Biology subjects

Lee, D.

Publications and source records attributed to Lee, D..

At least 235 records · Page 13Linked to original sources

bioRxiv2021

St18 specifies globus pallidus projection neuron identity in MGE lineage

The medial ganglionic eminence (MGE) is a progenitor domain in the subpallium that produces both locally-projecting interneurons which undergo tangential migration in structures such as the cortex as well as long-range projection neurons that occupy subcortical nuclei. Very little is known about the transcriptional mechanisms specifying the migratory behavior and axonal projection patterns of these two broad classes of MGE-derived neurons. In this study, we identify St18 as a novel transcriptional determinant specifying projection neuron fate in the MGE lineage. St18 is transiently expressed in the MGE subventricular zone (SVZ) and mantle, and we assessed its function using an ES cell-based model of MGE development. Induction of St18 is sufficient to direct ES-derived MGE neurons to adopt a projection neuron-like identity as defined by migration and morphology. Using genetic loss-of-function in mice, we find that St18 is required for the production of globus pallidus pars externa (GPe) prototypic projection neurons. Single cell RNA sequencing revealed that St18 regulates MGE output of specific neuronal populations: in the absence of St18, we observe a large expansion of cortical interneurons at the expense of putative GPe neurons. Through gene expression analysis we identified a downstream effector of St18, Cbx7, which is a component of Polycomb repressor complex 1. We find that Cbx7 is essential for projection neuron-like migration and is not involved in St18-mediated projection neuron-like morphology. Our results characterize a novel transcriptional determinant that directs GPe prototypic projection neuron identity. Further, we identified a downstream target of St18, Cbx7, which regulates only the migratory behavior of long-range projection neurons, suggesting that specific features of MGE projection neuron identity may be governed in a compartmentalized fashion by distinct transcriptional modules downstream of St18.

developmental biology↗

bioRxiv2021

The Mus musculus papillomavirus type 1 E7 protein binds to the retinoblastoma tumor suppressor - implications for viral pathogenesis

The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts specifically with RB1. We show that MmuPV1 E7 interacts through its C-terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 virus expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting non-canonical activities of RB1, which are independent of RB1s ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis. ImportancePapillomavirus infections cause a variety of epithelial hyperplastic lesions, warts. While most warts are benign, some papillomaviruses cause lesions that can progress to squamous cell carcinomas and approximately 5% of all human cancers are caused by human papillomavirus (HPV) infections. The papillomavirus E6 and E7 proteins are thought to function to reprogram host epithelial cells to enable viral genome replication in terminally differentiated, normally growth-arrested cells. E6 and E7 lack enzymatic activities and function by interacting and functionally altering host cell regulatory proteins. Many cellular proteins that can interact with E6 and E7 have been identified, but the biological relevance of these interactions for viral pathogenesis has not been determined. This is because papillomaviruses are species-specific and do not infect heterologous hosts. Here we use a recently established mouse papillomavirus (MmuPV1) model to investigate the role of the E7 protein in viral pathogenesis. We show that MmuPV1 E7 is necessary for papilloma formation. The retinoblastoma tumor suppressor protein (RB1) is targeted by many papillomaviral E7 proteins, including cancer-associated HPVs. We show that MmuPV1 E7 can bind RB1 and that infection with a mutant MmuPV1 virus that expresses an RB1 binding defective E7 mutant caused smaller and fewer papillomas that arise with delayed kinetics.

molecular biology↗

bioRxiv2021

SUITOR: selecting the number of mutational signatures through cross-validation

For de novo mutational signature analysis, the critical first step is to decide how many signatures should be expected in a cancer genomics study. An incorrect number could mislead downstream analyses. Here we present SUITOR (Selecting the nUmber of mutatIonal signaTures thrOugh cRoss-validation), an unsupervised cross-validation method that requires little assumptions and no numerical approximations to select the optimal number of signatures without overfitting the data. In vitro studies and in silico simulations demonstrated that SUITOR can correctly identify signatures, some of which were missed by other widely used methods. Applied to 2,540 whole-genome sequenced tumors across 22 cancer types, SUITOR selected signatures with the smallest prediction errors and almost all signatures of breast cancer selected by SUITOR were validated in an independent breast cancer study. SUITOR is a powerful tool to select the optimal number of mutational signatures, facilitating downstream analyses with etiological or therapeutic importance.

