Biology subjects

Su, D.

Publications and source records attributed to Su, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Cooperative IDR-folded domain interplay governs HP1α phase separation and underlies biphasic regulation by pathogenic poly-PR

HP1 undergoes liquid-liquid phase separation (LLPS), yet the prevailing model, which attributes condensation almost entirely to intrinsically disordered regions (IDRs), leaves the role of its folded domains unresolved. Using solution and solid-state NMR, we show that HP1 LLPS is instead governed by reciprocal interplay between IDRs and folded domains. Within the condensed phase, solid-state NMR reveals a dynamic hierarchy: the chromoshadow domain (CSD) assembles into polymorphic oligomers that form rigid cores, while the phosphorylation sites on the N-terminal extension (NTE) remain highly mobile, challenging their proposed role as stable multivalent hubs. Phosphorylation of the NTE instead promotes LLPS primarily by increasing local rigidity and releasing the NTE from the chromodomain (CD) to relieve autoinhibition, with electrostatic charge contributing secondarily. The C-terminal extension (CTE) suppresses phase separation through two routes: weakening the association between the hinge region (HR) and the CSD, and inhibiting interactions between CSD dimers. ALS-associated poly-PR targets this network at the NTE and exerts concentration-dependent biphasic regulation, promoting LLPS at low concentrations via multivalent bridging and dissolving condensates at high concentrations via saturation, a switch recapitulated in cells and in mouse brain. These findings reframe HP1 condensation from an IDR-driven process to one cooperatively governed by IDR-folded domain interplay.

biochemistry↗

bioRxiv2026

Testing Reversibility of Endosymbiotic Gene Transfer between Chloroplast and Nucleus

Most proteins targeted to the organelles of endosymbiotic origin are encoded in the nuclear genome, placing them under the regulatory dominance of the nucleus. For photosynthetic eukaryotes, nuclear-encoded chloroplast proteins arise via two routes: First, genes of cyanobacterial origin were relocated to the nucleus through endosymbiotic gene transfer (EGT). Second, proteins of eukaryotic origin emerged to support chloroplast function and structure. These proteins are reimported into the chloroplast via an import machinery. Reversing the transfer of such genes from the nucleus to the chloroplast genome may offer insights into chloroplast regulation and evolution. In this study, we established a highly efficient and accessible electroporation protocol for chloroplast transformation in the green alga Chlamydomonas reinhardtii, and used it to reverse-transfer two nuclear-encoded genes encoding proteins arising via the two routes described above: the cyanobacteria-derived chloroplast division protein FtsZ1 and the Rubisco-linker EPYC1 of eukaryotic origin. Regardless of origin, both chloroplast-encoded FtsZ1 and EPYC1 showed proper localization and functionality comparable to their nuclear-encoded counterparts. Together, our study provides a robust protocol for chloroplast transformation, a platform for investigating the evolutionary drivers of EGT, and a foundation for advancing chloroplast bioengineering. SIGNIFICANCE STATEMENTO_LIEndosymbiotic gene transfer has resulted in the mass migration of genes from the chloroplast genome to the nuclear genome. Reversing the gene transfer could reveal the evolutionary significance of genome partitioning. C_LIO_LIUsing the green alga Chlamydomonas reinhardtii, this study developed an efficient, electroporation-based protocol for chloroplast transformation. Relocating the genes encoding two chloroplast-targeted proteins, FTSZ1 and EPYC1, to the chloroplast genome showed that the proteins maintained normal localization and function. C_LIO_LIThe established transformation protocol facilitates systematic testing of reverse gene transfer to elucidate the potential evolutionary advantages of genome partitioning and opens new avenues for chloroplast bioengineering. C_LI

cell biology↗

bioRxiv2026

Septin-mediated coupling of protein import and division during chloroplast evolution

