1,283 research outputs found
Modifying the m6A brain methylome by ALKBH5-mediated demethylation: a new contender for synaptic tagging
Synaptic plasticity processes, which underlie learning and memory formation, require RNA to be translated local to synapses. The synaptic tagging hypothesis has previously been proposed to explain how mRNAs are available at specific activated synapses. However how RNA is regulated, and which transcripts are silenced or processed as part of the tagging process is still unknown. Modification of RNA by N6-methyladenosine (m6A/m) influences the cellular fate of mRNA. Here, by advanced microscopy, we showed that m6A demethylation by the eraser protein ALKBH5 occurs at active synaptic ribosomes and at synapses during short term plasticity. We demonstrated that at activated glutamatergic post-synaptic sites, both the YTHDF1 and YTHDF3 reader and the ALKBH5 eraser proteins increase in co-localisation to m6A-modified RNAs; but only the readers showed high co-localisation to modified RNAs during late-stage plasticity. The YTHDF1 and YTHFDF3 readers also exhibited differential roles during synaptic maturation suggesting that temporal and subcellular abundance may determine specific function. m6A-sequencing of human parahippocampus brain tissue revealed distinct white and grey matter m6A methylome profiles indicating that cellular context is a fundamental factor dictating regulated pathways. However, in both neuronal and glial cell-rich tissue, m6A effector proteins are themselves modified and m6A epitranscriptional and posttranslational modification processes coregulate protein cascades. We hypothesise that the availability m6A effector protein machinery in conjunction with RNA modification, may be important in the formation of condensed synaptic nanodomain assemblies through liquid-liquid phase separation. Our findings support that m6A demethylation by ALKBH5 is an intrinsic component of the synaptic tagging hypothesis and a molecular switch which leads to alterations in the RNA methylome, synaptic dysfunction and potentially reversible disease states
Training Memory: Exploring the Intersection of Plant Stress Signalling and DNA Methylation
Plants are sessile organisms living in a dynamic environment to
which they must continually acclimatize in order to maximise
their reproductive potential. This plasticity is achieved through
many complex and intricate signalling pathways that allow for the
continuous perception, response, and adjustments to new
environmental stimuli. A growing body of evidence suggests that
such pathways are not merely static but dynamic and can be primed
following repeated activation, thus affecting enhanced responses
to recurring stresses. Such examples of priming have led to a
notion that plants have some capacity to form stress memories of
past environmental perturbations. However, the full extent and
nature of such memory, and the machinery involved to store and
transmit these, remain enigmatic. One prospective mechanism is
the involvement of heritable, yet rapid and reversible, chromatin
marks that, theoretically, could be shaped by the environment to
convey a regulatory effect on the expression of the underlying
genotype, thus acting as an epigenetic layer of regulation.
This thesis explores the potential intersection of stress
signalling pathways and chromatin variation, specifically DNA
methylation, to co-ordinate plant stress responses. First,
mechanistic insights into the operation of a SAL1-PAP-XRN
retrograde signalling pathway to fine-tune plant physiology under
drought are presented. A key finding was that this pathway
complements canonical ABA signalling to induce stomatal closure,
thus minimising water-loss under water limited conditions.
Furthermore, the SAL1-PAP-XRN pathway was found to effect
chromatin patterns, specifically DNA methylation at short
transposable elements. These observations implicate cross-talk
with the RNA directed DNA methylation pathway, however, the exact
mechanism for this interaction remains to be identified.
Multiple investigations were performed to test for stress-induced
changes in DNA methylation that could potentially regulate
responses to recurring stress, thus conveying a memory. A
transgenerational recurring drought stress experiment tested
whether descendants of drought-exposed lineages displayed greater
drought tolerance (transgenerational memory). For the majority of
traits tested, including plant growth rate and drought survival,
offspring from plant lineages exposed to successive generations
of repeated drought stress performed comparably to those from
control lineages. However, memory was demonstrated in the form of
enhanced seed dormancy, in drought stressed lineages, that
persisted at least one generation removed from stress. Whether
this capacity for memory could be related to the type or severity
of stress applied, or species examined, remains to be
investigated further.
The transgenerational drought experiment was paired with a
recurring excess-light stress experiment to investigate memory
within a generation. Not only did this treatment lead to priming
of plant photosynthetic behaviour, indicative of a greater
capacity to withstand abrupt increases in light intensity, but
new leaves from stressed plants, developed in the absence of
stress, also showed altered photosynthetic characteristics
compared to unstressed counterparts. Such observations are
consistent with the mitotic transmission of stress-induced
traits.