bioinformatics↗

bioRxiv2021

Pooled screening of CAR T cells identifies non-native signaling domains for next- generation immunotherapies

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers, but have shown limited efficacy against solid tumors. Here, we introduce CAR Pooling, a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with diverse immune costimulatory domains were assessed in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several non-native domains from the TNF receptor family exhibited enhanced proliferation (CD40) or cytotoxicity (BAFF-R and TACI) relative to clinical benchmarks, and fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic and NK-cell-like innate phenotype previously associated with positive clinical outcomes. CAR Pooling enables efficient exploration of how CAR design affects cell activity and can be applied to optimize receptors across a range of applications and cell types.

immunology↗

bioRxiv2021

Single-orientation Quantitative Susceptibility Mapping identifies the Ventral Intermediate Nucleus of the Thalamus

IntroductionThe ventral intermediate nucleus (VIM) represents the primary target in the treatment of tremor. Accurate localization is extremely important given its proximity to other thalamic nuclei. We utilized single orientation quantitative susceptibility mapping (QSM) at 3T to directly visualize the VIM. MethodsFour adult volunteers, one adult cadaver, and an essential tremor patient were scanned on a 3T MRI using a multi-echo gradient echo sequence. QSM images were generated using the improved sparse linear equation and least-squares (iLSQR) algorithm. Two adult subjects underwent multiple head orientation imaging for multi-orientation QSM reconstruction. The VIM was prospectively identified with direct visualization as well as indirect landmark-based localization. ResultsThe bilateral VIM was consistently identified as a hypointense structure within the lateral thalamus, appearing similar on multi-orientation and single-orientation QSM, corresponding to the myelinated dentatorubrothalamic tract (DRTT). The indirect method resulted in a comparatively inferomedial localization, at times missing the VIM and DRTT. ConclusionSingle-orientation QSM offers a clinically feasible, non-invasive imaging-based approach to directly localize the VIM.

neuroscience↗

bioRxiv2021

Bacterial filamentation is an in vivo mechanism for cell-to-cell spreading

Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread from cell-to-cell in vivo. Here, we describe the discovery of a bacterial species that uses filamentation as an in vivo mechanism for intracellular spreading between the intestinal epithelial cells of its host, the rhabditid nematode Oscheius tipulae. In vitro and in vivo filamentation by this bacterium, Bordetella atropi, requires a highly conserved nutrient-sensing pathway used by divergent bacteria to detect rich conditions and inhibit the divisome. Thus, B. atropi uses a distinct mechanism for cell-to-cell spreading by coopting a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells.

microbiology↗

bioRxiv2021

CHD4 conceals aberrant CTCF-binding sites at TAD interiors by regulating chromatin accessibility in mESCs

CTCF plays a critical role in the 3D chromatin organization by determining the TAD borders. Although CTCF primarily binds at the TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking these putative CTCF-binding sites remains elusive. Here, we show that the ATP-dependent chromatin remodeler, CHD4, regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 SINEs in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible, and aberrant CTCF recruitment occurs at the TAD interiors, resulting in disorganization of the local TADs. Furthermore, RNA-binding intrinsically disordered domains of CHD4 is required to prevent the aberrant CTCF bindings. Lastly, CHD4 is required for the repression of B2 SINE transcripts. These results highlight the CHD4-mediated mechanism that safeguards the appropriate CTCF bindings and associated TAD organizations in mESCs.

molecular biology↗

bioRxiv2021

RNA surveillance controls 3D genome structure via stable cohesin-chromosome interaction

The 3D architecture that the genome is folded into is regulated by CTCF, which determines domain borders, and cohesin, which generates interactions within domains. However, organisms lacking CTCF have been reported to still have cohesin-mediated 3D structures with strong borders. How this can be achieved and precisely regulated are yet unknown. Using in situ Hi-C, we found that 3-end RNA processing factors coupled with proper transcription termination are a cis-acting determinant that regulates the localization and dynamics of cohesin on the chromosome arms. Loss of RNA processing factors, including nuclear exosome and Pfs2, destabilizes cohesin from the 3-ends of convergent genes and results in decreased cohesin-mediated domain boundaries. We observed the co-localization between Rad21 and a wide range of 3end RNA processing/termination factors. Further, deletion of Rrp6 leads to cohesin redistribution to facultative heterochromatin, resulting in improper domain boundaries. Importantly, we observed that knockdown of Rrp6Exosc10 caused a defect in cohesin binding and loss of local topologically associating domains (TADs) interactions in mouse embryonic stem cells. Based on these findings, we propose a novel function of the RNA surveillance pathway in 3D genome organization via cohesin complex, which provides the molecular basis underlying the dynamics of cohesin function.

