Academic literature on the topic 'Epigentics'

Many different factors are involved in the progression of cancers. Genes mutations and chromosomal abnormalities are normally considered main cause of cancers but there are some other reason for the development of cancers. Other cancer causing factors are known as epigenetic alterations [1,2]. Epigentic modification of genome is known as epigenetic alterations, lead toward cancer cells production. Epigentic modification does not cause change in sequences of nucleotide. Similar to genetic alteration epigenetic alteration can’t be ignored [3]. Basically mechanisms behind epigenetic modifications are deregulation of DNA proteins, change in CpG island methylation, change in histone, oncogenes activation and deactivation of tumor suppressor [4]. Epigenetic alterations is directly linked with functional alterations of genome. Alteration in DNA methylation, histone degeneration and functional and structural abnormalities of chromosomes are the major examples of epigenetic modifications [5]. The main function of all epigenetic alterations is to modulate gene expression with same DNA sequences. Means these changes never effect main basal sequence oF DNA [6], which remain same in cell division [7]. Many different types of cancers contains large number of epigenetic alterations, the most important of these are epigenetic alterations that occurs in DNA repair genes. These DNA repair genes drive slow expression of DNA proteins. These abnormalities cause genetic unreliability, which is mainly considered as characteristic of various cancers [8,9].

Epigenetic modifiers are the proteins involved in establishing and maintaining the epigenome of an organism. They are particularly important for development. Changes in epigenetic modifiers have been shown be lethal, or cause diseases. Our laboratory has developed an ENU mutagenesis screen to produce mouse mutants displaying altered epigenetic gene silencing. The screen relies on a GFP transgene that is expressed in red blood cells in a variegated manner. In the orginal transgenic FVB mice expression occurs in approximately 55% of red blood cells. During the course of my Masters, I characterised four different Mommes (Modifiers of murine metastable epiallele), MommeD32, MommeD33, MommeD35 and MommeD36. For each Momme, I identified the underlying mutation, and observed the corresponding phenotype. In MommeD32 the causative mutation is in Dnmt1, (DNA methyltransferase 1). This gene was previously identified in the screen, as MommeD2, and the new allele, MommeD32 has a change in the BAH domain of the protein. MommeD33 is the result of a change at the transgene itself. MommeD35 carries a mutation in Suv39h1 (suppressor of variegation 3-9 homolog 1). This gene has not previously been identified in the screen, but it is a known epigenetic modifier. MommeD36 had the same ENU treated sire as MommeD32, and I found that it has the same mutation as MommeD32. These mutant strains provide valuable tools that can be used to further our knowledge of epigenetic reprogramming. An example being the cancer study done with MommeD9 which has a mutation in Trim28. By crossing MommeD9+/- mutant mice with Trp53+/- mice, it can be seen if Trim28 has an effect on the rate of tumour genesis. However no clear effect of Trim28 haploinsufficiency can be observed in Trp53+/- mice.

Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X-chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals.¶ In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings.¶ The first paper reports a study of the imprinted locus responsible for the human Prader-Willi and Angelman syndromes (PWS and AS). A search for kangaroo and platypus orthologues of PWS-AS genes identified only the putative AS gene UBE3A, and showed it was in a completely different genomic context to that of humans and mice. The only PWS gene found in marsupials (SNRPN) was located in tandem with its ancient paralogue SNRPB, on a different chromosome to UBE3A. Monotremes apparently have no orthologue of SNRPN. The several intronless genes of the PWS-AS domain also have no orthologues in marsupials or monotremes or non-mammal vertebrates, but all have close paralogues scattered about the genome from which they evidently retrotransposed. UBE3A in marsupials and monotremes, and SNRPN in marsupials were found to be expressed from both alleles, so are not imprinted. Thus, the PWA-AS imprinted domain was assembled from many non-imprinted components relatively recently, demonstrating that the evolution of imprinting has been an ongoing process during mammalian radiation.¶ In the second paper, I examine the evolution of the X-inactivation centre, the key regulatory region responsible for X-chromosome inactivation in humans and mice, which is imprinted in mouse extraembryonic membranes. By sequencing and aligning flanking regions across the three mammal groups and non-mammal vertebrates, I discovered that the region homologous to the X-inactivation centre, though intact in birds and frogs, was disrupted independently in marsupial and monotreme mammals. I showed that the key regulatory RNA of this locus (X-inactive specific transcript or XIST) is absent, explaining why a decade-long search for marsupial XIST was unsuccessful. Thus, XIST is eutherian-specific and is therefore not a basic requirement for X-chromosome inactivation in all mammals.¶ The broader significance of the findings reported in these two papers is explored with respect to other current work regarding the evolution and construction of imprinted loci in mammals in the form of a review. This comparison enabled me to conclude that like the PWS-AS domain and the X-inactivation centre, many domains show unexpected construction from disparate genomic elements that correlate with their acquisition of imprinting.¶ The fourth and last paper examines the evolution of CCCTC-binding Factor (CTCF) and its parologue Brother Of Regulator of Imprinted Sites (BORIS) which contribute to the establishment and interpretation of genomic imprinting at the Insulin-Like Growth Factor 2/H19 locus. In this paper I show that the duplication of CTCF giving rise to BORIS occurred much earlier than previously recognised, and demonstrate that a major change in BORIS expression (restriction to the germline) occurred in concert with the evolution of genomic imprinting. The papers that form the bulk of this thesis show that the evolution of epigenetic traits such as genomic imprinting and X-chromosome inactivation is labile and has apparently responded rapidly to different selective pressures during the independent evolution of the three mammal groups. I have introduced these papers, and discussed them generally in terms of current theories of how and why these forms of monoallelic expression have evolved in mammals.

The mop2 gene is required for epigenetic silencing; it was originally defined as a mutation, Mop2-1, which when dominant prevented paramutation at b1. Paramutation is an allele communication that causes a mitotically and meiotically heritable change in gene expression. Mop2-1 was subsequently shown to be involved in maintaining the silenced paramutant state and to prevent dsRNA-mediated transcriptional gene silencing (activities revealed only when the mutation is homozygous). Understanding the product encoded by mop2 will help dissect the underlying mechanisms involved in paramutation and dsRNA-mediated transcriptional silencing. This dissertation describes map-based cloning and candidate gene approaches directed toward the eventual goal of identification of mop2.Initial mapping of mop2 placed it within a region delineated by the markers umc1823 and eks1. On the maize physical map this region contains 21 BAC (Bacteria Artificial Chromosome) clones, representing 2.9 Mb. Skim sequencing identified additional markers for mapping and revealed the gene content. Extensive candidate gene examinations, including gene sequencing, expression profiling with microarrays and RT-PCR, and complementation tests with mutant alleles did not identify any of the four chromatin and RNAi-related genes as mop2.The new markers developed from the skim sequence enabled further mapping and molecular genotyping, which revealed that the Mop2-1 mutation was unstable. Approxi¬mately 10% of phenotypic heterozygous plants were actually genotypic homozygous. Further mapping using only Mop2-1 homozygous plants reduced the mop2 interval to a region of nine BACs, containing 57 genes.The mop2 region is highly syntenic to a rice region of 1.25 Mb on chromosome 4. The gene alignment and repetitive sequence analyses between the syntenic regions in these two species revealed both syntenic and non-syntenic blocks of sequences. Analyses suggested several potential mechanisms for the collinearity breakage, including, but not limited to, tandem duplications of genes in one species but not the other and the presence of gene fragments in maize, but not in rice.The research described herein provides the basis for continued efforts to clone mop2. Fine-structure mapping with new markers and a larger population, as well as candidate gene sequencing in the Mop2-1 BAC library, should be pursued to clone mop2.