Chloroplast biogenesis depends on both protein import and organelle division, yet how their coordination emerged during evolution remains unclear. Here, we show that the single septin SEP1 links these pathways in the green alga Chlamydomonas reinhardtii. SEP1 forms a filamentous network on the chloroplast envelope during interphase and reorganizes into a ring at the chloroplast division site during cytokinesis. Loss of SEP1 selectively impairs import of chloroplast-division proteins and causes mispositioning of the division ring, without impairing bulk chloroplast protein import. SEP1 physically associates with outer-envelope TOC GTPases through evolutionarily related GTPase domains. Phylogenetic analysis places TOC GTPases within an algal septin-derived clade, and heterologous expression of SEP1 in land plants, in which septins are absent, shows conservation of its chloroplast targeting and TOC binding. Together, these findings identify septins as coordinators of plastid protein import and division and suggest that this coupling emerged early in chloroplast evolution.

cell biology↗

bioRxiv2026

G-quadruplexes regulate chromatin accessibility and gene expression in Bloom Syndrome

Bloom Syndrome (BS) is a recessive genetic disorder characterized by hyper-recombination and genome instability. It is caused by mutations in BLM, which encodes a conserved RecQ helicase that unwinds various aberrant DNA structures. One such structure is DNA G-quadruplexes (G4s), which have versatile regulatory potential in chromatin organization and gene expression. However, whether G4 profiles are altered in BS and how G4s contribute to disease-associated molecular changes remain unclear. Here, we profiled chromatin accessibility and gene expression using ATAC-seq and RNA-seq and mapped endogenous G4 by ChIP-seq in wild type (WT) and BS cell lines. We observed that in BS cell lines, differential G4 formation positively correlated with both differential chromatin accessibility and gene expression. To test the direct involvement of G4s in the molecular phenotypes in BS, we applied pyridostatin, a G4-stabilizing molecule, in WT cells and showed that G4 stabilization partially recapitulated BS-associated molecular phenotypes. Additionally, we found that regions with increased chromatin accessibility in BS individuals in a family were also enriched for G4-forming sequences. Together, our data substantiate a regulatory role for G4s in Bloom syndrome and support a molecular model in which unresolved G4s in BLM- /- cells enhance chromatin accessibility, thereby promoting gene expression. These findings reveal an expanded regulatory function of BLM mediated through G4 structures and previously underappreciated role of G4s in the molecular etiology of BS.

genomics↗

bioRxiv2026

Isthmin-1 is a Key Regulator of Induced Pluripotent Stem Cell-Derived Cardiomyocytes Maturation through Activation of p53 Signaling

AimsHuman induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibit an immature structural and metabolic phenotype that limits their utility for cardiac disease modeling and regenerative therapy. Although multiple extrinsic strategies have been proposed to enhance iPSC-CM maturation, intrinsic molecular regulators governing this process remain incompletely defined. This study aimed to identify and characterize a novel molecular factor that promotes cardiomyocyte maturation. Methods and ResultsCross-species transcriptomic analyses comparing adult versus fetal hearts and hypertrophic cardiomyopathy versus healthy myocardium identified Isthmin-1 (ISM1) as a conserved maturation-associated gene. In human iPSC-CMs, ISM1 overexpression enhanced sarcomere organization, mitochondrial oxidative phosphorylation, ATP production, and calcium-handling properties, whereas ISM1 knockdown impaired these maturation features. RNA sequencing revealed global transcriptional reprogramming toward an adult-like state, characterized by suppression of cell-cycle-related gene programs and activation of oxidative metabolic pathways. KEGG pathway enrichment, GSVA, and GSEA consistently identified p53 signaling as the most significantly activated pathway in ISM1-overexpressing iPSC-CMs. Mechanistically, ISM1 directly interacted with p53, enhanced its protein stability, promoted nuclear localization, and increased transcription of p53 downstream targets involved in metabolic remodeling and cell-cycle exit. Pharmacological inhibition of p53 abolished ISM1-induced structural and metabolic maturation, demonstrating that ISM1 promotes cardiomyocyte maturation in a p53-dependent manner. ConclusionsISM1 is a previously unrecognized molecular driver of cardiomyocyte maturation that promotes structural, metabolic, and functional maturation of iPSC-CMs through activation of p53-dependent transcriptional programs. These findings provide new mechanistic insight into cardiomyocyte maturation and identify ISM1 as a promising target for improving the maturity of stem cell-derived cardiomyocytes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/698535v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@15daca3org.highwire.dtl.DTLVardef@f1396dorg.highwire.dtl.DTLVardef@f13400org.highwire.dtl.DTLVardef@18d0b41_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Cortical potentials evoked by stimulation of cervical vagus vs. auricular nerve: a comparative, parametric study in nonhuman primates

