Biology subjects

Feil, R.

Publications and source records attributed to Feil, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Sugar signaling modulates SHOOT MERISTEMLESS expression and meristem function in Arabidopsis

In plants, development of all above-ground tissues is controlled by the shoot apical meristem (SAM) which balances cell proliferation and differentiation to allow life-long growth. To maximize fitness and survival, meristem activity is adjusted to the prevailing conditions through a poorly understood integration of developmental signals with environmental and nutritional information. Here, we show that sugar signals influence SAM function by altering the protein levels of SHOOT MERISTEMLESS (STM), a key regulator of meristem maintenance. STM is less abundant in the inflorescence meristems of plants grown or treated under limiting light conditions, with lower STM levels correlating with lower sugar content in these meristems. Additionally, sucrose but not light is sufficient to sustain STM accumulation in excised inflorescences. Plants overexpressing the 1-subunit of SUCROSE-NON-FERMENTING1-RELATED KINASE 1 (SnRK1) accumulate less STM protein under optimal light conditions, despite higher sugar accumulation in the meristem. Furthermore, SnRK11 interacts physically with STM, suggesting a direct local repression. Surprisingly, silencing SnRK1 in the meristem leads to reduced STM expression and severe developmental phenotypes previously associated with STM loss-of-function. Altogether, we demonstrate that sugars promote STM accumulation and that the SnRK1 sugar sensor plays a dual role in the SAM, limiting STM abundance under unfavorable conditions but being required for overall meristem organization and integrity. This highlights the importance of sugars and SnRK1 signaling for the proper coordination of meristem activities.

plant biology↗

bioRxiv2022

Diel fluctuations in in-vivo SnRK1 activity in Arabidopsis rosettes during light-dark cycles

SUCROSE-NON-FERMENTING1 (SNF1)-RELATED KINASE1 (SnRK1) is a central hub in carbon and energy signalling in plants, and is orthologous with SNF1 in yeast and the AMP-ACTIVATED PROTEIN KINASE (AMPK) in animals. Previous studies of SnRK1 relied on in-vitro activity assays or on monitoring the expression of putative marker genes. Neither approach gives unambiguous information about in-vivo SnRK1 activity. We have monitored in-vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with a SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle, and after perturbing this cycle. As expected, in vivo SnRK1 activity rose towards the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 hours into the light period. The sucrose signal trehalose 6-phosphate (Tre6P) has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harboured an inducible TREHALOSE-6-PHOSPHATE SYNTHASE construct. Elevated Tre6P decreased in-vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose 6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, and that Tre6P, hexose phosphates and the circadian clock contribute to regulation of SnRK1 activity in a context-dependent manner, and SnRK1-signalling is further modulated by factors that act downstream of SnRK1. One sentence summaryIn vivo SnRK1 activity shows an unexpected diel response and a complex relationship with trehalose 6-phosphate and other possible metabolic regulators.

plant biology↗

bioRxiv2022

Operation of Carbon-Concentrating Mechanisms in Cyanobacteria and Algae requires altered poising of the Calvin-Benson cycle

Cyanobacteria and eukaryotic algae make a major contribution to global photosynthetic productivity. To cope with the low availability of CO2 in aqueous systems they deploy inorganic carbon-concentrating mechanisms (CCMs). These concentrate CO2 in microcompartments that contain Rubisco (carboxysomes in cyanobacteria; pyrenoids in green algae). The rest of the Calvin-Benson cycle (CBC) is located outside these microcompartments. We hypothesized that this physical separation requires modified poising of the CBC. Hence, Rubisco is physically separated from the other CBC enzymes outside these microcompartments. To test the hypothesis that this physical separation requires appropriate poising of the CBC, we profiled CBC metabolites under ambient CO2 in the cyanobacterium Synechocystis sp. PCC 6803 and three eukaryotic algae (Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella ohadii). Comparison with recently reported profiles for a large set of terrestrial plants revealed that cyanobacteria and green algae have very distinctive CBC metabolite profiles, with low levels of pentose phosphates and, especially, high levels of ribulose 1,5-bisphosphate and 3-phosphoglycerate. We propose that large pools of the substrate and product of Rubisco are required to generate concentration gradients that drive movement into and out of the microcompartments. These observations raise questions about how CBC regulation was modified during the evolution of algal CCMs and their subsequent loss in terrestrial plants, and highlight that operation of CCMs requires co-evolution of the CBC. HighlightCBC metabolite profiles in the cyanobacterium Synechocystis and in three eukaryotic green algae at ambient CO2 concentration are very different to those in terrestrial plants, probably reflecting the operation of a carboxysome- or pyrenoid-based carbon concentrating mechanism.

