Wellington : a novel method for the accurate identification of digital genomic footprints from DNase-seq data

The expression of eukaryotic genes is regulated by cis-regulatory elements such as promoters and enhancers, which bind sequence-specific DNA-binding proteins. One of the great challenges in the gene regulation field is to characterise these elements. This involves the identification of transcription factor (TF) binding sites within regulatory elements that are occupied in a defined regulatory context. Digestion with DNase and the subsequent analysis of regions protected from cleavage (DNase footprinting) has for many years been used to identify specific binding sites occupied by TFs at individual cis-elements with high resolution. This methodology has recently been adapted for high-throughput sequencing (DNase-seq). In this study, we describe an imbalance in the DNA strand-specific alignment information of DNase-seq data surrounding protein–DNA interactions that allows accurate prediction of occupied TF binding sites. Our study introduces a novel algorithm, Wellington, which considers the imbalance in this strand-specific information to efficiently identify DNA footprints. This algorithm significantly enhances specificity by reducing the proportion of false positives and requires significantly fewer predictions than previously reported methods to recapitulate an equal amount of ChIP-seq data. We also provide an open-source software package, pyDNase, which implements the Wellington algorithm to interface with DNase-seq data and expedite analyses

The demarcation of transcription factor binding sites through the analysis of DNase-seq data

The expression of eukaryotic genes is controlled by non-coding regulatory elements such as promoters and enhancers, which bind sequence-specific DNA-binding proteins (transcription factors). In multicellular organisms, the characterisation of these elements is required in order to understand how a single genome is utilised to generate a multitude of cell types, and how aberrant regulation of transcription contributes to disease processes. This involves the identification of transcription factor binding sites within regulatory elements that are occupied in a defined regulatory context. Digestion with DNase I and the subsequent analysis of regions protected from digestion followed by high-throughput sequencing (DNase-seq footprinting), allows for the quantification of genome-wide transcription factor binding. However, the handful of methods for analysing DNase-seq data has not been extensively validated or benchmarked. This thesis describes a novel footprinting algorithm, Wellington, which is presented in the context of a comprehensive comparison of several other DNase-seq footprinting algorithms on a multitude of datasets. Wellington outperforms other methods in almost all situations. An open-source software package, pyDNase, that facilitates interacting with DNase-seq data and provides many tools for DNase-seq analysis is also presented. Wellington is used to perform footprinting on clinical samples to validate cell lines as a model system, and to identify the binding partners of the RUNX1/ETO fusion protein in t(8;21) AML. By expanding the Wellington method, differential footprinting is shown to be able to link differences in transcription factor binding at promoters to changes in gene expression. Applying this methodology to a range of haematopoietic cell types illustrates the ability for differential footprinting to identify key regulators in the haematopoietic lineage. These results represent advances in the methods available to analyse DNase-seq data (all of which have been released as free, opensource software) and demonstrate the power of integrating DNase-seq footprinting with other functional genomic assays to study transcriptional regulation

Plunging Floater Survival Causes Cryptic Population Decline in the Common Loon

Populations of many vertebrates are declining and geographic ranges contracting, largely as a consequence of anthropogenic threats. Many reports of such decline, however, lack the breadth and detail to narrow down its causes. Here we describe population decline in the Common Loon (Gavia immer), a charismatic aquatic bird, based on systematic resighting and measurement of a marked population. During our 27-year investigation, age-adjusted chick mass has fallen by 11%, mortality among young and old chicks has increased by 31% and 82%, respectively, and fledging success has declined by 26%. Meanwhile, the return rate of marked nonbreeders (“floaters”) has plunged by 53%, and the adult population overall has declined by 22%. Consistent with the thinning ranks of floaters, the rate of territory eviction has decreased by 52% during the study. Despite the decline in floaters, territory occupancy remains unchanged. However, a matrix model, updated with recent estimates for breeding success, juvenile survival, and senescence, yields a recalculated deterministic population growth rate (λ) of 0.94 for our study population, which suggests that declines in vital rates could lead to a loss of 52% of the current population and a decline of 37% in territory occupancy by 2031. Lack of data on floaters in other upper Midwest and New England loon populations leaves their status in doubt

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems

Altering the distribution of Foxp3+ regulatory T cells results in tissue-specific inflammatory disease