genomics↗

bioRxiv2021

Spatial and temporal autocorrelation weave human brain networks

High-throughput experimental methods in neuroscience have led to an explosion of techniques for measuring complex interactions and multi-dimensional patterns. However, whether sophisticated measures of emergent phenomena can be traced back to simpler low-dimensional statistics is largely unknown. To explore this question, we examine resting state fMRI (rs-fMRI) data using complex topology measures from network neuroscience. We show that spatial and temporal autocorrelation are reliable statistics which explain numerous measures of network topology. Surrogate timeseries with subject-matched spatial and temporal autocorrelation capture nearly all reliable individual and regional variation in these topology measures. Network topology changes during aging are driven by spatial autocorrelation, and multiple serotonergic drugs causally induce the same topographic change in temporal autocorrelation. This reductionistic interpretation of widely-used complexity measures may help link them to neurobiology.

neuroscience↗

bioRxiv2021

A connectomic study of a petascale fragment of human cerebral cortex

We acquired a rapidly preserved human surgical sample from the temporal lobe of the cerebral cortex. We stained a 1 mm3 volume with heavy metals, embedded it in resin, cut more than 5000 slices at [~]30 nm and imaged these sections using a high-speed multibeam scanning electron microscope. We used computational methods to render the three-dimensional structure containing 57,216 cells, hundreds of millions of neurites and 133.7 million synaptic connections. The 1.4 petabyte electron microscopy volume, the segmented cells, cell parts, blood vessels, myelin, inhibitory and excitatory synapses, and 104 manually proofread cells are available to peruse online. Many interesting and unusual features were evident in this dataset. Glia outnumbered neurons 2:1 and oligodendrocytes were the most common cell type in the volume. Excitatory spiny neurons comprised 69% of the neuronal population, and excitatory synapses also were in the majority (76%). The synaptic drive onto spiny neurons was biased more strongly toward excitation (70%) than was the case for inhibitory interneurons (48%). Despite incompleteness of the automated segmentation caused by split and merge errors, we could automatically generate (and then validate) connections between most of the excitatory and inhibitory neuron types both within and between layers. In studying these neurons we found that deep layer excitatory cell types can be classified into new subsets, based on structural and connectivity differences, and that chandelier interneurons not only innervate excitatory neuron initial segments as previously described, but also each others initial segments. Furthermore, among the thousands of weak connections established on each neuron, there exist rarer highly powerful axonal inputs that establish multi-synaptic contacts (up to [~]20 synapses) with target neurons. Our analysis indicates that these strong inputs are specific, and allow small numbers of axons to have an outsized role in the activity of some of their postsynaptic partners.

neuroscience↗

bioRxiv2021

Sequence-based correction of barcode bias in massively parallel reporter assays

Massively parallel reporter assays (MPRA) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream of a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag sequence-specific effects and demonstrate that our method can significantly reduce this source of variation, and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps to not only improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

bioinformatics↗

bioRxiv2021

A human brain vascular atlas reveals diverse cell mediators of Alzheimer's disease risk

The human brain vasculature is of vast medical importance: its dysfunction causes disability and death, and the specialized structure it forms--the blood-brain barrier--impedes treatment of nearly all brain disorders. Yet, no molecular atlas of the human brain vasculature exists. Here, we develop Vessel Isolation and Nuclei Extraction for Sequencing (VINE-seq) to profile the major human brain vascular and perivascular cell types through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 17 control and Alzheimers disease (AD) patients. We identify brain region-enriched pathways and genes divergent between humans and mice, including those involved in disease. We describe the principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum; but discover that many zonation and cell-type markers differ between species. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In AD, we observe a selective vulnerability of ECM-maintaining pericytes and gene expression patterns implicating dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 AD GWAS genes are expressed in the human brain vasculature, confirmed in situ. Vascular GWAS genes map to endothelial protein transport, adaptive immune, and ECM pathways. Many are microglia-specific in mice, suggesting an evolutionary transfer of AD risk to human vascular cells. Our work unravels the molecular basis of the human brain vasculature, informing our understanding of overall brain health, disease, and therapy.

