Dynamics Of Histone Modifications Research Articles

Dynamic histone modification signatures coordinate developmental programs in strawberry fruit ripening.

Chromatin structure plays a critical role in the regulation of dynamic gene expression in response to different developmental and environmental cues, but as yet their involvement in fruit ripening is not well understood. Here, we profile seven histone modifications in the woodland strawberry (Fragaria vesca) genome and analyze the histone modification signatures during ripening. Collectively, segments painted by the seven marks cover ~85% of the woodland strawberry genome. We report an eight-state chromatin structure model of the woodland strawberry based on the above histone marks, which reveals a diverse chromatin environment closely associated with transcriptional apparatus. Upon this model we build a chromatin-centric annotation to the strawberry genome. Expression of many genes essential for fruit ripening, such as abscisic acid catabolism, anthocyanin accumulation and fruit softening, are associated with shifts of active genic states and polycomb-associated chromatin states. Particularly, the expression levels of ripening-related genes are well correlated with histone acetylation, indicating a regulatory role of histone acetylation in strawberry ripening. Our identification of the chromatin states underpinning genome expression during fruit ripening not only elucidates the coordination of different pathways of morphological and metabolic development but also provides a framework to understand the signals that regulate fruit ripening.

Regulation of T helper cell differentiation by the interplay between histone modification and chromatin interaction

The development and function of the immune system are controlled by temporospatial gene expression programs, which are regulated by cis-regulatory elements, chromatin structure, and trans-acting factors. In this study, we cataloged the dynamic histone modifications and chromatin interactions at regulatory regions during T helper (Th) cell differentiation. Our data revealed that the H3K4me1 landscape established by MLL4 in naive CD4+ Tcells is critical for restructuring the regulatory interaction network and orchestrating gene expression during the early phase of Th differentiation. GATA3 plays a crucial role in further configuring H3K4me1 modification and the chromatin interaction network during Th2 differentiation. Furthermore, we demonstrated that HSS3-anchored chromatin loops function to restrict the activity of the Th2 locus control region (LCR), thus coordinating the expression of Th2 cytokines. Our results provide insights into the mechanisms of how the interplay between histone modifications, chromatin looping, and trans-acting factors contributes to the differentiation of Th cells.

Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation

The placenta serves as an essential organ for fetal growth throughout pregnancy. Histone modification is a crucial regulatory mechanism involved in numerous biological processes and development. Nevertheless, there remains a significant gap in our understanding regarding the epigenetic regulations that influence trophoblast lineage differentiation, a fundamental aspect of placental development. Here, through comprehensive mapping of H3K4me3, H3K27me3, H3K9me3, and H3K27ac loci during the differentiation of trophoblast stem cells (TSCs) into syncytiotrophoblasts (STs) and extravillous trophoblasts (EVTs), we reveal dynamic reconfiguration in H3K4me3 and H3K27ac patterns that establish an epigenetic landscape conducive to proper trophoblast lineage differentiation. We observe that broad H3K4me3 domains are associated with trophoblast lineage-specific gene expression. Unlike embryonic stem cells, TSCs lack robust bivalent domains. Notably, the repression of ST- and EVT-active genes in TSCs is primarily attributed to the weak H3K4me3 signal rather than bivalent domains. We also unveil the inactivation of TSC enhancers precedes the activation of ST enhancers during ST formation. Our results provide a comprehensive global map of diverse histone modifications, elucidating the dynamic histone modifications during trophoblast lineage differentiation.

The Dynamics of Histone Modifications during Mammalian Zygotic Genome Activation.

Mammalian fertilization initiates the reprogramming of oocytes and sperm, forming a totipotent zygote. During this intricate process, the zygotic genome undergoes a maternal-to-zygotic transition (MZT) and subsequent zygotic genome activation (ZGA), marking the initiation of transcriptional control and gene expression post-fertilization. Histone modifications are pivotal in shaping cellular identity and gene expression in many mammals. Recent advances in chromatin analysis have enabled detailed explorations of histone modifications during ZGA. This review delves into conserved and unique regulatory strategies, providing essential insights into the dynamic changes in histone modifications and their variants during ZGA in mammals. The objective is to explore recent advancements in leading mechanisms related to histone modifications governing this embryonic development phase in depth. These considerations will be useful for informing future therapeutic approaches that target epigenetic regulation in diverse biological contexts. It will also contribute to the extensive areas of evolutionary and developmental biology and possibly lay the foundation for future research and discussion on this seminal topic.

