Biology subjects

Folkes, L.

Publications and source records attributed to Folkes, L..

6 recordsLinked to original sources

bioRxiv2024

Environmentally induced variation in sperm sRNAs is linked to gene expression and transposable elements in zebrafish offspring

Environmental factors affect not only paternal condition but may translate into the following generations where sperm-mediated small RNAs (sRNAs) can contribute to the transmission of paternal effects. SRNAs play a key role in the male germ line in genome maintenance and repair, and particularly in response to environmental stress and the resulting increase in transposable element (TE) activity. Here, we investigated how the social environment (high competition, low competition) of male zebrafish Danio rerio affects small RNAs in sperm and how these are linked to gene expression and TE activity in their offspring. In a first experiment, we collected sperm samples after exposing males to each social environment for two weeks to test for differentially expressed sperm micro-(miRNA) and piwi-interacting RNAs (piRNA). In a separate experiment, we performed in vitro fertilisations after one two-week period using a split-clutch design to control for maternal effects and collected embryos at 24 hours to test for differentially expressed genes and transposable elements. We developed new computational prediction tools to link sperm sRNAs with differentially expressed TEs and genes in the embryos. Our results support the idea that the molecular stress response in the male germ line has significant down-stream effects on the molecular pathways, and we provide a direct link between sRNAs, TEs and gene expression. Author summaryThe discovery that sperm transmit more than just the fathers genome to the next generation is relatively recent and the potential implications are far reaching. What we do not know is whether these non-genetic components contained in sperm play a role in adaptation to changing environments or whether they are simply a result of the fathers stress response that also affects the germ cells. Environmentally induced stress is known to trigger a response to maintain and repair the germ cells to ensure the production of high quality sperm and key factors in this response are small RNAS. Small RNAs are guardians of the germ line and protect the germline genome against the activity of selfish genetic elements. We investigated how the social environment in male zebrafish affects the expression level of small RNAs in sperm and how these changes in small RNAs are linked to changes in the gene epxression and the activity of selfish genetic elements in the offspring. We developed specialised bioinformatic pipelines to provide a clear link between the response in the fathers germline to changes in the environment and gene expresssion in their offspring.

evolutionary biology↗

bioRxiv2023

The miR-144/Hmgn2 regulatory axis orchestrates chromatin organization during erythropoiesis

SUMMARYDifferentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation. Among the several targets of miR-144 that influence chromatin organization, the miR-144-dependent regulation of Hmgn2 is conserved from fish to humans. Our genetic probing of the miR-144/Hmgn2 regulatory axis established that intact miR-144 target sites in the Hmgn2 3UTR are necessary for the proper maturation of erythrocytes in both zebrafish and human iPSC-derived erythroid cells while loss of Hmgn2 rescues in part the miR-144 null phenotype. Altogether, our results uncover miR-144 and its target Hmgn2 as the backbone of the genetic regulatory circuit that controls the terminal differentiation of erythrocytes in vertebrates.

developmental biology↗

bioRxiv2022

Muscle stem cell function is impaired in absence of Talpid3 - a gene required for primary cilia formation

Skeletal muscle stem cells (MuSC) are crucial for tissue homeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells selfrenew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their role in muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Repeat injury exacerbates the regeneration phenotype in TA3iSC-KO mice, indicating depletion of MuSCs. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data suggest that TA3 and primary cilia are important for MuSC self-renewal, and that pharmacological treatment can efficiently restore muscle regeneration.

cell biology↗

bioRxiv2021

Small-RNA sequencing reveals altered skeletal muscle microRNAs and snoRNAs signatures in weanling male offspring from mouse dams fed a low protein diet during lactation