Given multiple demonstrations of memory, comparisons were made to
unstressed controls to test for any correlating changes in DNA
methylation that might explain the phenomena observed. However,
in both experiments, observations of memory were found to be
independent of large-scale conserved changes in DNA methylation
discounting it as a conveyor of plant stress memories, under
these conditions, raising questions regarding the mechanism(s)
responsible for the examples of memory observed herein.
Ultimately, this thesis systematically evaluates the notion that
plants are able to form genuine memories, potentially underpinned
by reversible chromatin marks, that may facilitate acclimation to
local environments on a relatively rapid scale compared to the
fixation of adaptive genetic polymorphisms. Any capacity for
plant stress memories may provide avenues for further epigenomic
based agronomic tools to improve crop stress tolerance. However,
the nature of such memories observed here appear subtle and
nuanced, and are forgotten beyond a generation. Further
characterisation and mechanistic understanding of mitotic memory
mechanisms, however, may still hold potential. It was also
observed that stress signalling pathways can interact with those
involved in chromatin modification, giving novel insight into
their mechanistic functioning and the how the onset of stress may
induce chromatin changes. Despite this potential, the DNA
methylome was found to be relatively impervious to stress-induced
changes and, thus, is an unlikely memory mechanism
Current epigenetic aspects the clinical kidney researcher should embrace
Chronic kidney disease (CKD), affecting 10-12% of the world's adult population, is associated with a considerably elevated risk of serious comorbidities, in particular, premature vascular disease and death. Although a wide spectrum of causative factors has been identified and/or suggested, there is still a large gap of knowledge regarding the underlying mechanisms and the complexity of the CKD phenotype. Epigenetic factors, which calibrate the genetic code, are emerging as important players in the CKD-associated pathophysiology. In this article, we review some of the current knowledge on epigenetic modifications and aspects on their role in the perturbed uraemic milieu, as well as the prospect of applying epigenotype-based diagnostics and preventive and therapeutic tools of clinical relevance to CKD patients. The practical realization of such a paradigm will require that researchers apply a holistic approach, including the full spectrum of the epigenetic landscape as well as the variability between and within tissues in the uraemic milieu
Enhancing resolution of natural methylome reprogramming behavior in plants
We have developed a novel methylome analysis procedure, Methyl-IT, based on information thermodynamics and signal detection. Methylation analysis involves a signal detection problem, and the method was designed to discriminate methylation regulatory signal from background noise induced by thermal fluctuations. Comparison with three commonly used programs and various available datasets to furnish a comparative measure of resolution by each method is included. To confirm results, methylation analysis was integrated with RNAseq and network enrichment analyses. Methyl-IT enhances resolution of genome methylation behavior to reveal network-associated responses, offering resolution of gene pathway influences not attainable with previous methods
Enhancing resolution of natural methylome reprogramming behavior in plants
We have developed a novel methylome analysis procedure, Methyl-IT, based on information thermodynamics and signal detection. Methylation analysis involves a signal detection problem, and the method was designed to discriminate methylation regulatory signal from background noise induced by thermal fluctuations. Comparison with three commonly used programs and various available datasets to furnish a comparative measure of resolution by each method is included. To confirm results, methylation analysis was integrated with RNAseq and network enrichment analyses. Methyl-IT enhances resolution of genome methylation behavior to reveal network-associated responses, offering resolution of gene pathway influences not attainable with previous methods
Enhancing resolution of natural methylome reprogramming behavior in plants
We have developed a novel methylome analysis procedure, Methyl-IT, based on information thermodynamics and signal detection. Methylation analysis involves a signal detection problem, and the method was designed to discriminate methylation regulatory signal from background noise induced by thermal fluctuations. Comparison with three commonly used programs and various available datasets to furnish a comparative measure of resolution by each method is included. To confirm results, methylation analysis was integrated with RNAseq and network enrichment analyses. Methyl-IT enhances resolution of genome methylation behavior to reveal network-associated responses, offering resolution of gene pathway influences not attainable with previous methods
Enhancing resolution of natural methylome reprogramming behavior in plants
We have developed a novel methylome analysis procedure, Methyl-IT, based on information thermodynamics and signal detection. Methylation analysis involves a signal detection problem, and the method was designed to discriminate methylation regulatory signal from background noise induced by thermal fluctuations. Comparison with three commonly used programs and various available datasets to furnish a comparative measure of resolution by each method is included. To confirm results, methylation analysis was integrated with RNAseq and network enrichment analyses. Methyl-IT enhances resolution of genome methylation behavior to reveal network-associated responses, offering resolution of gene pathway influences not attainable with previous methods
