Biology subjects

Hernando-Herraez, I.

Publications and source records attributed to Hernando-Herraez, I..

7 recordsLinked to original sources

bioRxiv2024

Using CRISPR barcoding as a molecular clock to capture dynamic processes at single-cell resolution

Biological processes are fundamentally dynamic, yet existing methods for capturing these temporal changes are limited. We present scDynaBar, a novel approach that integrates CRISPR-Cas9 dynamic barcoding with single-cell sequencing to enable the recording of temporal cellular events. In this system, genetic barcodes accumulate mutations over a 25-day period and then are sequenced together with the transcriptome of each single cell. We propose that this gradual accumulation of genetic diversity can be exploited to create an ordered record of a cellular event. We apply this approach to track the transition from a pluripotent state to a two-cell (2C)-like state in mouse embryonic stem cells (mESCs). The results provide compelling evidence for the transient nature of the 2C-like state. Additionally, our system shows consistent mutation rates across diverse cell types in a mouse gastruloid model, underscoring its robustness and versatility across various biological contexts. This technique not only improves our ability to study single-cell dynamics but also creates new opportunities for recording other temporal signals--in other words, using dynamic barcoding as a molecular clock in individual cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/618192v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1665361org.highwire.dtl.DTLVardef@ec3114org.highwire.dtl.DTLVardef@8a3123org.highwire.dtl.DTLVardef@a0235f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Multi-omic rejuvenation of human cells by maturation phase transient reprogramming

Ageing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Somatic cell reprogramming, the process of converting somatic cells to induced pluripotent stem cells (iPSCs), can reverse these age-associated changes. However, during iPSC reprogramming somatic cell identity is lost, and can be difficult to reacquire as re-differentiated iPSCs often resemble foetal rather than mature adult cells. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of reprogramming, which suggests full iPSC reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first "maturation phase transient reprogramming" (MPTR) method, where reprogramming factors are expressed until this rejuvenation point followed by withdrawal of their induction. Using dermal fibroblasts from middle age donors, we found that cells reacquire their fibroblast identity following MPTR, possibly as a result of persisting epigenetic memory at enhancers. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome, including H3K9me3 histone methylation levels and the DNA methylation ageing clock, was rejuvenated to a similar extent. The magnitude of rejuvenation instigated by MTPR is substantially greater than that achieved in previous transient reprogramming protocols. MPTR fibroblasts produced youthful levels of collagen proteins, suggesting functional rejuvenation. Overall, our work demonstrates that it is possible to separate rejuvenation from pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies. HighlightsO_LIWe developed a novel method by which human fibroblasts are reprogrammed until the maturation phase of iPSCs and are then returned to fibroblast identity C_LIO_LIDNA methylation memory in fibroblast enhancers may allow recovery of cell identity when fibroblast gene expression programmes are already extinct C_LIO_LIMolecular measures of ageing including transcriptome and DNA methylation clocks and H3K9me3 levels reveal robust and substantial rejuvenation C_LIO_LIFunctional rejuvenation of fibroblasts by MPTR is suggested by reacquisition of youthful levels of collagen proteins C_LI

molecular biology↗

bioRxiv2020

Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation

Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.

molecular biology↗

bioRxiv2020

Specialisation and plasticity in a primitively social insect

Biological systems not only have the remarkable capacity to build and maintain complex spatio-temporal structures in noisy environments, they can also rapidly break up and rebuild such structures. How such systems can simultaneously achieve both robust specialisation and plasticity is poorly understood. Here we use primitive societies of Polistes wasps as a model system where we experimentally perturb the social structure by removing the queen and follow the re-establishment of the social steady state over time. We combine a unique experimental strategy correlating time-resolved measurements across vastly different scales with a theoretical approach. We show that Polistes integrates antagonistic processes on multiple scales to distinguish between extrinsic and intrinsic perturbations and thereby achieve both robust specialisation and rapid plasticity. The long-term stability of the social structure relies on dynamic DNA methylation which controls transcriptional noise. Such dynamics provide a general principle of how both specialization and plasticity can be achieved in biological systems. One Sentence SummaryA primitive social insect simultaneously achieves specialisation and plasticity by integrating antagonistic dynamics on different scales. HighlightsO_LIWe employ a unique experimental approach correlating dynamics of societies, individuals, and epigenetic gene regulation C_LIO_LIA social insect simultaneously achieves specialisation and plasticity by integrating antagonistic processes on different spatial scales C_LIO_LIRegulation of population-level noise by DNA methylation ensures long-term stability of phenotypic specialisation C_LI