La dépression est fréquente, sa prise en charge difficile et les connaissances de sa physiopathologie très incomplètes. Il est établi qu’il existe une composante familiale et héréditaire au trouble dépressif mais le substrat biologique de cette vulnérabilité est inconnu et souvent les études souffrent d’un manque de reproductibilité. Par ailleurs, il n’existe pas de bio-marqueur validé utilisable en pratique courante.Nous proposons dans ce travail de thèse d’explorer les ARN périphériques chez les patients souffrant de dépression de façon à explorer s’ils peuvent définir des bio-marqueurs et si leur étude peut nous permettre de mieux comprendre le processus physiopathologique.Nous avons recruté des patients souffrant de dépression sévère dans plusieurs études pour répondre à nos objectifs. Nous avons été attentif dans ces études à des problèmes méthodologiques importants, en particulier à propos du choix des gènes de contrôle pour les PCR en temps réel, du choix de critères statistiques dans l’étude pan-génomique et de la prise en compte de prélèvements répétés chez les sujets sains pour contrôler toute variation due à des facteurs non contrôlés. En accord avec une abondante littérature sur le sujet, nous avons pu mettre en évidence des gènes déjà décrits dont l’expression transcriptionnelle est dérégulée chez les patients par rapport aux sujets contrôles ou au cours de l’évolution de la dépression, dans des études centrées sur des gènes candidats et une étude pan-génomique.Nous avons pu également mettre en évidence de façon nouvelle l’expression dérégulée chez les patients déprimés de gènes impliqués dans la régulation chromatinienne ou l’expression des gènes.Nous avons également pu nous rendre compte que les approches pan-génomiques complétaient l’approche gène candidat avec une meilleure convergence que ne le laissait supposer la littérature.Nous avons également étudié les micro-ARN et mis en évidence qu’un certain nombre d’entre eux étaient des marqueurs traits ou des marqueurs liés à l’état au cours de la dépression.L’ensemble des données issues de notre étude pan-génomique et de notre étude sur les micro-ARN montre qu’il existe des interactions probables entre les micro-ARN et les ARNm dérégulés et ces données confirment le rôle possible des gènes régulant la chromatine ou l’expression des gènes dans la dépression.Enfin, l’étude des variabilités inter- et intra-individuelles de l’expression génétique confirme l’absence d’altération globale de la transcription au cours de la dépression et souligne l’importance d’un ensemble de molécules régulant la transcription dont l’expression est contrainte c’est à dire très peu variable d’un individu à l’autre et d’un moment à l’autre chez un même sujet.Si nous n’avons pas pu mener une étude validant des marqueurs biologiques, nos résultats ouvrent la voie à l’exploration à plus grande échelle de ces marqueurs potentiels comme à l’étude d’hypothèses originales sur la physiopathologie de la dépression
Major depression is a frequent and severe disease whose treatment is often inconsistent and patients care remains insufficient. Despite some hypothesis which implicate mono-amine and genetic factors, the pathophysiology of major depression remains unclear. Moreover, no biological marker is available in current clinical practice.Our work aims to propose methodological tools and offers preliminary results to develop such biological markers by studying gene expression in peripheral blood mononuclear cells from severe depressive patients and sex and age-matched controls in different comparative prospective studies. Candidate gene and pangenomic approaches were combined. Moreover, we explored for the first time human microRNA transcription variation in major depression by multiplex RT-qPCR.Among our main findings, we demonstrate that some well-known candidate gene such as serotonin transporter mRNA could be interesting biomarkers of major depression evolution or prognosis. In addition, pangenomic study highlights the implication of genes related to chromatin structure and gene expression regulation like histone family. We also identified variations in the expression of a set of microRNAs during a major depressive episode and, with in silico approaches, we propose putative functional interactions between candidate miRNAs and mRNAs.Overall, our work underlines the feasibility and the relevance of studying the level of expression of RNAs in a psychiatric disorder using peripheral tissues. We obtained both convergent and novel results in regard to previous investigations opening the way to better knowledge of major depression pathophysiology as well as biomarkers development

