Biology subjects

Dong, P.

Publications and source records attributed to Dong, P..

31 records · Page 2Linked to original sources

bioRxiv2021

From compartments to gene loops: Functions of the 3D genome in the human brain

The 3D genome plays a key role in the regulation of gene expression. However, little is known about the spatiotemporal organization of chromatin during human brain development. We investigated the 3D genome in human fetal cortical plate and in adult prefrontal cortical neurons and glia. We found that neurons have weaker compartments than glia that emerge during fetal development. Furthermore, neurons form loop domains whereas glia form compartment domains. We show through CRISPRi on CNTNAP2 that transcription is coupled to loop domain insulation. Gene regulation during neural development involves increased use of enhancer-promoter and repressor-promoter loops. Finally, transcription is associated with gene loops. Altogether, we provide novel insights into the relationship between gene expression and different scales of chromatin organization in the human brain.

genomics↗

bioRxiv2021

Neuronal mechanism of a BK channelopathy in absence epilepsy and movement disorders

A growing number of gain-of-function (GOF) BK channelopathy have been identified in patients with epilepsy and paroxysmal movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and movement disorders. We found that the BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits. BK-D434G mutation causes hyperexcitability of cortical pyramidal neurons and cerebellar Purkinje cells, which contributes to the pathogenesis of absence seizures and the motor defects, respectively. A BK channel blocker paxilline potently suppresses BK-D434G-induced hyperexcitability and effectively mitigates absence seizures in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and provided the evidence that BK inhibition is a promising therapeutic strategy to mitigate BK GOF-induced neurological disorders. SignificanceDysfunction of BK channels or BK channelopathy has been increasingly implicated in diverse neurological disorders including epilepsy, movement, cognitive and neurodevelopmental disorders. However, precision medicine to treat BK channelopathy is lacking. Here we characterized a mouse model carrying a gain-of-function BK channelopathy D434G from a large family of patients with absence epilepsy and involuntary movement disorders. The BK-D434G mice resemble the clinical manifestations of absence seizures and exhibit severe motor defects. The hyperexcitability in BK-D434G cortical neurons and cerebellar Purkinje cells underscores the neuronal mechanism of BK gain-of-function induced absence epilepsy and movement disorders. The effectiveness of a BK channel blocker on preventing absence seizures suggests that BK inhibition is a promising strategy to treat gain-of-function BK channelopathy.

neuroscience↗

bioRxiv2021

Acetylated Chromatin Domains Link Chromosomal Organization to Cell- and Circuit-level Dysfunction in Schizophrenia and Bipolar Disorder

To explore modular organization of chromosomes in schizophrenia (SCZ) and bipolar disorder (BD), we applied population-scale correlational structuring of 739 histone H3-lysine 27 acetylation and H3-lysine 4 trimethylation profiles, generated from the prefrontal cortex (PFC) of 568 cases and controls. Neuronal histone acetylomes and methylomes assembled as thousands of cis-regulatory domains (CRDs), revealing fine-grained, kilo-to megabase scale chromatin organization at higher resolution but firmly integrated into Hi-C chromosomal conformations. Large clusters of domains that were hyperacetylated in disease shared spatial positioning within the nucleus, predominantly regulating PFC projection neuron function and excitatory neurotransmission. Hypoacetylated domains were linked to inhibitory interneuron- and myelination-relevant genes. Chromosomal modular architecture is affected in SCZ and BD, with hyperacetylated domains showing unexpectedly strong convergences defined by cell type, nuclear topography, genetic risk, and active chromatin state across a wide developmental window.

genomics↗

bioRxiv2021

Nucleocapsid mutation R203K/G204R increases the infectivity, fitness and virulence of SARS-CoV-2

In addition to the mutations on the spike protein (S), co-occurring mutations on nucleocapsid (N) protein are also emerging in SARS-CoV-2 world widely. Mutations R203K/G204R on N, carried by high transmissibility SARS-CoV-2 lineages including B.1.1.7 and P.1, has a rapid spread in the pandemic during the past year. In this study, we performed comprehensive population genomic analyses and virology experiment concerning on the evolution, causation and virology consequence of R203K/G204R mutations. The global incidence frequency (IF) of 203K/204R has rose up from nearly zero to 76% to date with a shrinking from August to November in 2020 but bounced later. Our results show that the emergence of B.1.1.7 is associated with the second growth of R203K/G204R mutants. We identified positive selection evidences that support the adaptiveness of 203K/204R variants. The R203K/G204R mutant virus was created and compared with the native virus. The virus competition experiments show that 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly in relation to the ribonucleocapsid (RNP) assemble during the virus replication. Moreover, the 203K/204R virus increased the infectivity in a human lung cell line and induced an enhanced damage to blood vessel of infected hamsters lungs. In consistence, we observed a positive association between the increased severity of COVID-19 and the IF of 203K/204R from in silicon analysis of global clinical and epidemic data. In combination with the informatics and virology experiment, our work suggested the contribution of 203K/204R to the increased transmission and virulence of the SARS-CoV-2. In addition to mutations on the S protein, the mutations on the N protein are also important to virus spread during the pandemic.