Post-Translational Modifications in Histones and Their Role in Abiotic Stress Tolerance in Plants.

Abiotic stresses profoundly alter plant growth and development, resulting in yield losses. Plants have evolved adaptive mechanisms to combat these challenges, triggering intricate molecular responses to maintain tissue hydration and temperature stability during stress. A pivotal player in this defense is histone modification, governing gene expression in response to diverse environmental cues. Post-translational modifications (PTMs) of histone tails, including acetylation, phosphorylation, methylation, ubiquitination, and sumoylation, regulate transcription, DNA processes, and stress-related traits. This review comprehensively explores the world of PTMs of histones in plants and their vital role in imparting various abiotic stress tolerance in plants. Techniques, like chromatin immune precipitation (ChIP), ChIP-qPCR, mass spectrometry, and Cleavage Under Targets and Tag mentation, have unveiled the dynamic histone modification landscape within plant cells. The significance of PTMs in enhancing the plants' ability to cope with abiotic stresses has also been discussed. Recent advances in PTM research shed light on the molecular basis of stress tolerance in plants. Understanding the intricate proteome complexity due to various proteoforms/protein variants is a challenging task, but emerging single-cell resolution techniques may help to address such challenges. The review provides the future prospects aimed at harnessing the full potential of PTMs for improved plant responses under changing climate change.

Novel underlying regulatory mechanism of the MsDAD2-mediated salt stress response in alfalfa

Alfalfa (Medicago sativa L.), a crucial and widely grown forage legume, faces yield and quality challenges due to salinity stress. The defender against apoptotic death (DAD) gene, recognized initially as an apoptosis suppressor in mammals, plays a pivotal role in catalyzing N-glycosylation, acting as a positive regulator for protein folding and endoplasmic reticulum (ER) export. Here, we found that the MsDAD2 gene was specially induced in the salt-tolerant alfalfa cultivar (DL) under salinity stress, but not in the salt-sensitive cultivar (SD). Overexpression of MsDAD2 enhanced the salinity resistance of transgenic alfalfa by promoting NAD(P)H-quinone oxidoreductase (NQO1) and cytochrome b6f complex subunit (Cyt b6/f) expression, thereby mitigating reactive oxygen species (ROS) production. ChIP-qPCR analysis suggested that the differential expression of MsDAD2 in DL and SD under salinity stress may be linked to dynamic histone modifications in its promoter. Therefore, our findings elucidate a novel regulatory mechanism of MsDAD2 in alfalfa's response to salinity stress, underscoring its significance as a target for alfalfa breeding to enhance salt tolerance.

Beyond heat waves: Unlocking epigenetic heat stress memory in Arabidopsis.

Plants remember their exposure to environmental changes and respond more effectively the next time they encounter a similar change by flexibly altering gene expression. Epigenetic mechanisms play a crucial role in establishing such memory of environmental changes and fine-tuning gene expression. With the recent advancements in biochemistry and sequencing technologies, it has become possible to characterize the dynamics of epigenetic changes on scales ranging from short term (minutes) to long term (generations). Here, our main focus is on describing the current understanding of the temporal regulation of histone modifications and chromatin changes during exposure to short-term recurring high temperatures and reevaluating them in the context of natural environments. Investigations of the dynamics of histone modifications and chromatin structural changes in Arabidopsis after repeated exposure to heat at short intervals have revealed the detailed molecular mechanisms of short-term heat stress memory, which include histone modification enzymes, chromatin remodelers, and key transcription factors. In addition, we summarize the spatial regulation of heat responses. Based on the natural temperature patterns during summer, we discuss how plants cope with recurring heat stress occurring at various time intervals by utilizing 2 distinct types of heat stress memory mechanisms. We also explore future research directions to provide a more precise understanding of the epigenetic regulation of heat stress memory.

Transcription factors direct epigenetic reprogramming at specific loci in human cancers.

The characterization of epigenetic changes during cancer development and progression led to notable insights regarding the roles of cancer-specific epigenetic reprogramming. Recent studies showed that transcription factors (TFs) are capable to regulate epigenetic reprogramming at specific loci in different cancer types through their DNA-binding activities. However, the causal association of dynamic histone modification change mediated by TFs is still not well elucidated. Here we evaluated the impacts of 636 transcription factor binding activities on histone modification in 24 cancer types. We performed Instrumental Variables analysis by using genetic lesions of TFs as our instrumental proxies, which previously discovered to be associated with histone mark activities. As a result, we showed a total of 6 EpiTFs as strong directors of epigenetic reprogramming of histone modification in cancers, which alters the molecular and clinical phenotypes of cancer. Together our findings highlight a causal mechanism driven by the TFs and genome-wide histone modification, which is relevant to multiple status of oncogenesis.