Stimulation of sensory vagal pathways is typically delivered via invasive, cervical vagus nerve stimulation (cVNS) or noninvasive, trans-auricular nerve stimulation (taNS). While both methods are investigated therapeutically, their effects on brain physiology remain poorly understood, hindering mechanistic understanding and stimulus optimization. In 6 awake nonhuman primates, we recorded cortical vagal-evoked potentials (VEPs) from subdural electrodes placed in prefrontal, sensorimotor and parietal cortical areas, in response to cVNS or taNS. Across 478 different taNS and cVNS protocols, we varied stimulation side, intensity, frequency, pulse count, and pulse width (PW) and assessed independent effects on amplitude and latency of early (EC; 30-100 ms), intermediate (IC; 101-200 ms) and late components (LC; 201-500 ms) of VEPs. Fixed and random effects of stimulation parameters and subjects, respectively, on VEP measurements, were assessed using a linear mixed-effects model. Overall, cVNS elicits more robust VEPs than taNS, with larger EC, IC and LC amplitudes in both hemispheres. cVNS-elicited ECs and LCs are largest in PFC and PC areas, whereas ICs are largest in SM areas. On the other hand, taNS generally does not elicit area-specific responses. cVNS-elicited ECs have slower latency than ta-NS elicited ECs. Higher stimulation frequencies and intensities and a longer pulse width elicit larger ECs and ICs for cVNS, and to some extent for taNS. Both short and long cVNS trains elicit stronger ECs, and long trains elicit slower ICs. Earlobe stimulation elicits VEPs that partially overlap with those from taNS. In conclusion, cVNS and taNS elicit cortical VEPs in a manner consistent with distinct engagement of ascending vagal pathways, and with similarities and differences in the effects of stimulation parameters on evoked responses.

neuroscience↗

bioRxiv2025

Inhibitory GABAergic neuron loss due to oxidative damage during ex vivo acute brain slice preparation influences genesis and dynamics of epileptiform activity

Ex vivo acute brain slice is a popular technique in neuroscience research. Since its inception five decades ago, many variations of the brain slice preparation method have emerged. While all variations are currently used by many labs throughout the world, no study has comprehensively examined the impact of these variation on the quality of the acute brain slice preparation. In this study, we comprehensively examined the effect of animal sacrifice methods (decapitation or transcardial perfusion) and cutting solution (normal or sucrose artificial cerebrospinal fluid) on brain slice preparation. Neuronal population was quantified by immunohistochemistry against various neuronal markers. Neuronal dynamics was evaluated by in vitro electrophysiology using two acute epilepsy models - zero-magnesium and 4-aminopyridine. To modulate the stress incurred in acute brain slice preparation, we administered antioxidants or HIF-1 inhibitor, chrysin, to the cutting solution. The method of brain slice preparation significantly affected the quality of the brain slice preparation. In general, the combination of transcardial perfusion and sucrose artificial cerebrospinal fluid produces the optimal brain slice preparation. This is evidenced by a preservation of inhibitory GABAergic neurons in slices prepared with this combination. We subsequently found that this loss of inhibitory GABAergic neurons significantly influenced the genesis and dynamics of induced acute epileptiform activity. The slices with preserved inhibition had less successful induction of acute epileptiform activity and a seizure-like event that is typical of those induced in brain with preserved inhibition. Finally, we found that loss of inhibitory GABAergic neurons during brain slice preparation is primarily due to oxidative damage. Limiting oxidative stress is an effective neuroprotection strategy to prevent loss of inhibition in brain slice preparation. In conclusion, consideration of brain slice preparation method is crucial in preservinginhibitory GABAergic neurons and the degree of inhibition in ex vivo acute brain slice preparation.