evolutionary biology↗

bioRxiv2021

Transcribed enhancers in the human brain identify novel disease risk mechanisms

Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding the population-level variation of enhancers in the human brain. Besides regulating tissue- and cell-type-specific transcription of target genes, enhancers themselves can be transcribed. We expanded the catalog of known human brain transcribed enhancers by an order of magnitude by generating and jointly analyzing large-scale cell-type-specific transcriptome and regulome data. Examination of the transcriptome in 1,382 brain samples in two independent cohorts identified robust expression of transcribed enhancers. We explored gene-enhancer coordination and found that enhancer-linked genes are strongly implicated in neuropsychiatric disease. We identified significant expression quantitative trait loci (eQTL) for 25,958 enhancers which mediate 6.8% of schizophrenia heritability, mostly independent from standard gene eQTL. Inclusion of enhancer eQTL in transcriptome-wide association studies enhanced functional interpretation of disease loci. Overall, our study characterizes the enhancer-gene regulome and genetic mechanisms in the human cortex in both healthy and disease states.

neuroscience↗

bioRxiv2021

The three-dimensional landscape of chromatin accessibility in Alzheimer's disease

Much is still unknown about the neurobiology of Alzheimers disease (AD). To better understand AD, we generated 636 ATAC-seq libraries from cases and controls to construct detailed genomewide chromatin accessibility maps of neurons and non-neurons from two AD-affected brain regions, the entorhinal cortex and superior temporal gyrus. By analyzing a total of 19.6 billion read pairs, we expanded the known repertoire of regulatory sequences in the human brain. Multi-omic data integration associated global patterns of chromatin accessibility with gene expression and identified cell-specific enhancer-promoter interactions. Using inter-individual variation in chromatin accessibility, we define cis-regulatory domains capturing the 3D structure of the genome. Multifaceted analyses uncovered disease associated perturbations impacting chromatin accessibility, transcription factor regulatory networks and the 3D genome, and implicated transcriptional dysregulation in AD. Overall, we applied a systematic approach to understand the role of the 3D genome in AD and to illuminate novel disease biology that can advance diagnosis and therapy.

neuroscience↗

bioRxiv2019

Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin

Access to cis-regulatory elements packaged in chromatin is essential for directing gene expression and cell viability. Here, we report a super-resolution imaging strategy, 3D ATAC-PALM, that enables direct visualization of the entire accessible genome. We found that active chromosomal segments are organized into spatially-segregated accessible chromatin domains (ACDs). Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced enhanced ACD clustering, reduced physical separation between intrachromosomal ACDs, and differentially regulated ACD compaction. Experimental perturbations and polymer modeling suggest that dynamic protein-protein and protein-DNA interactions within ACDs couple with loop extrusion to organize ACD topology. Dysorganization of ACDs upon CTCF or Cohesin loss alters transcription factor binding and target search dynamics in living cells. These results uncover fundamental mechanisms underpinning the formation of 3D genome architecture and its pivotal function in transcriptional regulation.

molecular biology↗

bioRxiv2019

Quantifying E2F1 protein dynamics in single cells

The Rb/E2F pathway plays a central role in regulating cell-fate decisions and cell-cycle progression. The E2F1 protein, a major effector of the pathway, is regulated via a combination of transcriptional, translational and posttranslational constraints. Elucidating the regulation and impact of the Rb/E2F pathway requires direct measurement of E2F1 dynamics in single cells. To this end, we have engineered fluorescent E2F1 protein reporters to enable live detection and quantification in single cells. The reporter constructs expressed an E2F1-Venus fusion protein under the regulation of the mouse or human E2F1 promoter and contained or excluded the 3UTR of the E2F1 gene, a sequence that contains miRNA regulatory regions that modulate expression of the protein. Expression of the reporter protein was highly dynamic during the cell cycle: there was no or little fluorescent signal in G0, but levels steadily increased during late G1 and peaked during mid to late S phase before returning to baseline before the onset of mitosis. The absence of the E2F1 3UTR in the constructs led to considerably higher steady-state levels of the fusion protein, which although normally regulated, exhibited a slightly less complex dynamic profile during the cell cycle or genotoxic stress. Lastly, the presence or absence of Rb failed to impact in substantial ways the overall detection and levels of the reporters.