Uncovering the mechanisms and information content of CpG-resolved DNA methylation programming during hematopoietic differentiation
DNA methylome remodeling is an essential molecular mechanism underlying all stages of hematopoietic differentiation. However, current datasets only cover a fraction of the genome and are often limited to specific hematopoietic cell types. A comprehensive, genome-wide atlas of the DNA methylation dynamics during hematopoietic differentiation is still missing. Preliminary evidence suggests that the single-cell landscape of the hematopoietic stem and progenitor cell (HSPC) compartment is characterized by a structured continuum of epigenetically-defined cell states. Significant advances in charting this epigenetic state manifold have recently been achieved for the chromatin accessibility and histone modification layers. However, despite its potential importance, the landscape of single-cell DNA methylome states in the HSPC compartment remains largely unexplored. This project aimed to comprehensively map the genome-wide DNA methylation dynamics during hematopoietic differentiation and leverage this atlas as a reference to analyze the single-cell DNA methylome landscape in the HSPC compartment and among mature hematopoietic cells. The functional importance and rich information content of differentially methylated regions (DMRs) are well-established. However, the DNA methylation layer inherently possesses the capability to encode information at CpG resolution. The role and extent of differentially methylated CpG (DMCpG) programming within DMR regions is largely unexplored. This project therefore aimed to evaluate the role and mechanisms of DMCpG programming during hematopoietic differentiation. Using high-coverage tagmentation-based whole-genome bisulfite sequencing data for 25 hematopoietic populations, I have compiled a genome-wide, dual-layer DMR/DMCpG atlas, which maps, annotates, and integrates DMR and DMCpG programming during hematopoietic differentiation. Loss of stemness was associated with lineage-independent gain of DNA methylation, while lineage specification was accompanied by hierarchical DNA methylation dynamics, characterized by unidirectional loss of DNA methylation. Different DMCpGs within focal DMR intervals were often distinctly programmed and thus contained heterogeneous information content. In particular, most of the DMRs were seeded and progressively expanded through subsequent programming of specific DMCpGs at different stages of differentiation. Mature hematopoietic cells exhibited systematic seed DMCpG hypomethylation in DMRs associated with alternative cell fates. This seed hypomethylation likely represents epigenetic memory of alternative fate explorations in progenitor cells. Collectively, these findings suggest a hierarchical model of DNA methylation programming, in which information is encoded through DMR programming and through DMCpG programming within DMR regions. This model represents a significant extension of the commonly accepted paradigm of regional DNA methylation programming. Using the dual-layer DMR/DMCpG atlas as a reference, single-cell methylome states for 312 HSPCs, as well as for a total of 136 mature B cells, T cells, CFU-Es, and monocytes, could be dissected with high resolution. The HSPC compartment was characterized by a structured continuum of single-cell DNA methylome states. Multiple lines of evidence suggested that differentiation starts from apex HSCs possessing a lineage-naive DNA methylome state. Exit from the apex HSC state was initiated by balanced, multi-lineage DMR seeding. This early DMR programming was strictly restricted to specific DMR seeding regions, which often comprised only one or two DMCpGs. This contrasts with the conventional paradigm that functionally relevant DMRs always contain at least several DMCpGs. Further differentiation within the HSPC compartment was accompanied by continuous, gradually more lineage-specific accumulation of hypomethylation, leading to progressive DMR expansion. The dual-layer DMR/DMCpG atlas provides an essential resource for studying the epigenetic regulation of the hematopoietic differentiation process and serves as a valuable reference for the analysis of single-cell bisulfite sequencing data. This work highlights the highly-resolved, progressive, and stable nature of DNA methylome remodeling during hematopoietic differentiation and reveals several aspects of the structure and information content of the DNA methylome layer which go beyond the currently accepted paradigms. It appears likely that the DNA methylome remodeling mechanisms active in other differentiation systems and related processes, such as tumor evolution, share the same principles of hierarchical DNA methylation programming with CpG resolution. However, in many systems, the information content of the DNA methylome may be convoluted by a combination of this programming mechanism and other programming mechanisms characterized by stochastic regional accumulation of DNA methylation alterations. The analysis strategies presented in this work provide a basis for the further development of computational methods capable of dissecting the rich but complex information content of the DNA methylome with high resolution
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