Diverse modes of H3K36me3-guided nucleosomal deacetylation by Rpd3S

Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation1–4. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions5–7. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3–Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal DNA and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic centre of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated histone H3 lysine 4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered histone H3 acetylated lysine 9. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.

Incomplete erasure of histone marks during epigenetic reprogramming in medaka early development.

Epigenetic modifications undergo drastic erasure and reestablishment after fertilization. This reprogramming is required for proper embryonic development and cell differentiation. In mammals, some histone modifications are not completely reprogrammed and play critical roles in later development. In contrast, in nonmammalian vertebrates, most histone modifications are thought to be more intensively erased and reestablished by the stage of zygotic genome activation (ZGA). However, histone modifications that escape reprogramming in nonmammalian vertebrates and their potential functional roles remain unknown. Here, we quantitatively and comprehensively analyzed histone modification dynamics during epigenetic reprogramming in Japanese killifish, medaka (Oryzias latipes) embryos. Our data revealed that H3K27ac, H3K27me3, and H3K9me3 escape complete reprogramming, whereas H3K4 methylation is completely erased during cleavage stage. Furthermore, we experimentally showed the functional roles of such retained modifications at early stages: (i) H3K27ac premarks promoters during the cleavage stage, and inhibition of histone acetyltransferases disrupts proper patterning of H3K4 and H3K27 methylation at CpG-dense promoters, but does not affect chromatin accessibility after ZGA; (ii) H3K9me3 is globally erased but specifically retained at telomeric regions, which is required for maintenance of genomic stability during the cleavage stage. These results expand the understanding of diversity and conservation of reprogramming in vertebrates, and unveil previously uncharacterized functions of histone modifications retained during epigenetic reprogramming.

Development of homozygous tetraploid potato and whole genome doubling-induced the enrichment of H3K27ac and potentially enhanced resistance to cold-induced sweetening in tubers.

Polyploid plants typically display advantages on some agronomically important traits over their diploid counterparts. Extensive studies have shown genetic, transcriptomic, and epigenetic dynamics upon polyploidization in multiple plant species. However, few studies have unveiled those alternations imposed only by ploidy level, without any interference from heterozygosity. Cultivated potato is highly heterozygous. Thus, in this study, we developed two homozygous autotetraploid lines and one homozygous diploid line in parallel from a homozygous diploid potato. We confirmed their ploidy levels using chloroplast counting and karyotyping. Oligo-FISH and genome re-sequencing validated that these potato lines are nearly homozygous. We investigated variations in phenotypes, transcription, and histone modifications between two ploidies. Both autotetraploid lines produced larger but fewer tubers than the diploid line. Interestingly, each autotetraploid line displayed ploidy-related differential expression for various genes. We also discovered a genome-wide enrichment of H3K27ac in genic regions upon whole-genome doubling (WGD). However, such enrichment was not associated with the differential gene expression between two ploidies. The tetraploid lines may exhibit better resistance to cold-induced sweetening (CIS) than the diploid line in tubers, potentially regulated through the expression of CIS-related key genes, which seems to be associated with the levels of H3K4me3 in cold-stored tubers. These findings will help to understand the impacts of autotetraploidization on dynamics of phenotypes, transcription, and histone modifications, as well as on CIS-related genes in response to cold storage.

Coordination of zygotic genome activation entry and exit by H3K4me3 and H3K27me3 in porcine early embryos.