cell biology↗

bioRxiv2019

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

Loss of function mutations in Methyl-CpG-binding Protein 2 (MeCP2) cause the severe neurological disorder Rett Syndrome. MeCP2 is a highly abundant nuclear protein particularly enriched in neurons. Although biochemical and genomic analyses of MeCP2-DNA interaction and genomic distribution demonstrate that MeCP2 binding on chromatin is dependent on DNA modification state, the dynamic behavior of individual MeCP2 proteins in live neurons has not been explored. Here we use live-cell single-molecule imaging to assess the detailed kinetic features of MeCP2 in distinct sub-nuclear regions at high spatial and temporal resolution. Surprisingly, we found that, in granule cell nuclei, MeCP2 has unique diffusion and chromatin binding kinetics that are distinct from highly mobile sequence-specific transcription factors (TF) and immobile histone proteins. Approximately, half of MeCP2 is bound to DNA in a transiently stable mode that is similar to TF binding to their cognate sites. The binding of meCP2 to DNA requires its methyl-binding domain (MBD) and is sensitive to the levels of both DNA methylation and hydroxymethylation. However, when not stably bound, MeCP2 moves slowly in the nucleus most closely resembling histone H1.0. The rate of MeCP2 diffusion in compact, granule cell nuclei is determined by weak, transient DNA interactions mediated primarily by the MBD and three AT-hook domains located in the C-terminal portion of the protein. Both the fraction of stably bound MeCP2 and its rate of diffusion depend on the level of chromatin compaction and neuronal cell type. Our data reveal new features of MeCP2 that dictate its dynamic behavior in neuronal nuclei and suggest that the limited nuclear diffusion of MeCP2 in live neurons may contribute to its local impact on chromatin structure and gene expression.

molecular biology↗

bioRxiv2019

Tissue-specific Hi-C analyses of rice, foxtail millet and maize suggest non-canonical function of plant chromatin domains

Chromatins are not randomly packaged in the nucleus and their organization plays important roles in transcription regulation. Using in situ Hi-C, we have compared the 3D chromatin architectures of rice mesophyll and endosperm, foxtail millet bundle sheath and mesophyll, and maize bundle sheath, mesophyll and endosperm tissues. We have also profiled their DNA methylation, open chromatin, histone modification and gene expression to investigate whether chromatin structural dynamics are associated with epigenome features changes. We found that plant global A/B compartment partitions are stable across tissues, while local A/B compartment has tissue-specific dynamic that is associated with differential gene expression. Plant domains are largely stable across tissues, while rare domain border changes are often associated with gene activation. Genes inside plant domains are not conserved across species, and lack significant co-expression behavior unlike those in mammalian cells. When comparing synteny gene pairs, we found those maize genes involved in gene island chromatin loops have shorter genomic distances in smaller genomes without gene island loops such as rice and foxtail millet, suggesting that they have conserved functions. Our study revealed that the 3D configuration of the plant chromatin is also complex and dynamic with unique features that need to be further examined.

genomics↗

bioRxiv2018

High-Speed Imaging of Paw Withdrawal Reflex to Objectively Assess Pain State in Mice

Rodents are often used for studying chronic pain mechanisms and developing new pain therapeutics, but objectively determining the animals pain state is a major challenge. To improve the precision of using reflexive withdrawal behaviors for interpreting the mouse pain state, we adopted high-speed videography to capture sub-second movement features of mice upon hind paw stimulation. We identified several parameters that are significantly different between behaviors evoked by innocuous and noxious stimuli, and combined them to map the mouse pain state through statistical modeling and machine learning. To test the utility of this approach, we determined the pain state triggered by von Frey hairs (VFHs) and optogenetic activation of two nociceptor populations. Our method reliably assesses the \"pain-like\" probability for each mouse paw withdrawal reflex under all scenarios, highlighting the improved precision of using this high resolution behavior-centered composite methodology to determine the mouse pain state from reflexive withdrawal assays.

neuroscience↗

bioRxiv2017

Dynamic and Selective Low-Complexity Domain Interactions Revealed by Live-Cell Single-Molecule Imaging

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs) but how they perform transactivation functions remains unclear. Recent studies report that TF-LCDs can undergo hydrogel formation or liquid-liquid phase separation in vitro. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II) and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid reversible and multivalent LCD-LCD interactions occur between TFs and the Pol II machinery, which underpins a central mechanism for transactivation and plays a key role in gene expression and disease.

biophysics↗

bioRxiv2017

Stochastic Protein Labeling Enables Long-term Single Molecule Observation In Vivo

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

biophysics↗