Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells, bioRxiv, 2019-02-02
In situ assays of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.
biorxiv genomics 200-500-users 2019SynQuant An Automatic Tool to Quantify Synapses from Microscopy Images, bioRxiv, 2019-02-02
AbstractMotivationSynapses are essential to neural signal transmission. Therefore, quantification of synapses and related neurites from images is vital to gain insights into the underlying pathways of brain functionality and diseases. Despite the wide availability of synaptic punctum imaging data, several issues are impeding satisfactory quantification of these structures by current tools. First, the antibodies used for labeling synapses are not perfectly specific to synapses. These antibodies may exist in neurites or other cell compartments. Second, the brightness for different neurites and synaptic puncta is heterogeneous due to the variation of antibody concentration and synapse-intrinsic differences. Third, images often have low signal to noise ratio due to constraints of experiment facilities and availability of sensitive antibodies. These issues make the detection of synapses challenging and necessitates developing a new tool to easily and accurately quantify synapses.ResultsWe present an automatic probability-principled synapse detection algorithm and integrate it into our synapse quantification tool SynQuant. Derived from the theory of order statistics, our method controls the false discovery rate and improves the power of detecting synapses. SynQuant is unsupervised, works for both 2D and 3D data, and can handle multiple staining channels. Through extensive experiments on one synthetic and three real data sets with ground truth annotation or manual labeling, SynQuant was demonstrated to outperform peer specialized synapse detection tools as well as generic spot detection methods, including 4 unsupervised and 11 variants of 3 supervised methods.AvailabilityJava source code, Fiji plug-in, and test data available at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comyu-lab-vtSynQuant>httpsgithub.comyu-lab-vtSynQuant<jatsext-link>.Contactyug@vt.edu
biorxiv neuroscience 0-100-users 2019Freeze-frame imaging of synaptic activity using SynTagMA, bioRxiv, 2019-02-01
AbstractInformation within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. To tag active synapses in a user-defined time window, we developed SynTagMA. Upon 405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we developed software to identify and track the fluorescence of thousands of individual synapses in tissue. Together, these tools provide a high throughput method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.
biorxiv neuroscience 100-200-users 2019The Arabidopsis thaliana pan-NLRome, bioRxiv, 2019-02-01
Disease is both among the most important selection pressures in nature and among the main causes of yield loss in agriculture. In plants, resistance to disease is often conferred by Nucleotide-binding Leucine-rich Repeat (NLR) proteins. These proteins function as intracellular immune receptors that recognize pathogen proteins and their effects on the plant. Consistent with evolutionarily dynamic interactions between plants and pathogens, NLRs are known to be encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define the majority of the Arabidopsis thaliana species-wide 'NLRome'. From NLR sequence enrichment and long-read sequencing of 65 diverse A. thaliana accessions, we infer that the pan-NLRome saturates with approximately 40 accessions. Despite the high diversity of NLRs, half of the pan-NLRome is present in most accessions. We chart the architectural diversity of NLR proteins, identify novel architectures, and quantify the selective forces that act on specific NLRs, domains, and positions. Our study provides a blueprint for defining the pan-NLRome of plant species.
biorxiv plant-biology 100-200-users 2019Ensuring meiotic DNA break formation in the mouse pseudoautosomal region Data File S1, bioRxiv, 2019-01-31
Sex chromosomes in males share only a diminutive homologous segment, the pseudoautosomal region (PAR), wherein meiotic double-strand breaks (DSBs), pairing, and crossing over must occur for correct segregation. How cells ensure PAR recombination is unknown. Here we delineate cis- and trans-acting factors that control PAR ultrastructure and make the PAR the hottest area of DSB formation in the male mouse genome. Prior to DSB formation, PAR chromosome axes elongate, sister chromatids separate, and DSB-promoting factors hyperaccumulate. These phenomena are linked to mo-2 minisatellite arrays and require ANKRD31 protein. We propose that the repetitive PAR sequence confers unique chromatin and higher order structures crucial for DSB formation, X-Y pairing, and recombination. Our findings establish a mechanistic paradigm of mammalian sex chromosome segregation during spermatogenesis.
biorxiv molecular-biology 100-200-users 2019