biophysics↗

bioRxiv2019

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming processes and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics to systematically probe candidate regulators of ZGA. We screened 230 epigenetic and transcriptional regulators by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNA-sequencing (scRNA-seq) of CRISPRa-perturbed cells, we generated approximately 200,000 single-cell transcriptomes, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using integrative dimensionality reduction of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 44 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. Upon upregulation of these factors, including the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, ESCs adopt an early embryonic-like state. Supporting their roles as ZGA regulators, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes, however, overexpression of Dppa2 in Smarca5 knock-out ESCs, but not vice versa, rescues ZGA-like expression, suggesting that Smarca5 regulates ZGA upstream and via Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA.\n\nHighlightsO_LIFirst large-scale screen combining pooled CRISPRa with scRNA-seq.\nC_LIO_LIMulti-omics factor analysis identifies a ZGA-like signature for 44 of the candidate regulators.\nC_LIO_LIDppa2, Smarca5 and Patz1 were validated as strong inducers of ZGA gene expression.\nC_LIO_LISmarca5 regulates zygotic genome activation in a Dppa2-dependent manner.\nC_LI

developmental biology↗

bioRxiv2018

Ageing affects DNA methylation drift and transcriptional cell-to-cell variability in muscle stem cells

Age-related tissue alterations have been associated with a decline in stem cell number and function1. Although increased cell-to-cell variability in transcription or epigenetic marks has been proposed to be a major hallmark of ageing2-5, little is known about the molecular diversity of stem cells during ageing. Here, by combined single-cell transcriptome and DNA methylome profiling in mouse muscle stem cells, we show a striking global increase of uncoordinated transcriptional heterogeneity together with context-dependent alterations of DNA methylation with age. Importantly, promoters with increased methylation heterogeneity are associated with increased transcriptional heterogeneity of the genes they drive. Notably, old cells that change the most with age reveal alterations in the transcription of genes regulating cell-niche interactions. These results indicate that epigenetic drift, by accumulation of stochastic DNA methylation changes in promoters, is a substantial driver of the degradation of coherent transcriptional networks with consequent stem cell functional decline during ageing.

genomics↗

bioRxiv2018

A comparison of gene expression and DNA methylation patterns across tissues and species

Previously published comparative functional genomic data sets from primates using frozen tissue samples, including many data sets from our own group, were collected and analyzed using non-optimal study designs and analysis approaches. In addition, when samples from multiple tissues were studied in a comparative framework, individual and tissue were confounded. We designed a multi-tissue comparative study of gene expression and DNA methylation in primates that minimizes confounding effects by using a balanced design with respect to species, tissues, and individuals. We also developed a comparative analysis pipeline that minimizes biases due to sequence divergence. We thus present the most comprehensive catalog of similarities and differences in gene expression and methylation levels between livers, kidneys, hearts, and lungs, in humans, chimpanzees, and rhesus macaques. We estimate that overall, only between 7 to 11% (depending on the tissue) of inter-species differences in gene expression levels can be accounted for by corresponding differences in promoter DNA methylation. However, gene expression divergence in conserved tissue-specific genes can be explained by corresponding inter-species methylation changes more often. We end the paper by providing recommendations for effective study design and best practices for meta-data recording for comparative functional genomic studies in primates.

genomics↗