neuroscience↗

bioRxiv2025

Targeted Ablation of Canonical Disulfide Bonds Mitigates TCR Mismatching in Engineered T Cells

T cell receptor-engineered T (TCR-T) cell therapy holds distinct advantages over chimeric antigen receptor (CAR)-T cell therapy in treating solid tumors by exploiting intracellular targets through MHC-mediated antigen presentation. Despite this potential, only one TCR-T therapy has received FDA approval to date. A key challenge lies in chain mispairing between exogenous therapeutic TCR chains and endogenous TCR chains, which compromises membrane localization efficiency and elevates off-target toxicity risks. While multiple engineering strategies have been developed to enhance TCR surface expression, our systematic evaluation using a novel mispairing assessment platform revealed that conventional approaches may paradoxically exacerbate chain mismatching. Through targeted cysteine mutations that disrupt the native interchain disulfide bonds in TCR constant domains, we achieved a substantial reduction in exogenous-endogenous TCR mispairing. Importantly, these structural modifications preserved both high surface expression level and potent tumor-specific cytotoxicity. This innovative engineering approach establishes a critical foundation for clinical-grade manufacturing of TCR-T therapies with enhanced safety profiles.

immunology↗

bioRxiv2025

Concentration- and time-dependence of phosphorylation events stimulated by fibroblast growth factor-4 (FGF-4) in Rama 27 fibroblasts

Gradients of FGFs are an essential feature of many developmental processes. Thus, FGF2 stimulates different responses at high and low concentrations, while FGF4 gradients are critical, e.g., in limb development., but it is not known if responses to FGF4 differ in a concentration-dependent manner. Employing rat mammary (Rama) 27 fibroblasts, we therefore measured the FGF4 concentration- and time-dependence of the simulation of phosphorylation of FRS2, MAPK1 and MAPK3 which are downstream of the FGF receptor (FGFR1c). At 10 pg/mL FGF4 caused a very weak phosphorylation of FRS2 at Y435 and Y196 and a stronger one of MAPK1 and MAPK3 that oscillated with maximum levels after 30 min and 240 min stimulation. The phosphorylation of these proteins at 1 ng/mL FGF4 was considerably stronger and showed a similar oscillation. At 10 ng/mL FGF4, the phosphorylation of FRS2 reached an early peak after 5 min, declined at 15 min and then rose again at 30 min before declining to the end of the time-course at 240 min, whereas the phosphorylation of MAPK1 and MAPK3 was strong after 5 minutes, reached a maximum after 30 minutes and then declined gradually. At 1 {micro}g/mL and 3 {micro}g/mL FGF4 after 15 min and 60 min the phosphorylation of FRS2, MAPK1 and MAPK3 was much lower at 60 min compared to that observed at lower concentrations of FGF4. The data indicate that at low concentrations FGF4 elicits an oscillatory response at the level of phosphorylation of FRS2 and of MAPK1 and MAPK3, whereas a bell-shaped dose response may occur at the highest concentrations of FGF4. The addition of exogenous heparin, an effective mimic of endogenous heparan sulfate, suggests that there may be an influence of the interaction of FGF4 with pericellular matrix and the dose-and time-dependence of the phosphorylation of FRS2 and MAPK1and MAPK3.

biochemistry↗

bioRxiv2025

Tbr2-Dependent Parallel Pathways Regulate the Development of Distinct ipRGC Subtypes