neuroscience↗

bioRxiv2021

Widespread multi-targeted therapy resistance via drug-induced secretome fucosylation

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post-translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In both cancer cell cultures and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes revealed that fucosylation of the antioxidant PON1 is a critical component of the therapy-induced secretome. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Non-specific and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

cancer biology↗

bioRxiv2021

Single-cell immune profiling with TCR clonotype barcoding identifies biomarker signatures that predict response to immune checkpoint blockade

The identification of predictive biomarkers for patient treatment response is urgently needed to increase the probability of success of existing and novel experimental therapies. Single-cell profiling has provided novel biological insights into drug responses in the tumor microenvironment, but its potential for biomarker discovery has not been fully explored for therapeutic purposes. We describe a novel approach to discover predictive response biomarkers from single-cell data from a small patient cohort using the T cell receptor sequence intrinsic to each T cell to match clonotypes between pre- and post-treatment tumor samples. As a result, we have identified a predictive gene expression signature for immune checkpoint blockade and validated its predictive performance using data from three larger clinical studies. Our results demonstrated that applying clonotyping with single-cell genomic profiling is a promising novel approach for biomarker identification that does not require data collected from large patient cohorts. This could increase success rates, reduce clinical trial size, and significantly impact future clinical developments of immunomodulatory therapeutics.

immunology↗

bioRxiv2021

Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.

microbiology↗

bioRxiv2021

The chromatin remodeler Ino80 mediates alternative RNAPII pausing site determination

Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step in early transcription elongation for the precise regulation of gene expression. Here, we provide evidence of promoter-proximal pausing-like distributions of RNAPII in S. cerevisiae. We found that genes bearing an alternative pausing site utilize Ino80p to properly localize RNAPII pausing at the first pausing site and to suppress the accumulation of RNAPII at the second pausing site, which is tightly associated with the +1 nucleosome. This alternative pausing site determination was dependent on the remodeling activity of Ino80p to modulate the +1 nucleosome position and might be controlled synergistically with Spt4p. Furthermore, we observed similar Ino80-dependent RNAPII pausing in mouse embryonic stem cells (mESCs). Based on our collective results, we hypothesize that the chromatin remodeler Ino80 plays a highly conserved role in regulating early RNAPII elongation to establish intact pausing.

molecular biology↗

bioRxiv2021

DECODE: A Deep-learning Framework for Condensing Enhancers and Refining Boundaries with Large-scale Functional Assays

SummaryMapping distal regulatory elements, such as enhancers, is the cornerstone for investigating genome evolution, understanding critical biological functions, and ultimately elucidating how genetic variations may influence diseases. Previous enhancer prediction methods have used either unsupervised approaches or supervised methods with limited training data. Moreover, past approaches have operationalized enhancer discovery as a binary classification problem without accurate enhancer boundary detection, producing low-resolution annotations with redundant regions and reducing the statistical power for downstream analyses (e.g., causal variant mapping and functional validations). Here, we addressed these challenges via a two-step model called DECODE. First, we employed direct enhancer activity readouts from novel functional characterization assays, such as STARR-seq, to train a deep neural network classifier for accurate cell-type-specific enhancer prediction. Second, to improve the annotation resolution ([~]500 bp), we implemented a weakly-supervised object detection framework for enhancer localization with precise boundary detection (at 10 bp resolution) using gradient-weighted class activation mapping. ResultsOur DECODE binary classifier outperformed the state-of-the-art enhancer prediction methods by 24% in transgenic mouse validation. Further, DECODE object detection can condense enhancer annotations to only 12.6% of the original size, while still reporting higher conservation scores and genome-wide association study variant enrichments. Overall, DECODE improves the efficiency of regulatory element mapping with graphic processing units for deep-learning applications and is a powerful tool for enhancer prediction and boundary localization. Contactpi@gersteinlab.org

bioinformatics↗

bioRxiv2021

Complement-associated loss of CA2 inhibitory synapses in the demyelinated hippocampus impairs memory

The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer, and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we further found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by a reduced spike output. Ultimately, we show that these electrophysiological changes were associated with an impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS.

neuroscience↗