The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

During gene regulation, DNA accessibility is thought to limit the availability of transcription factor (TF) binding sites, while TFs can increase DNA accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events in the modulation of gene expression remain unknown for the vast majority of genes. We utilized deeply sequenced ATAC-Seq data and site-specific knock-in reporter genes to investigate the relationship between the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of Cebpa during macrophage-neutrophil differentiation. While the enhancers upregulate reporter expression during the earliest stages of differentiation, there is little corresponding increase in their total accessibility. Conversely, total accessibility peaks during the last stages of differentiation without any increase in enhancer activity. The accessibility of positions neighboring C/EBP-family TF binding sites, which indicates TF occupancy, does increase significantly during early differentiation, showing that the early upregulation of enhancer activity is driven by TF binding. These results imply that a generalized increase in DNA accessibility is not sufficient, and binding by enhancer-specific TFs is necessary, for the upregulation of gene expression. Additionally, high-coverage ATAC-Seq combined with time-series expression data can infer the sequence of regulatory events at binding-site resolution.

LucFlow: A method to measure Luciferase reporter expression in single cells.

Reporter assays, in which the expression of an inert protein is driven by gene regulatory elements such as promoters and enhancers, are a workhorse for investigating gene regulation. Techniques for measuring reporter gene expression vary from single-cell or single-molecule approaches having low throughput to bulk Luciferase assays that have high throughput. We developed a Luciferase Reporter Assay using Flow-Cytometry (LucFlow), which measures reporter expression in single cells immunostained for Luciferase. We optimized and tested LucFlow with a murine cell line that can be differentiated into neutrophils, into which promoter-reporter and enhancer-promoter-reporter constructs have been integrated in a site-specific manner. The single-cell measurements are comparable to bulk ones but we found that dead cells have no detectable Luciferase protein, so that bulk assays underestimate reporter expression. LucFlow is able to achieve a higher accuracy than bulk methods by excluding dead cells during flow cytometry. Prior to fixation and staining, the samples are spiked with stained cells that can be discriminated during flow cytometry and control for tube-to-tube variation in experimental conditions. Computing fold change relative to control cells allows LucFlow to achieve a high level of precision. LucFlow, therefore, enables the accurate and precise measurement of reporter expression in a high throughput manner.

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

The upregulation of gene expression by enhancers depends upon the interplay between the binding of sequence-specific transcription factors (TFs) and DNA accessibility. DNA accessibility is thought to limit the ability of TFs to bind to their sites, while TFs can increase accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events underlying the modulation of gene expression during cellular differentiation remain unknown for the vast majority of genes. We investigated the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of an important neutrophil gene, Cebpa, during macrophage-neutrophil differentiation. Reporter genes were integrated in a site-specific manner in PUER cells, which are progenitors that can be differentiated into neutrophils or macrophages in vitro by activating the pan-leukocyte TF PU.1. Time series data show that two enhancers upregulate reporter expression during the first 48 hours of neutrophil differentiation. Surprisingly, there is little or no increase in the total accessibility, measured by ATAC-Seq, of the enhancers during the same time period. Conversely, total accessibility peaks 96 hrs after PU.1 activation-consistent with its role as a pioneer-but the enhancers do not upregulate gene expression. Combining deeply sequenced ATAC-Seq data with a new bias-correction method allowed the profiling of accessibility at single-nucleotide resolution and revealed protected regions in the enhancers that match all previously characterized TF binding sites and ChIP-Seq data. Although the accessibility of most positions does not change during early differentiation, that of positions neighboring TF binding sites, an indicator of TF occupancy, did increase significantly. The localized accessibility changes are limited to nucleotides neighboring C/EBP-family TF binding sites, showing that the upregulation of enhancer activity during early differentiation is driven by C/EBP-family TF binding. These results show that increasing the total accessibility of enhancers is not sufficient for upregulating their activity and other events such as TF binding are necessary for upregulation. Also, TF binding can cause upregulation without a perceptible increase in total accessibility. Finally, this study demonstrates the feasibility of comprehensively mapping individual TF binding sites as footprints using high coverage ATAC-Seq and inferring the sequence of events in gene regulation by combining with time-series gene expression data.

Development of a polyclonal anti-dugong immunoglobulin G (IgG) antibody with evaluation of total plasma IgG in a living dugong (Dugong dugon) population.

Species-specific antibodies (Ab) for the measurement of immunoglobulins (Ig) are valuable tools for determining the humoral immune status of threatened and endangered wildlife species such as dugongs. However, no studies have reported antibody reagents against dugong immunoglobulin. The object of this study was to develop an Ab with specificity for dugong IgG and apply this tool to survey total IgG levels in plasma samples from a live wild population of dugongs in southern Queensland, Australia. Dugong IgG was isolated from plasma by protein A/G column chromatography and a polyclonal antiserum was successfully raised against the dugong IgG through immunization of mice. The anti-dugong antiserum was reactive with dugong serum but not immunoglobulin from other species such as rats and humans. When tested against a panel of dugong plasma samples, relative IgG levels from dugongs (n = 116) showed biologically relevant relationships with pregnancy status and a principal component of Body Mass Index (BMI)/globulin/fecal glucocorticosteroid (chronic stress) levels combined, which together accounted for 9.2% of the variation in total Ig levels. Together these data suggest that dugongs show variation in total IgG and that this correlates with some physiological parameters of dugong health.