The intrinsically photosensitive retinal ganglion cells (ipRGC) are the conduit between the retinas and brain regions responsible for non-image-forming and image-forming vision. In mice, six ipRGC subtypes have been discovered based on morphological characteristics, functions, and molecular profiles. All ipRGCs arise from Tbr2-expressing RGCs during developmental stages and subsequently diverge and differentiate into the six mature, distinct subtypes in adult retinas. However, the cellular and molecular mechanisms controlling the formation and maturation of the six ipRGC subtypes remain elusive. Here, we demonstrate that two Tbr2-dependent transcription factors, Iroquois-related homeobox 1 (Irx1) and T-box containing factor 20 (Tbx20), are key downstream transcription factors guiding lineage segregations of Tbr2-expressing RGC into distinct adult ipRGC subtypes. Both factors also control Opn4 expression. Irx1 is expressed in the M3, M4, and M5 subtypes, while Tbx20 is predominantly expressed in M1, M2, M6, and subgroups of M3 and M5. When Irx1 is ablated during retinal development, Opn4 expression is significantly reduced in the M3, M4, and M5 ipRGC groups; however, the formation of Irx1-expressing ipRGCs is not affected. In contrast, when Tbx20 is deleted, a significant number of Tbx20-expressing cells fail to develop while Opn4 expression is down-regulated. These findings reveal two parallel transcription cascades downstream of Tbr2 for controlling ipRGC subtype formation, fate divergence, and maintenance in the adult retina.

developmental biology↗

bioRxiv2025

High-throughput single-cell DNA methylation and chromatin accessibility co-profiling with SpliCOOL-seq

DNA methylation and chromatin accessibility are fundamental epigenetic mechanisms that orchestrate gene expression programs, define cellular states, and drive developmental trajectories. scCOOL-seq has enabled simultaneously measuring the two modalities in the same single cells, but in quite a low throughput manner. We present single-cell split-pool ligation-based multi-omics sequencing technology (SpliCOOL-seq), which improves the throughput to thousands of cells by combining split-pool ligation based single-cell indexing after in situ tagmentation with universal Tn5 transposase and scCOOL-seq. SpliCOOL-seq achieved higher sensitivity than previous high throughput single-cell DNA methylation sequencing methods and can clearly distinguish different lung cancer cells based on both genetic and multiple epigenetic modalities. We show that the two DNMT inhibitors, 5-Azacitidine and Decitabine, both cause large scale demethylation but in distinct patterns. Applied to the primary lung tumor, SpliCOOL-seq clearly captured subclones within the tumor lesion and revealed candidate genes related to tumorigenesis. Furthermore, we presented the first report on the heterogeneity of scDNAm age acceleration among tumor subclones as predicted from a single-cell perspective. In conclusion, SpliCOOL-seq achieves parallel profiling of whole genome DNA methylation and chromatin accessibility in the same individual cells in a high-throughput manner and is hopefully used to illustrate regulatory interactions under different cell states.

genetics↗

bioRxiv2024

Single-Cell full-length Isoform Sequencing Unveils Transcriptional Dysregulation in Autism Spectrum Disorder During Cerebral Cortex Development

Regulation of RNA splicing is pivotal in neural development, with established gene isoform expression patterns (1, 2). However, the specific roles of isoform diversity across cell types in both healthy and diseased brains warrant further investigation. Here, we employed a combination of metabolic RNA labeling using 4-thiouridine (4sU) and long-read sequencing based single-cell full-length transcriptome sequencing to capture newly synthesized transcripts within the developing mouse cortices. This approach allowed us to identify predetermined cell states supported by new RNAs and the driving isoforms of transcription factors that regulate the development of deep-and upper-layer neurons. Through detailed single-cell isoform expression analysis, we discovered novel cell type-specific isoforms and uncovered isoform switch events that modulate neuron differentiation. Additionally, we investigated isoform regulation associated with Autism Spectrum Disorder (ASD) during embryonic development of BTBR T+ Itpr3tf (BTBR) mice. Notably, our findings indicate a premature emergence of cortical projection neurons (CPNs) with an immature identity in ASD-affected cortices. These CPNs exhibit the highest degree of differential transcript usage (DTU), significantly overlapping with their cell type markers and being enriched in RNA splicing processes. Exon inclusion was significantly enriched in ASD and the related RNA binding proteins (RBPs) were extracted, nearly 60% of which have been reported as ASD risk genes. Lastly, we revealed a reduction in isoform diversity in ASD, potentially linked to H3K27ac dysregulation in the associated genes. Collectively, our study marks a substantial advancement in understanding the molecular basis of cortical development and function, paving the way for future studies on neurodevelopmental disorders.