CD4+Foxp3+ regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4+CD103hi phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4+Foxp3+ T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease

Inducible chromatin priming is associated with the establishment of immunological memory in T cells

Immunological memory is a defining feature of vertebrate physiology, allowing rapid responses to repeat infections. However, the molecular mechanisms required for its establishment and maintenance remain poorly understood. Here, we demonstrated that the first steps in the acquisition of T-cell memory occurred during the initial activation phase of naïve T cells by an antigenic stimulus. This event initiated extensive chromatin remodeling that reprogrammed immune response genes toward a stably maintained primed state, prior to terminal differentiation. Activation induced the transcription factors NFAT and AP-1 which created thousands of new DNase I-hypersensitive sites (DHSs), enabling ETS-1 and RUNX1 recruitment to previously inaccessible sites. Significantly, these DHSs remained stable long after activation ceased, were preserved following replication, and were maintained in memory-phenotype cells. We show that primed DHSs maintain regions of active chromatin in the vicinity of inducible genes and enhancers that regulate immune responses. We suggest that this priming mechanism may contribute to immunological memory in T cells by facilitating the induction of nearby inducible regulatory elements in previously activated T cells

The transcription factor Nfix is essential for normal brain development

Background: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nficdeficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice. Results: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals. Conclusion: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects

COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal

The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β’-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β’-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway

Loss of NFIX transcription factor biases postnatal stem/progenitor cells towards oligodendrogenesis

Murine postnatal neural stem cells (NSCs) give rise to neurons, astrocytes, or oligodendrocytes (OLs); however, our knowledge of the genes that control this lineage specification is incomplete. In this study, we show that nuclear factor I X (NFIX), a transcription factor known to regulate NSC quiescence, also suppresses oligodendrogenesis (ODG) from NSCs. Immunostaining reveals little or no expression of NFIX in OL lineage cells both in vivo and in vitro. Loss of NFIX from subventricular zone (SVZ) NSCs results in enhanced ODG both in vivo and in vitro, while forced expression of NFIX blocks NSC differentiation into OLs in vitro. RNA-seq analysis shows that genes previously shown to be differentially expressed in OL progenitors are significantly enriched in RNA from Nfix(-/-) versus wild-type NSCs. These data indicate that NFIX influences the lineage specification of postnatal SVZ NSCs, specifically suppressing ODG

Morphology of late Quaternary submarine landslides along the U.S. Atlantic continental margin

This paper is not subject to U.S. copyright. The definitive version was published in Marine Geology 264 (2009): 4-15, doi:10.1016/j.margeo.2009.01.009.The nearly complete coverage of the U.S. Atlantic continental slope and rise by multibeam bathymetry and backscatter imagery provides an opportunity to reevaluate the distribution of submarine landslides along the margin and reassess the controls on their formation. Landslides can be divided into two categories based on their source areas: those sourced in submarine canyons and those sourced on the open continental slope and rise. Landslide distribution is in part controlled by the Quaternary history of the margin. They cover 33% of the continental slope and rise of the glacially influenced New England margin, 16% of the sea floor offshore of the fluvially dominated Middle Atlantic margin, and 13% of the sea floor south of Cape Hatteras. The headwall scarps of open-slope sourced landslides occur mostly on the lower slope and upper rise while they occur mostly on the upper slope in the canyon-sourced ones. The deposits from both landslide categories are generally thin (mostly 20–40 m thick) and comprised primarily of Quaternary material, but the volumes of the open-slope sourced landslide deposits can be larger (1–392 km3) than the canyon-sourced ones (1–10 km3). The largest failures are located seaward of shelf-edge deltas along the southern New England margin and near salt domes that breach the sea floor south of Cape Hatteras. The spatial distribution of landslides indicates that earthquakes associated with rebound of the glaciated part of the margin or earthquakes associated with salt domes were probably the primary triggering mechanism although other processes may have pre-conditioned sediments for failure. The largest failures and those that have the potential to generate the largest tsunamis are the open-slope sourced landslides.The U.S. Nuclear Regulatory Commission and the U.S. Geological Survey are acknowledged for their support of this research.Work was funded by US Nuclear Regulatory Commission grant N6480 Physical study of tsunami sources