In dieser Arbeit wurde die funktionelle Rolle und Regulation des murinen Transkriptionsfaktor Foxp3 untersucht. Der erste wesentliche Teil zur Analyse der funktionellen Rolle war dabei die Erzeugung einer BAC- transgenen Maus. Hierfür wurde ein Zielgenvektor mit der kodierenden Region des eYFPs und einer dualen Selektionskassette sowie die Methode des ET- Klonierens verwendet. Leider war die homologe Rekombination des Zielgenvektors in den BAC nicht erfolgreich. Es kam zu einer ungeklärten Rekombination mit Fremd- DNS. Die Erzeugung der transgenen Maus wurde nach diesem Ergebnis eingestellt, und es wurde mit einer von unserem Kooperationspartner zur Verfügung gestellten BAC- transgenen Maus weitergearbeitet. Diese Maus, die DEREG- Maus, wurde nach dem gleichen Prinzip erstellt, wie die in dieser Arbeit gestartete transgene Maus, an Stelle des eYFPs trägt die DEREG- Maus die kodierenden Region des GFPs und des Diphtheria- Toxin- Rezeptors. Mit dieser Maus wurden erste Analysen zur Überprüfung der transgenen Maus unternommen. Es wurde die Koexpression von GFP und Foxp3, sowie die Depletion der Foxp3+ T- Zellen mittels Diphtheria- Toxin analysiert. Als nächstes wurde die funktionelle Rolle des Transkriptionsfaktors Foxp3 analysiert. Als einer der ersten Schritte wurde die Stabilität von Foxp3 in vivo überprüft und gezeigt, dass T- Zellen, die das Foxp3- Protein exprimieren, bis zu 14 Tage in vivo stabil sind. Weiterhin wurde die Stabilität der Foxp3- Expression in in vitro Kulturen nach Induktion durch TGF-beta untersucht. Die induzierten Tregs zeigten keine stabile Foxp3- Expression und auch bei der Methylierungsanalyse der TSDR zeigten diese T- Zellen nicht das für ex vivo isolierte Foxp3+ T- Zellen beschriebene Methylierungsmuster. Die Stabilität scheint mit der Demethylierung der TSDR zu korrelieren. Die induzierten Tregs zeigten neben dem nicht stabilen Foxp3- Phänotyp auch eine von der Foxp3- Expression abhängige Suppression von naiven Zellen im in vitro Proliferations- Test. Im dritten Teil der Arbeit wurde die Struktur und Regulation des Transkriptionsfaktors Foxp3 untersucht. Der Lokus wurde auf konservierte Regionen im Vergleich zu den Spezies Maus, Mensch, Ratte, Huhn, Schimpanse, Hund und Frosch untersucht. Die in Floess*, Freyer* et al. (63) gefundenen Region TSDR enthält einen hochkonservierten Bereich. Die Region wurde auf mögliche Transkriptionsfaktor- Bindungsstellen hin analysiert, und ebenfalls wurden in diesem Bereich Histon- Modifikationen für die Acetylierung der Histone H3 und H4, sowie Tri- Methylierung des Lysin4 des Histons H3 gefunden. Die TSDR wurde in Luciferase- Tests auf ihre transkriptionelle Aktivität hin getestet und zeigte einem Enhancer ähnliche unterstützende Aktivität. Die Methylierung der TSDR in den Luciferase- Tests führte zu einer Reduktion der transkriptionellen Aktivität. Deletionsmutanten der TSDR konnten den Bereich für die transkriptionelle Aktivität weiter einschränken und zeigten ein 275pb großes Fragment auf, in welchem viele interessante, mögliche Transkriptionsfaktor- Bindungsstellen und auch die größte Anzahl der differentiell methylierten CpG- Motive liegen.
The aim of the study was to analyze the function and regulation of the transcription factor Foxp3. In a first step we designed a BAC-transgenic mouse with eYFP under the control of the Foxp3 promoter. For creating these mice we use the ET- cloning method. The step of homologous recombination of the target vector into the BAC failed. Because of that, we decided to work in cooperation with the group of Tim Sparwasser from Munich and their BAC- transgenic mouse called DEREG- mouse. This mouse expresses the coding region of eGFP fused to the diphtheria- toxin- receptor under the control of the Foxp3 promoter. Therefore Foxp3+ T cells can be easily detected by eGFP expression and can even be depleted by diphtheria- toxin- application. We confirmed the co- expression of Foxp3 and eGFP and furthermore tested the functionality of the depletion- process of Foxp3+ T cells by treatment with diphtheria- toxin. In a second study, we analyzed the stability of Foxp3 expressing cells in vivo. Therefore we transferred Foxp3+ T cells in syngenic mice and analyzed these cells after 14 days for their Foxp3- expression. Furthermore, we tested the induction of Foxp3 expression through TGF-beta and the suppressive activity of these cells. We also analyzed those cells for their methylation pattern, comparing cells, which showed an induction of Foxp3- expression after one week of culture with TGF-beta to cells, which received TGF-beta for one week and were then restimulated in the absence of TGF-beta. The stability of Foxp3 expression seems to correlate with the demethylated state of the TSDR (Treg Specific Demethylated Region). To get a closer look on the region called TSDR in the murine foxp3 locus, we decided to analyze this region under different aspects. First, we checked for putative binding sites of transcription factors by database analysis of the TSDR. We also analysed histon modifications, such as acetylation of histon H3 and H4 and tri- methylation of lysine 4 at histon3, in this region. Presence of these modifications hinted an epigenetic regulation of Foxp3 involving the TSDR. In a last step, the transcriptional activity of TSDR was tested to delineate whether the TSDR serves as an alternative promoter or acts as a regulative element like an enhancer. Luciferase assays showed that TSDR is a regulative enhancer element, which loses transcriptional activity when methylated. Deletion mutants determined the most important fragment of the TSDR.