developmental biology↗

bioRxiv2024

Genetic determinants of distinct CD8+ α/β-TCR repertoires in the genus Mus

The adaptive immune systems efficacy relies on the diversity of T cell receptors and the ability to distinguish between self and foreign antigens. Analysis of the paired heterodimeric {beta}-TCR chains of individual T cells requires single-cell resolution, but existing single-cell approaches offer limited coverage of the vast TCR repertoire diversity. Here we introduce CITR-seq, a novel, instrument-free, high-throughput method for single-cell TCR sequencing with >88% {beta}-TCR pairing precision. We analyzed the TCR repertoires of CD8+ T cells originated from 32 inbred mice using CITR-seq, comprising four evolutionary divergent sister species and their F1 hybrids. Overall, we identified more than 5 million confidently paired TCRs. We found that V(D)J gene usage patterns are highly specific to the genotype and that V{beta}-gene usage is strongly impacted by thymic selection. Using F1 hybrids, we show that differences in gene segment usage across species are likely caused by cis-acting factors prior to thymic selection, which imposed strong allelic biases. At the greatest divergence, this led to increased rates of TCR depletion through rejection of particular V{beta}-genes. TCR repertoire overlap analysis across all mice revealed that sharing of identical paired CDR3 amino acid motifs is four times more frequent than predicted by random pairing of TCR and TCR{beta} chains, with significantly increased sharing rates among related individuals. Collectively, we show that beyond the stochastic nature of TCR repertoire generation, genetic factors contribute significantly to the shape of an individuals repertoire.

genomics↗

bioRxiv2024

Distinct evolution at TCRα and TCRβ loci in the genus Mus

T cells recognize an immense spectrum of pathogens to initiate immune responses by means of a large repertoire of T cell receptors (TCRs) that arise from somatic rearrangements of variable, diversity and joining gene segments at the TCR loci. These gene segments have emerged from a limited number of ancestral genes through a series of gene duplication events, resulting in a greatly variable number of such genes across different species. Apart from the complete V(D)J gene annotations in the human and mouse reference assemblies, little is known about the structure of TCR loci in other species. Here, we performed a comprehensive comparison of the TCR and TCR{beta} gene segment clusters in mice and three of its closely related sister species. We show that the TCR variable gene cluster is frequently rearranged, leading to deletions and sequence inversions in this region. The resulting complexity of TCR loci severely complicates the assembly of these loci and the annotation of gene segments. By jointly utilizing genomic and transcriptomic data, we show that in Mus musculus castaneus the variable gene cluster at the locus has undergone a recent major locus contraction, leading to the loss of 74 variable gene segments. Additionally, we validated the expression of functional variable genes, including atypical ones with inverted orientation relative to other such segments. Disentangling the fine-scale structure of TCR loci in different species can provide valuable insights in the evolution and diversity of TCR repertoires.

genomics↗

bioRxiv2024

A unique mechanism of snRNP core assembly

The assembly of spliceosomal snRNP cores involves seven Sm proteins (D1/D2/F/E/G/D3/B) forming a ring around each snRNA and typically requires several essential assembly chaperones, particularly the SMN-Gemins complex implicated in spinal muscular atrophy (SMA). Strikingly, in budding yeast, snRNP core assembly only involves Brr1, a nonessential homolog of Gemin2. To unravel this enigma, we systematically investigated snRNP core assembly in budding yeast using biochemical and genetic approaches. We discovered two distinct pathways in budding yeast: chaperone-mediated and direct. The chaperone-mediated pathway involves two proteins, Brr1 and a novel protein, Lot5, but is inefficient. Lot5 binds D1/D2/F/E/G to form a heterohexameric ring (6S). Brr1 binds D1/D2/F/E/G and 6S but cannot displace Lot5 to facilitate assembly. Disruption of both BRR1 and LOT5 genes caused mild growth retardation, but LOT5 overexpression substantially impeded growth. The direct pathway uniquely involves F/E/G as a trimer and a stable D1/D2/F/E/G intermediate complex, explaining the non-essentiality of assembly chaperones. These findings unveil a unique assembly mechanism of snRNP cores, illuminate the evolution of assembly chaperones, and suggest avenues for studying SMA pathophysiology.

