.washington.edu/proj/segment.

Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers

DNA methylation and chromatin DNaseI sensitivity were analyzed in and adjacent to D4Z4 repeat arrays, which consist of 1 to ∼100 tandem 3.3-kb units at subtelomeric 4q and 10q. D4Z4 displayed hypomethylation in some cancers and hypermethylation in others relative to normal tissues. Surprisingly, in cancers with extensive D4Z4 methylation there was a barrier to hypermethylation spreading to the beginning of this disease-associated array (facioscapulohumeral muscular dystrophy, FSHD) despite sequence conservation in repeat units throughout the array. We infer a different chromatin structure at the proximal end of the array than at interior repeats, consistent with results from chromatin DNaseI sensitivity assays indicating a boundary element near the beginning of the array. The relative chromatin DNaseI sensitivity in FSHD and control myoblasts and lymphoblasts was as follows: a non-genic D4Z4-adjacent sequence (p13E-11, array-proximal)> untranscribed gene standards > D4Z4 arrays> constitutive heterochromatin (satellite 2; P < 10−4 for all comparisons). Cancers displaying D4Z4 hypermethylation also exhibited a hypermethylation-resistant subregion within the 3.3-kb D4Z4 repeat units. This subregion contains runs of G that form G-quadruplexes in vitro. Unusual DNA structures might contribute to topological constraints that link short 4q D4Z4 arrays to FSHD and make long ones phenotypically neutral.

Imprinting of mammalian male gametes is gene specific and does not occur at a single stage of differentiation

Epigenetic modifications such as DNA methylation and alterations to chromatin structure have been proposed as hallmarks of imprinting in somatic cells after fertilization. In the germ cell line, gene imprinting needs to be reset in order to transmit the correct sex-specific imprinting pattern to the next generation. The precise timing of imprint erasure and re-establishment for many genes remains to be determined and precise molecular mechanisms of genomic imprinting have not yet been fully characterized. Here, we have analysed the methylation state and DNase-I sensitivity of two genes with reciprocal genomic imprinting (U2af1-rs1 and H19 genes) in a male mouse primordial germ cell (PGC) derived cell line (EG-1), isolated post-natal spermatogonia and mature sperm cells. Our results show that establishment of imprinting of the U2af1-rs1 and H19 genes during male germ cell differentiation occurs at different stages of differentiation. Furthermore, the presence of DNase-I hypersensitive sites may constitute a molecular marker to identify alleles and subsequently acquire the appropriate methylation imprint. We propose that this molecular identifier may be present or absent for a specific gene according to the sex of the gamete.

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self-assembles into virus-like particles (VLPs) of approximately 40 nm in diameter when over-expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro, here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40-nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40-nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40-nm particles. In addition, sucrose gradient sedimentation analysis and DNase I-sensitivity assays indicated that DNA of up to 2,000 bp is packaged into the 40-nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.

Noncoding transcription controls downstream promoters to regulate T-cell receptor α recombination

The T early alpha (TEA) promoter in the murine Tcra locus generates noncoding transcripts that extend across the 65 kb Jalpha array. Here, we have analyzed the significance of TEA transcription for Tcra locus regulation through the targeted introduction of a transcription terminator downstream of the TEA promoter. We demonstrate that noncoding transcription driven by this single promoter can instruct both positively and negatively the activity of downstream Jalpha promoters, and can similarly instruct alterations in Jalpha chromatin structure and Jalpha recombination. TEA transcription activates promoters associated with relatively proximal Jalpha segments and stimulates histone acetylation, histone methylation and chromatin accessibility in this region. In contrast, at more distal locations, TEA transcription inhibits promoter activity through transcriptional interference and suppresses chromatin accessibility. In combination, these effects target initial Valpha-to-Jalpha recombination to TEA-proximal Jalpha segments and promote the ordered usage of the Jalpha array. The ability of TEA transcription to coordinate the activity of multiple downstream promoters maximizes the biological potential of the Jalpha array and diversifies the Tcra repertoire.

Tracking chromatin states using controlled DNase I treatment and real-time PCR

A novel approach to DNase I-sensitivity analysis was applied to examining genes of the spermatogenic pathway, reflective of the substantial morphological and genomic changes that occur during this program of differentiation. A new real-time PCR-based strategy that considers the nuances of response to nuclease treatment was used to assess the nuclease susceptibility through differentiation. Data analysis was automated with the K-Lab PCR algorithm, facilitating the rapid analysis of multiple samples while eliminating the subjectivity usually associated with Ct analyses. The utility of this assay and analytical paradigm as applied to nuclease-sensitivity mapping is presented.

In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

For a bacterium, Streptomyces coelicolor A3(2) contains a relatively large genome (8.7 Mb) with a complex and adaptive pattern of gene regulation. We discovered a correlation between the physical structure of the S.coelicolor genome and the transcriptional activity of the genes therein. Twelve genes were surveyed throughout 72 h of growth for both in vivo sensitivity to DNase I digestion and levels of transcription. DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA. The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome. This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.