Histone modifications are critical epigenetic indicators of chromatin state associated with gene expression. Although the reprogramming patterns of H3K4me3 and H3K27me3 have been elucidated in mouse and human preimplantation embryos, the relationship between these marks and zygotic genome activation (ZGA) remains poorly understood. By ultra-low-input native chromatin immunoprecipitation and sequencing, we profiled global H3K4me3 and H3K27me3 in porcine oocytes and in vitro fertilized (IVF) embryos. We observed sharp H3K4me3 peaks in promoters of ZGA genes in oocytes, and these peaks became broader after fertilization and reshaped into sharp peaks again during ZGA. By simultaneous depletion of H3K4me3 demethylase KDM5B and KDM5C, we determined that broad H3K4me3 domain maintenance impaired ZGA gene expression, suggesting its function to prevent premature ZGA entry. In contrast, broad H3K27me3 domains underwent global removal upon fertilization, followed by a re-establishment for H3K4me3/H3K27me3 bivalency in morulae. We also found that bivalent marks were deposited at promoters of ZGA genes, and inhibiting this deposition was correlated with the activation of ZGA genes. It suggests that promoter bivalency contributes to ZGA exit in porcine embryos. Moreover, we demonstrated that aberrant reprogramming of H3K4me3 and H3K27me3 triggered ZGA dysregulation in somatic cell nuclear transfer (SCNT) embryos, whereas H3K27me3-mediated imprinting did not exist in porcine IVF and SCNT embryos. Our findings highlight two previously unknown epigenetic reprogramming modes coordinated with ZGA in porcine preimplantation embryos. Finally, the similarities observed between porcine and human histone modification dynamics suggest that the porcine embryo may also be a useful model for human embryo research.

Lysine demethylase 2B regulates angiogenesis via Jumonji C dependent suppression of angiogenic transcription factors

Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.

Transcriptional and epigenetic control of early life cell fate decisions.

Global epigenetic reprogramming of the parental genomes after fertilization ensures the establishment of genome organization permissive for cell specialization and differentiation during development. In this review, we highlight selected, well-characterized relationships between epigenetic factors and transcriptional cell fate regulators during the initial stages of mouse development. Blastomeres of the mouse embryo are characterized by atypical and dynamic histone modification arrangements, noncoding RNAs and DNA methylation profiles. Moreover, asymmetries in epigenomic patterning between embryonic cells arise as early as the first cleavage, with potentially instructive roles during the first lineage allocations in the mouse embryo. Although it is widely appreciated that transcription factors and developmental signaling pathways play a crucial role in cell fate specification at the onset of development, it is increasingly clear that their function is tightly connected to the underlying epigenetic status of the embryonic cells in which they act. Findings on the interplay between genetic, epigenetic and environmental factors during reprogramming and differentiation in the embryo are crucial for understanding the molecular underpinnings of disease processes, particularly tumorigenesis, which is characterized by global epigenetic rewiring and progressive loss of cellular identity.

Investigating Histone Modification Dynamics by Mechanistic Computational Modeling.

The maintenance of transcriptional states regulated by histone modifications and controlled switching between these states are fundamental concepts in our understanding of nucleosome-mediated epigenetic memory. Any approach relying on genome-wide bioinformatic analyses alone offers limited scope for dissecting the molecular mechanisms involved in maintenance and switching. Mechanistic mathematical models-describing the dynamics of histone modifications at individual genomic loci-offer an alternative way to investigate these mechanisms. These models, in conjunction with quantitative experimental data-ChIP data, quantification of mRNA levels, and single-cell fluorescence tracking in clonal lineages-can generate predictions that drive more targeted experiments, allowing us to understand mechanisms that would be challenging to unravel by a purely experimental approach. In this chapter, we describe a generic stochastic modeling framework that can be used to capture histone modification dynamics and associated molecular processes-including transcription and read-write feedback by chromatin modifying complexes-at individual genomic loci. Using a specific example-transcriptional silencing by Polycomb-mediated H3K27 methylation-we demonstrate how to construct and simulate a stochastic histone modification model. We provide a step-by-step guide to programming simulations for such a model and discuss how to analyze the simulation output.

High-spatial and colourimetric imaging of histone modifications in single senescent cells using plasmonic nanoprobes

Histones are closely related to the state of chromatin, and epigenetic modification of their tail results in regulation in cells. Therefore, developing various analytical tools to map the changes in position and distribution of histone modifications is helpful in studying underlying mechanisms. Herein, we propose a high-spatial and colourimetric imaging method using plasmonic nanoparticles as probes to visualize heterochromatin histone markers in a single nucleus. We visualized the reorganization between repressive histone markers, H3K9me3 and H3K27me3, caused by oncogene-induced senescence based on the scattering colours and spectral shift of plasmonic nanoprobes to longer wavelengths using their distance-dependent coupling effect. The measured scattering profiles were correlated with the computation results simulating the scattering spectra according to the arrangements and distances among the plasmonic nanoprobes. The plasmonic nanoprobe-based high-spatial hyperspectral imaging provides an advanced way to study the dynamics of histone modifications for predicting the progression of diseases or senescence.