molecular biology↗

bioRxiv2024

Copy number normalization distinguishes differential signals driven by copy number differences in ATAC-seq and ChIP-seq

A common objective across ATAC-seq and ChIP-seq analyses is to identify differential signals across contrasted conditions. However, in differential analyses, the impact of copy number variation is often overlooked. Here, we demonstrated copy number differences among samples could drive, if not dominate, differential signals. To address this, we propose a pipeline featuring copy number normalization. By comparing the averaged signal per gene copy, it effectively segregates differential signals driven by copy number differences from other factors. Further applying it to Down syndrome, we unveiled distinct dosage-dependent and -independent changes on chromosome 21. Thus, we recommend normalization as a general approach.

genomics↗

bioRxiv2024

Flexible and high-throughput simultaneous profiling of gene expression and chromatin accessibility in single cells

Gene regulation underpins development and is an intricate biological process involving transcription, typically at promoters within accessible chromatin. To understand cell-type specific regulatory networks, the ability to capture both transcription and chromatin accessibility simultaneously is crucial. However, joint measurements are technically challenging and current methodologies still face adoption challenges. Here, we present easySHARE-seq, an improvement on SHARE-seq, for the simultaneous measurement of ATAC- and RNA-seq in single cells. We address several limitations of the previous method by improving the barcode and streamlining the protocol. As a result, easySHARE-seq libraries have a usable sequence of up to 300bp (+200bp increase), making it suitable for e.g. investigation of allele-specific signals or variant discovery. Furthermore, easySHARE-seq libraries do not require a dedicated sequencing run thus saving costs. We applied easySHARE-seq to murine liver nuclei and recovered 19,664 nuclei with joint chromatin and expression profiles. By benchmarking against other combinatorial indexing-based techniques, we showed we can recover over 1.5 fold more transcripts per cell while retaining high scalability and low cost. To showcase our method, we identified cell types, exploited the multiomic measurements to link cis-regulatory elements to their target genes and investigated liver-specific micro-scale changes. We conclude that easySHARE-seq improves upon previous methods and can produce high-quality multiomic datasets. We expect it to be applicable to a wide range of study designs.

genomics↗

bioRxiv2023

Activation of a cortical neurogenesis transcriptional program during NEUROD1-induced astrocyte-to-neuron conversion

NEUROD1-induced astrocyte-to-neuron (AtN) conversion has garnered significant attention as a potential therapeutic intervention to neurological disorders. To gain insight into the molecular regulations underlying this neuronal reprogramming process, we applied single-cell multiomics analyses on in vitro ND1-induced AtN conversion to systematically investigate how ND1 changed the fate of astrocytes at transcriptomic and epigenetic levels. Our findings reveal that the initial immature astrocytes go through an intermediate state where both astrocytic and neuronal genes are activated at early stage of AtN conversion. ND1 directly reshapes the chromatin accessibility landscape of astrocytes to that of neurons, promoting expression of endogenous Neurod1 and other neurogenic genes such as Hes6, Insm1 etc. Interestingly, cell proliferation status is highly correlated with conversion rate, and inhibition of cell division significantly reduces the conversion ratio. Moreover, in comparison with another AtN reprogramming transcription factor, ASCL1, external ND1 can activate endogenous Neurod1 and directly promote neuronal gene transcription; whereas external ASCL1 hardly activates endogenous Ascl1, leading to slower and inefficient conversion. Together, our studies demonstrate that in vitro AtN conversion mimics neurogenic transcriptional program in embryonic neurogenesis.

neuroscience↗