biochemistry↗

bioRxiv2022

The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Ongoing work has identified several transcriptional regulators of nitrate signaling, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN 7 (NLP7), but additional regulators likely remain to be identified. Here, we characterized Arabidopsis NLP2 as a master upstream transcriptional regulator of early nitrate responses that interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, a DNA binding motif, and some target genes with NLP7. Additional genetic, genomic and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multi-tiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production. One-sentence summaryNLP2 and NLP7 orchestrate plant responses to nitrate supply and control nitrate- dependent regulation of carbon and energy metabolism.

plant biology↗

bioRxiv2021

Optogenetic manipulation of cGMP highlights PDE5 as the predominant cGMP-hydrolyzing PDE in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatiotemporal regulation in megakaryocytes (MKs) is lacking. We expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide (NO) triggered cGMP generation in BM MKs. In summary, we established for the first time optogenetics in primary MKs and identified PDE5 as the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

cell biology↗

bioRxiv2021

Trehalose-6-phosphate links singlet oxygen-induced signalling with metabolic signalling in Chlamydomonas reinhardtii

Using a novel mutant screen, we identified TREHALOSE 6-PHOSPHATE PHOSPHATASE 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose in Chlamydomonas reinhardtii. The tspp1 knock-out results in reprogramming of the cell metabolism via significantly altered transcriptome. As a secondary effect, tspp1 also shows impairment in 1O2-induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect 1O2-signalling. 1O2-inducible GLUTATHIONE PEROXIDASE 5 (GPX5) gene expression is suppressed by increased content of fumarate, an intermediate in the tricarboxylic acid cycle (TCA cycle) in mitochondria and dicarboxylate metabolism in the cytosol, while it is promoted by another TCA cycle intermediate, aconitate. Furthermore, genes encoding known essential components of chloroplast-to-nucleus 1O2-signalling, PSBP2, MBS, and SAK1, show decreased transcript levels in the tspp1 mutant, which can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving 1O2 depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to 1O2.

cell biology↗

bioRxiv2021

Energy status-promoted growth and development of Arabidopsis require copper deficiency response transcriptional regulator SPL7

Copper (Cu) is a cofactor of around 300 Arabidopsis proteins including photosynthetic and mitochondrial electron transfer chain enzymes critical for ATP production and carbon fixation. Plant acclimation to Cu deficiency requires the transcription factor SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7). We report that in the wild type and in the spl7-1 mutant, respiratory electron flux via Cu-dependent cytochrome c oxidase remained unaffected under both normal and low-Cu cultivation conditions. Contrary to the wild type, supplementing Cu-deficient media with exogenous sugar failed to stimulate growth of spl7-1. The spl7-1 mutant accumulated carbohydrates including the signaling sugar trehalose 6-phosphate, as well as ATP and NADH, also under normal Cu supply and without sugar supplementation. Late flowering of spl7-1 was in agreement with its attenuated sugar responsiveness. Functional TOR and SnRK1 kinase signaling in spl7-1 suggested against fundamental defects in these energy-signaling hubs. Sequencing of chromatin immunoprecipitates combined with transcriptomics identified direct targets of SPL7-mediated positive regulation, including FE SUPEROXIDE DISMUTASE1 (FSD1), COPPER-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR1 (CITF1) and uncharacterized bHLH23 (CITF2), as well as an enriched upstream GTACTRC motif. In summary, transducing energy availability into growth and reproductive development requires the function of SPL7. Our results could help to increase crop yields, especially on Cu-deficient soils.

