Biology subjects

Chialastri, A.

Publications and source records attributed to Chialastri, A..

8 recordsLinked to original sources

bioRxiv2025

Photolabile oligonucleotides combined with topologically imposed light gradients enable spatially resolved single-cell transcriptomics and epigenomics

The organization of cells within a tissue plays a critical role in tuning cellular function. Several methods have recently been developed to capture the transcriptome of cells while retaining spatial information. However, these genome-wide sequencing methods typically lack the spatial resolution of individual cells and are confined to quantifying positional information within predefined lattice locations, thereby failing to capture large sections of a tissue outside these regions. Further, these methods are generally limited to profiling fixed cells with reduced mRNA capture efficiency compared to standard scRNA-seq. In addition, existing methods lack modularity and cross-platform compatibility, thereby limiting most of these techniques from jointly profiling the epigenetic and transcriptomic state of individual cells. To overcome these limitations, we present scSTAMP-seq (single-cell Spatial Transcriptomic And Multiomic Profiling), an approach that employs cholesterol-tagged photolabile oligonucleotides that incorporate into cell membranes, enabling us to "stamp" the position of cells using spatially imposed light gradients prior to tissue dissociation and single-cell sequencing. Applied to live cells, scSTAMP-seq efficiently captures spatially resolved single-cell transcriptomes at high resolution for all cells within a field of view. Further, we demonstrate that light patterning enables dynamic spatial resolution, including the ability to map the position of individual cells. Finally, we show that scSTAMP-seq is modular and can be seamlessly integrated with various downstream single-cell sequencing technologies. We demonstrate this by performing scRNA-seq using plate- and droplet-based methods, and by performing joint epigenome and transcriptome sequencing from the same cell while preserving positional information. Collectively, these results demonstrate that scSTAMP-seq is a sensitive and high-throughput technology for mapping single-cell transcriptomes and epigenomes at the spatial resolution of individual cells.

genomics↗

bioRxiv2025

Joint profiling of 5mC, 5hmC, and the transcriptome in single cells identifies factors responsible for genome-wide DNA methylation erasure in human primordial germ cell maturation

Reprogramming of DNA methylation plays a vital role in the establishment of cell identity during early mammalian development. To gain deeper mechanistic insights into this process requires capturing the entire dynamics of DNA methylation - both 5-methylcytosine (5mC) and its downstream oxidation product 5-hydroxymethylcytosine (5hmC) - in individual cells. Therefore, in this work, we report a new single-cell genome-wide strand-specific sequencing method, scMHT-seq, to jointly profile 5mC, 5hmC, and the transcriptome from individual cells. Using human embryonic stem cells (hESCs), we first show that scMHT-seq can accurately detect both 5mC and 5hmC from the same cell with minimal crosstalk in quantifying these two DNA modifications, and that the multi-modal measurements are in close agreement with individual measurements of 5mC and 5hmC in single cells. After establishing the method, we next applied scMHT-seq to gain insights into human primordial germ cell (hPGC) development. After specification, hPGCs undergo rapid global demethylation as they mature, and this reprogramming is critical for normal development of gametes. However, it has not been possible to fully overcome this key epigenetic barrier in culture, thereby limiting our ability to generate mature hPGC-like cells (hPGCLCs) and accomplish in vitro gametogenesis. To gain deeper understanding of the molecular factors involved in germ cell maturation, we applied scMHT-seq to an extended in vitro culture system for generating hPGCLCs and observed partial and heterogeneous erasure of the methylome across single cells that is mechanistically predominantly driven by passive demethylation due to reduced DNMT1-mediated maintenance methylation activity. Notably, we discover that hPGCLCs in extended culture can be transcriptionally classified into two distinct states, with one population enriched with more mature hPGCLCs exhibiting genome-wide loss of DNA methylation. Moreover, analysis of these two cell states identifies DND1 and SOX15 as two factors that are potentially key drivers of hPGCLC demethylation and maturation. Overall, we demonstrate that scMHT-seq is a robust and high-throughput technology that can provide insights into the mechanisms driving DNA methylation dynamics and their effect on cell states.