Nutrition plays a key role in pre- and postnatal growth of the musculoskeletal system. Maternal diet during gestation and lactation affects the development of skeletal muscles in the offspring and determines muscle health in later life, however, the molecular mechanisms that govern these effects are largely unknown. In this study, we aim to describe the association between maternal low protein diet-induced changes in offspring skeletal muscle and the differential expression (DE) of small non-coding RNAs (sncRNAs). We used a mouse model of maternal protein restriction to characterise the impact of early-life undernutrition on skeletal muscle morphology in male offspring at weaning. Mouse dams were fed either a normal (N, 20%) or a low protein (L, 8%) diet during gestation and newborn pups were cross-fostered to N or L lactating dams, resulting in the generation of NN, NL and LN offspring groups. Total body and tibialis anterior (TA) weights were decreased in NL males but not different in the LN group, as compared to NN, although neonates from low protein fed dams were smaller at birth than those born to dams fed a normal protein. However, histological evaluation of TA muscle revealed reduced muscle fibre size in both groups at the end of lactation. Small RNA-seq analysis demonstrated DE of multiple classes of sncRNAs, including miRs, snoRNAs and snRNAs. Bioinformatic analyses of miRs-15a, -34a, -122 and -199a, in combination with known myomiRs, confirmed their implication in key muscle-specific biological processes and cellular functions and suggest a promising set of miRs in muscle physiology studies. To our knowledge, this is the first comprehensive report for the DE of sncRNAs in nutrition-associated programming of skeletal muscle development, highlighting the need for further research.

molecular biology↗

bioRxiv2020

IGF targeting perturbs global replication through ribonucleotide reductase dysfunction

IGF receptor (IGF-1R) inhibition delays repair of radiation-induced DNA double-strand breaks (DSBs), prompting us to investigate whether IGF-1R influences endogenous DNA damage. We demonstrate that IGF-1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. We detect delayed replication fork progression in IGF-inhibited or IGF-1R depleted cancer cells, with activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflects unanticipated regulation of global replication by IGF-1, mediated via AKT, MEK/ERK and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. IGF-1R inhibition or depletion downregulate RRM2, compromising RNR function and dNTP supply. The resulting delay in fork progression and hallmarks of replication stress are rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF-1 regulates replication. Following targeted compound screens, we identify synergy between IGF inhibition and ATM loss, with evidence that IGF inhibition compromises growth of ATM null cells and xenografts. This synthetic lethal effect reflects conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. These data implicate IGF-1R in alleviating replication stress, and the identified reciprocal IGF:ATM co-dependence provides an approach to exploit this effect in ATM-deficient cancers.

cancer biology↗

bioRxiv2020

Characterising open chromatin identifies novel cis-regulatory elements important for paraxial mesoderm formation and axis extension

The development of multicellular organisms is exquisitely regulated through differential gene activity, which governs cell differentiation programs. However, many details of spatiotemporal control of gene regulation are still poorly understood. We used the accessibility of chick embryos to examine genome-wide signatures characterizing the progressive differentiation of paraxial mesoderm along the head-to-tail axis. Paraxial mesoderm becomes organized into repetitive units, termed somites, the hallmark of the segmented vertebrate body plan. New somite pairs form periodically as the axis extends at the posterior end. This process generates a developmental gradient within a single embryo, with anterior somites more advanced in their differentiation compared to posterior somites. Following somite formation, cell rearrangements generate compartments, comprising lineages of the musculoskeletal system, including cartilage of the vertebral column and ribs, and skeletal muscle cells of the trunk and limbs. To examine how paraxial mesoderm becomes regionalized and patterned to eventually generate these discrete lineages, we investigated dynamic changes of the transcriptome and of chromatin accessibility using RNA-seq and ATAC-seq across a spatiotemporal series along the embryonic axis. Footprint analysis uncovers differential coverage of binding sites for a number of key transcription factors known to be involved in axial patterning and differentiation, including HOX genes. Furthermore, associating accessible chromatin with nearby expressed genes identifies candidate cis-regulatory elements (CRE). As exemplars we use TCF15 and MEOX1, which are crucial for somite formation and differentiation, to experimentally validate CREs in vivo using fluorescent reporters. Time-lapse microscopy reveals CRE spatiotemporal activity and mutation analysis uncovers necessary upstream regulators. The CRE for MEOX1 is conserved and recognized in Xenopus. In addition, a human element is active in chicken. In vivo epigenome editing of TCF15 and MEOX1 CREs disrupts gene expression regulation and recapitulates phenotypic abnormalities of anterior-posterior axis extension.

developmental biology↗