plant biology↗

bioRxiv2021

Recruitment of an ancient branching program to suppress carpel development in maize flowers

Floral morphology is immensely diverse. One developmental process acting to shape this diversity is growth suppression. For example, grass flowers exhibit extreme diversity in floral sexuality, arising through differential suppression of stamens or carpels. In maize, carpels undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen, and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1; ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ~160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes, and show how an ancient developmental program was redeployed to sculpt floral form.

plant biology↗

bioRxiv2021

cGMP signaling: probing antagonistic cyclic nucleotide platelet signals by modeling and experiment

BackgroundThe cyclic nucleotides cAMP and cGMP inhibit platelet activation. ResultsWe extended an older model and systematically integrated drugs as external stimuli. Data driven modeling allowed us to design models that provide a quantitative output for quantitative input information. This relies on condensed information about involved regulation and modeling of pharmacological interventions by systematic optimization methods. By multi-experiment fitting, we validated our model optimizing the parameters of the model. In addition, we show how the output of the developed cGMP model can be used as input for a modular model of VASP phosphorylation and for the activity of cAMP and cGMP pathways in platelets. ConclusionsWe present a model for cGMP signaling and VASP phosphorylation, that allows to estimate drug action on any of the inhibitory cyclic nucleotide pathways (cGMP, cAMP) and has been validated by experimental data.

systems biology↗

bioRxiv2021

The pathway of starch synthesis in Arabidopsis thaliana leaves

Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling, Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the classical pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts. Significance statementMutant analysis shows that sucrose synthase makes no significant contribution to transitory starch synthesis in Arabidopsis leaves, resolving a 20-year old controversy about one of the most important pathways of photosynthetic metabolism.

plant biology↗

bioRxiv2020

The role of trehalose 6-phosphate in shoot branching - local and non-local effects on axillary bud outgrowth in arabidopsis rosettes

- Trehalose 6-phosphate (Tre6P) is a sucrose signalling metabolite that has been implicated in regulation of shoot branching, but its precise role is not understood. - We expressed tagged forms of TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) to determine where Tre6P is synthesized in arabidopsis (Arabidopsis thaliana), and investigated the impact of localized changes in Tre6P levels, in axillary buds or vascular tissues, on shoot branching in wild-type and branching mutant backgrounds. - TPS1 is expressed in axillary buds and the subtending vasculature, as well as in the leaf and stem vasculature. Expression of a heterologous trehalose-6-phosphate phosphatase (TPP) to lower Tre6P in axillary buds strongly delayed bud outgrowth in long days and inhibited branching in short days. TPP expression in the vasculature also delayed lateral bud outgrowth and decreased branching. Increased Tre6P in the vasculature enhanced branching and was accompanied by higher expression of FLOWERING LOCUS T (FT) and up-regulation of sucrose transporters. Increased vascular Tre6P levels enhanced branching in branched1 but not in ft mutant backgrounds. - These results provide direct genetic evidence of a local role for Tre6P in regulation of axillary bud outgrowth within the buds themselves, and also connect Tre6P with systemic regulation of shoot branching via FT.

plant biology↗

bioRxiv2020

Cooperation between Caenorhabditis elegans COMPASS and condensin in germline chromatin organization

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters by SET1/COMPASS is associated with context-dependent effects on gene expression and local changes in chromatin organization. Whether SET1/COMPASS also contributes to higher-order chromosome structure has not been investigated. Here, we address this question by quantitative FRET (Forster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on C. elegans germ cells expressing histones H2B-eGFP and H2B-mCherry. We find that SET1/COMPASS subunits strongly influence meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of SET-2, the C. elegans homologue of SET1, or CFP-1, the chromatin targeting subunit of COMPASS, strongly enhance chromosome organization defects and loss of fertility resulting from depletion of condensin-II. Defects in chromosome morphology resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes, were also aggravated in the absence of SET-2. Combined, our in vivo findings suggest a model in which the SET1/COMPASS histone methyltransferase complex plays a role in shaping meiotic chromosome in cooperation with the non-histone proteins condensin-II and topoisomerase.