genomics↗

bioRxiv2024

YAP dysregulation triggers hypertrophy by CCN2 secretion and TGFβ uptake in human pluripotent stem cell-derived cardiomyocytes

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGF{beta}. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/597045v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@3357aforg.highwire.dtl.DTLVardef@c1c210org.highwire.dtl.DTLVardef@abb72forg.highwire.dtl.DTLVardef@187aa80_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity

Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the genome-wide activity of the DNA methylation maintenance machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.

genomics↗

bioRxiv2022

Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape

The Integrated Stress Response (ISR) is a conserved signaling network that detects cellular damage and computes adaptive or terminal outcomes. Understanding the mechanisms that underly these computations has been difficult because natural stress inputs activate multiple parallel signaling pathways and classical ISR inducers have pleiotropic effects. To overcome this challenge, we engineered photo-switchable control over the ISR stress sensor kinase PKR (opto-PKR), which allows virtual control of the ISR. Using controlled light inputs to activate opto-PKR we traced information flow in the ISR both globally, in the transcriptome, and for key ISR effectors. Our analyses revealed a biphasic, input-proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal ISR outcomes. Using this data, we constructed an ordinary differential equation (ODE) model of the ISR which predicted system hysteresis dependent on prior stress durations and that stress memory encoding may lead to resilience. Our results demonstrate that the input dynamics of the ISR encode information in stress levels, durations, and the timing between stress encounters.

systems biology↗

bioRxiv2022

Integrated single-cell sequencing reveals principles of epigenetic regulation of human gastrulation and germ cell development in a 3D organoid model

The emergence of different cell types and the role of the epigenome in regulating transcription is a key yet understudied event during human gastrulation. Investigating these questions remain infeasible due to the lack of availability of embryos at these stages of development. Further, human gastrulation is marked by dynamic changes in cell states that are difficult to isolate at high purity, thereby making it challenging to map how epigenetic reprogramming impacts gene expression and cellular phenotypes. To overcome these limitations, we describe scMAT-seq, a high-throughput one-pot single-cell multiomics technology to simultaneously quantify DNA methylation, DNA accessibility and the transcriptome from the same cell. Applying scMAT-seq to 3D human gastruloids, we characterized the epigenetic landscape of major cell types corresponding to the germ layers and primordial germ cell-like cells (hPGCLC). As the identity of the progenitors that give rise to human PGCLCs remain unclear, we used this system to discover that the progenitors emerge from epiblast cells and show transient characteristics of both amniotic- and mesoderm-like cells, before getting specified towards hPGCLCs. Finally, as cells differentiate along different lineages during gastrulation, we surprisingly find that while changes in DNA accessibility are tightly correlated to both upregulated and downregulated genes, reorganization of gene body DNA methylation is strongly related to only genes that get downregulated, with genes that turn on displaying a lineage trajectory-dependent correlation with DNA methylation. Collectively, these results demonstrate that scMAT-seq is a high-throughput and sensitive approach to elucidate epigenetic regulation of gene expression in complex systems such as human gastrulation that are marked by rapidly transitioning cell states.

genomics↗

bioRxiv2019

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

DNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

genomics↗

bioRxiv2019

Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells

The epigenome plays a critical role in regulating gene expression in mammalian cells. However, understanding how cell-to-cell heterogeneity in the epigenome influences gene expression variability remains a major challenge. Here we report a novel method for simultaneous single-cell quantification of protein-DNA contacts with DamID and transcriptomics (scDamID&T). This method enables quantifying the impact of protein-DNA contacts on gene expression from the same cell. By profiling lamina-associated domains (LADs) in human cells, we reveal different dependencies between genome-nuclear lamina (NL) association and gene expression in single cells. In addition, we introduce the E. coli methyltransferase, Dam, as an in vivo marker of chromatin accessibility in single cells and show that scDamID&T can be utilized as a general technology to identify cell types in silico while simultaneously determining the underlying gene-regulatory landscape. With this strategy the effect of chromatin states, transcription factor binding, and genome organization on the acquisition of cell-type specific transcriptional programs can be quantified.

molecular biology↗