Hematology of dugongs (Dugong dugon) in southern Queensland.

BACKGROUND: Little is known of the hematology of the dugong (Dugong dugon), a secretive and endangered coastal marine mammal. OBJECTIVES: This paper reports hematologic reference intervals (RI) for dugongs and characterizes morphologic, cytochemical, and ultrastructural features of dugong leukocytes. METHODS: Blood was collected from live, apparently healthy dugongs and analyzed using Cell-Dyn 3700 or Sysmex XT-2000iV hematology analyzers. Blood films were subjected to a series of cytochemical stains, and leukocyte structure was examined using transmission electron microscopy. RESULTS: Reference intervals were established for 14 hematologic variables, total solids, and fibrinogen for 92 dugongs. Significant differences in some variables were found for animal size class, sex, and pregnancy status, and between analyzers. Subadults had higher leukocyte and lymphocyte counts than adults. Males had higher total solids and fibrinogen than females. Pregnant females had higher HCT, MCV, and circulating nucleated RBC, and lower platelet counts than nonpregnant females. Lymphocytes were usually the predominant circulating leukocyte. Heterophil cytoplasmic granules were abundant, fine, round to ovoid, and intensely eosinophilic, and round to ovoid or rod-shaped, and variably electron dense in electron microscopy. Eosinophils contained larger round eosinophilic to orange cytoplasmic granules, which ultrastructurally were bicompartmental with a round eccentric electron-dense core. Cytochemical staining of dugong heterophils suggests biochemical similarity to those of manatees and elephants, and for eosinophils, similarity to those of elephants, ruminants, and equids. CONCLUSIONS: Generation of hematologic RI and characterization of leukocyte morphology improves evaluation of dugong health across this population and serves as a reference for other populations outside southern Queensland.

Serum biochemistry reference intervals of live wild dugongs (Dugong dugon) from urban coastal Australia.

BACKGROUND: Little is known about the baseline clinical pathology of the dugong (Dugong dugon), a vulnerable marine mammal found in tropical coastal marine systems. OBJECTIVES: The purpose of the study was to collect and determine reference intervals (RI) for select serum biochemical variables for dugongs, and to analyze differences between males and females and different age groups. METHODS: Reference intervals were established from 103 apparently healthy, wild-caught dugongs for 31 analytes using a Beckman Coulter AU400 Automated Chemistry Analyzer and an Olympus AU680 Chemistry-Immuno Analyzer. RESULTS: Significant differences (P < .05) in some of the variables were found related to size class, sex, and pregnancy status. Adult dugongs had higher serum sodium, potassium, bicarbonate, glucose, and l-lactate concentrations and higher anion gap, compared to sub-adults. Male dugongs had higher triglyceride and l-lactate concentrations than females. Pregnant females displayed higher l-lactate levels compared to nonpregnant animals. Statistical differences in variables within the population contributed to better understanding of the physiologic differences between cohorts. Some serum biochemistry changes observed in this study here also potentially include some effects of pursuit on dugongs (eg, higher l-lactate); however, as all dugongs were subject to similar capture and handling, serum biochemistry RI should be considered as normal for captured dugongs. CONCLUSIONS: The serum biochemical RI documented here are considered representative of a population of healthy captured dugongs. They provide a baseline for health surveillance of this and other dugong populations.

Evaluation of exertion and capture stress in serum of wild dugongs (Dugong dugon).

Seven hundred fifty-one dugongs (Dugong dugon) were pursued, captured, and handled for up to 20 min for population sampling. Fifty of these dugongs were then removed from the water for up to 55 min for comprehensive medical examination. Fifty whole blood and separated serum samples were analyzed for potassium, sodium, chloride, creatinine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), urea, creatinine, glucose, anion gap, and total blood CO2. Serum biochemical variables of the dugong were compared with those obtained in previous studies of the related West Indian manatee, a mammal that does not appear to experience capture myopathy based on available data. Differences between these species included higher blood sodium and chloride in dugongs, which may reflect differences in salt balance and renal function, and higher blood lactate and CO2. Some biochemical analytes such as CK and AST, which may be indicative of rhabdomyolysis associated with capture stress myopathy (a potentially fatal condition for which dugongs have been thought to be highly susceptible) were high compared with levels previously measured in wild West Indian manatees (Trichechus latirostris). One of the 50 dugongs had marked elevations of CK and AST but showed no other clinical indications of rhabdomyolysis associated with capture myopathy such as hyperthermia. Rather, generally high levels of lactate, CK, and AST most probably reflect metabolic acidosis resulting from the exertion involved in the pursuit prior to capture. Earlier observations suggesting that dugongs were probably susceptible to capture stress myopathy (based on high serum potassium levels) were not supported by this study. Capture and handling methods currently used on dugongs in this research program do not appear to result in acute capture stress.