genetics↗

bioRxiv2019

Differences in berry primary and secondary metabolisms identified by transcriptomic and metabolic profiling of two table grape color somatic variants

Anthocyanins are flavonoids responsible for the color of berries in skin-pigmented grapevine (Vitis vinifera L.). Due to the widely adopted vegetative propagation of this species, somatic mutations occurring in meristematic cell layers can be fixed and passed into the rest of the plant when cloned. In this study we focused on the transcriptomic and metabolic differences between two color somatic variants. Using microscopic, metabolic and mRNA profiling analyses we compared the table grape cultivar (cv.) Red Globe (RG, with purplish berry skin) and cv. Chimenti Globe (CG, with a contrasting reddish berry skin color). As expected, significant differences were found in the composition of flavonoids and other phenylpropanoids, but also in their upstream precursors shikimate and phenylalanine. Among primary metabolites, sugar phosphates related with sucrose biosynthesis were less accumulated in cv. CG. The red-skinned cv. CG only contained di-hydroxylated anthocyanins (i.e. peonidin and cyanidin) while the tri-hydroxylated derivatives malvidin, delphinidin and petunidin were absent, in correlation to the reddish cv. CG skin coloration. Transcriptomic analysis showed alteration in flavonoid metabolism and terpenoid pathways and in primary metabolism such as sugar content. Eleven flavonoid 35-hydroxylase gene copies were down-regulated in cv. CG. This family of cytochrome P450 oxidoreductases are key in the biosynthesis of tri-hydroxylated anthocyanins. Many transcription factors appeared down-regulated in cv. CG in correlation to the metabolic and transcriptomic changes observed. The use of molecular markers and its confirmation with our RNA-seq data showed the exclusive presence of the null MYBA2 white allele (i.e. homozygous in both L1 and L2 layers) in the two somatic variants. Therefore, the differences in MYBA1 expression seem sufficient for the skin pigmentation differences and the changes in MYBA target gene expression in cv. Chimenti Globe.

plant biology↗

bioRxiv2019

Proteomic and metabolomic profiling underlines the stage- and time-dependent effects of high temperature on grape berry metabolism

Climate change scenarios predict an increase in mean air temperatures and in the frequency, intensity, and length of extreme temperature events in many wine-growing regions worldwide. Because elevated temperature has detrimental effects on the berry growth and composition, it threatens the economic and environmental sustainability of wine production. Using Cabernet Sauvignon fruit-bearing cuttings, we investigated the effects of high temperature (HT) on grapevine berries through a label-free shotgun proteomic analysis coupled to a complementary metabolomic study. Among the 2279 proteins identified, 592 differentially abundant proteins were found in berries exposed to HT. The gene ontology categories \"Stress\", \"Protein\", \"Secondary metabolism\" and \"Cell wall\" were predominantly altered under HT. High temperatures strongly impaired carbohydrate and energy metabolism, and the effects depended on the stage of development and duration of treatment. Transcript amounts correlated poorly with protein expression levels in HT berries, highlighting the value of proteomic studies in the context of heat stress. Furthermore, this work reveals that HT alters key proteins driving berry development and ripening. Finally, we provide a list of differentially abundant proteins that can be considered as potential markers for developing or selecting grape varieties that are better adapted to warmer climates or extreme heat waves.

plant biology↗

bioRxiv2019

Phytochrome light receptors control metabolic flux, and their action during seedling development sets the trajectory for biomass production