Histone Modifying Enzymes as Targets for Therapeutic Intervention in Oesophageal Adenocarcinoma.

Simple SummaryDespite advances in both the surgical and medical management of oesophageal adenocarcinoma (OAC), overall prognosis remains poor, with less than 1 in 5 patients surviving more than 5 years from diagnosis. Patients suitable for treatment with curative intent receive pre-operative chemotherapy, but few (<20%) gain a clinically meaningful response. Epigenetic targets have been identified as key drivers in OAC. With therapies targeting histone-modifying enzymes (HME) already in clinical use in several cancer types, they may be a potential novel therapeutic in OAC. This review outlines the current literature surrounding the roles of HMEs in OAC tumorignesis and therapeutic efficacy.Oesophageal adenocarcinoma (OAC) has a dismal prognosis, where curable disease occurs in less than 40% of patients, and many of those with incurable disease survive for less than a year from diagnosis. Despite the widespread use of systematic chemotherapy in OAC treatment, many patients receive no benefit. New treatments are urgently needed for OAC patients. There is an emerging interest in epigenetic regulators in cancer pathogenesis, which are now translating into novel cancer therapeutic strategies. Histone-modifying enzymes (HMEs) are key epigenetic regulators responsible for dynamic covalent histone modifications that play roles in both normal and dysregulated cellular processes including tumorigenesis. Several HME inhibitors are in clinical use for haematological malignancies and sarcomas, with numerous on-going clinical trials for their use in solid tumours. This review discusses the current literature surrounding HMEs in OAC pathogenesis and their potential use in targeted therapies for this disease.

Per1/Per2-Igf2 axis-mediated circadian regulation of myogenic differentiation.

Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter-enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.

Histone modification dynamics at H3K27 are associated with altered transcription of in planta induced genes in Magnaporthe oryzae.

Transcriptional dynamic in response to environmental and developmental cues are fundamental to biology, yet many mechanistic aspects are poorly understood. One such example is fungal plant pathogens, which use secreted proteins and small molecules, termed effectors, to suppress host immunity and promote colonization. Effectors are highly expressed in planta but remain transcriptionally repressed ex planta, but our mechanistic understanding of these transcriptional dynamics remains limited. We tested the hypothesis that repressive histone modification at H3-Lys27 underlies transcriptional silencing ex planta, and that exchange for an active chemical modification contributes to transcription of in planta induced genes. Using genetics, chromatin immunoprecipitation and sequencing and RNA-sequencing, we determined that H3K27me3 provides significant local transcriptional repression. We detail how regions that lose H3K27me3 gain H3K27ac, and these changes are associated with increased transcription. Importantly, we observed that many in planta induced genes were marked by H3K27me3 during axenic growth, and detail how altered H3K27 modification influences transcription. ChIP-qPCR during in planta growth suggests that H3K27 modifications are generally stable, but can undergo dynamics at specific genomic locations. Our results support the hypothesis that dynamic histone modifications at H3K27 contributes to fungal genome regulation and specifically contributes to regulation of genes important during host infection.

Modulation of cell cycle progression and chromatin dynamic as tolerance mechanisms to salinity and drought stress in maize.

Salinity and drought are the major abiotic stresses that disturb several aspects of maize plants growth at the cellular level, one of these aspects is cell cycle machinery. In our study, we dissected the molecular alterations and downstream effectors of salinity and drought stress on cell cycle regulation and chromatin remodeling. Effects of salinity and drought stress were determined on maize seedlings using 200 mM NaCl (induced salinity stress), and 250 mM mannitol (induced drought stress) treatments, then cell cycle progression and chromatin remodeling dynamics were investigated. Seedlings displayed severe growth defects, including inhibition of root growth. Interestingly, stress treatments induced cell cycle arrest in S-phase with extensive depletion of cyclins B1 and A1. Further investigation of gene expression profiles of cell cycle regulators showed the downregulation of the CDKA, CDKB, CYCA, and CYCB. These results reveal the direct link between salinity and drought stress and cell cycle deregulation leading to a low cell proliferation rate. Moreover, abiotic stress alters chromatin remodeling dynamic in a way that directs the cell cycle arrest. We observed low DNA methylation patterns accompanied by dynamic histone modifications that favor chromatin decondensation. Also, the high expression of DNA topoisomerase 2, 6 family was detected as consequence of DNA damage. In conclusion, in response to salinity and drought stress, maize seedlings exhibit modulation of cell cycle progression, resulting in the cell cycle arrest through chromatin remodeling.