The phytochromes (phys) photoreceptors are known to be major regulators of plastic growth responses to vegetation shade. Recent reports have begun to uncover an important role for phys in carbon resource management. Our earlier work showed that phy mutants had a distinct metabolic profile with elevated levels of metabolites including TCA intermediates, amino acids and sugars. Here we show that in seedlings phy regulates the balance between glucose and starch. Multi-allele phy mutants have excess glucose and low starch levels, which is conducive to hypocotyl elongation. 13C-CO2 labelling demonstrates that metabolic flux balance in adult plants is markedly altered in phy mutants. Phytochrome reduces synthesis rates of stress metabolites, including raffinose and proline and several typical stress-induced biosynthetic genes related to these metabolites show higher expression in phy mutants.\n\nSince growth and metabolism are typically inter-connected, we investigated why phy mutants have severely reduced biomass. Quantification of carbon fixation, biomass accumulation, and 13C labelling of cell wall polysaccharides established that relative growth rate is impaired in multi allele phy mutants for the first 2.5 weeks after germination but equivalent to the WT thereafter. Mathematical modelling predicts that the altered growth dynamics and final biomass deficit can be explained by the smaller cotyledon size of the multiple phy mutants. This indicates that the established role of phy in promoting seedling establishment has enduring effects that govern adult plant biomass.

plant biology↗

bioRxiv2019

Trehalose 6-phosphate Controls Seed Filling by Inducing Auxin Biosynthesis

Plants undergo several developmental transitions during their life cycle. One of these, the differentiation of the young embryo from a meristem-like structure into a highly-specialized storage organ, is vital to the formation of a viable seed. For crops in which the seed itself is the end product, effective accumulation of storage compounds is of economic relevance, defining the quantity and nutritive value of the harvest yield. However, the regulatory networks underpinning the phase transition into seed filling are poorly understood. Here we show that trehalose 6-phosphate (T6P), which functions as a signal for sucrose availability in plants, mediates seed filling processes in seeds of the garden pea (Pisum sativum), a key grain legume. Seeds deficient in T6P are compromised in size and starch production, resembling the wrinkled seeds studied by Gregor Mendel. We show also that T6P exerts these effects by stimulating the biosynthesis of the pivotal plant hormone, auxin. We found that T6P promotes the expression of the auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE RELATED2 (TAR2), and the resulting effect on auxin levels is required to mediate the T6P-induced activation of storage processes. Our results suggest that auxin acts downstream of T6P to facilitate seed filling, thereby providing a salient example of how a metabolic signal governs the hormonal control of an integral phase transition in a crop plant.

plant biology↗

bioRxiv2019

The 20S proteasome activator PA28γ controls the compaction of HP1β-linked heterochromatin

PA28{gamma}, a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28{gamma} controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, similarly to that observed upon the knockdown of HP1{beta}, a key factor in heterochromatin structure. We show that PA28{gamma} is present at HP1{beta}-containing repetitive-DNA sequences abundant in heterochromatin and importantly, that HP1{beta} on its own is unable to drive chromatin compaction without the presence of PA28{gamma}. At the molecular level, we show that this novel function of PA28{gamma} is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are both involved in heterochromatin formation. Overall, our results implicate PA28{gamma} as a key factor involved in the higher order structuration of chromatin.

cell biology↗

bioRxiv2019

CTCF controls imprinted gene activity at the mouse Dlk1-Dio3 and Igf2- H19 domains by modulating allele-specific sub-TAD structure

Mammalian genomic imprinting is essential for development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. Here, we compared chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints--the Igf2-H19 and the Dlk1-Dio3 domains--and assessed the involvement of the insulator protein CTCF. At both domains, CTCF binds the maternal allele of a differentially-methylated region (DMR), in addition to multiple instances of bi-allelic CTCF binding in their surrounding TAD (Topologically Associating Domain). On the paternal chromosome, bi-allelic CTCF binding alone is sufficient to structure a first level of sub-TAD organization. Maternal-specific CTCF binding at the DMRs adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal sub-TAD organisation. Genome-editing experiments at the Dlk1-Dio3 locus confirm that the maternal sub-TADs are essential during development to maintain the imprinted Dlk1 gene in an inactive state on the maternal chromosome.

molecular biology↗