The Australian Zebra Finch (Taeniopygia guttata) is an important animal model for vertebrate development and behavior. New research initiatives in the fields of epigenetics rely heavily on injecting hormones and environmental toxins directly into the eggs of different bird species such as zebra finches and other passerine songbirds to replicate the effects maternal condition on offspring. However, the widely used method of egg-injections does not accurately replicate physiological conditions, as the injected substances remain concentrated at the injection site for extended periods and do not diffuse into the developing tissues. Therefore, we propose an alternative method to injection protocols that takes advantage of the porous nature of eggs. Corticosterone (CORT), a major vertebrate stress hormone, dissolved in ethyl alcohol was applied to the surface of zebra finch eggs daily. The effect of this treatment on decreasing hatching success shows that topical hormonal treatments are a viable alternative to egg injection.

Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X-chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals.¶ In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings.¶ ...

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour which secretes ACTH and other related peptides. Contrary to normal production by the pituitary, ACTH production is not inhibited by glucocorticoids (Gcs) in SCLC. This insensitivity to Gc action can be attributed to impaired Gc receptor (GR) expression in these cells. Over-expression of the GR induces apoptosis both in vitro and in vivo. Evasion of GR signalling thus confers a significant survival advantage to SCLC cells. Re-expression of endogenous GR in SCLC cells may provoke the same effect. Many tumours silence the expression of tumour suppresser genes by epigenetic mechanisms. Recent evidence suggests that the GR in SCLC cells is epigenetically silenced by hypermethylation of its promoter. The overall aim of this study was to determine whether endogenous GR re-expression induces apoptosis of SCLC cells. The DMS 79 SCLC cell line, and the control HEK and non-SCLC A549 cell lines were treated with the DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (5-aza), to determine whether treatment with 5-aza results in re-expression of endogenous GR. Conflicting results were thought to result from the use of possibly degraded 5-aza. However, a quantitative real-time PCR analysis using newly purchased, freshly prepared 5-aza indicated that 5-aza treatment up-regulated GR mRNA expression in the DMS 79 cells (p<0.0005). No significant changes in GR expression were seen in the HEK and/or A549 cells, suggesting that the GR in these cell lines is not methylated. Contrary to expectations and possibly due to the use of degraded stock, Western blot analysis revealed that 5-aza had no effect on GR protein expression in DMS 79 cells, yet affected GR protein expression in HEK and A549 cells (p=0.003 and p=0.042, respectively). Cell viability assays indicated that treatment with varying concentrations of 5-aza had no effect on the viability of DMS 79 and A549 cells, but had a minimal effect on HEK cell (p<0.0005) viability. These data reinforce the hypothesis that stock 5-aza had degraded as 5-aza is known to exert cytotoxic effects at higher concentrations. Using newly purchased, freshly prepared 5-aza, flow cytometry and/or microscopy were performed to establish whether endogenous GR re-expression was sufficient to kill the SCLC cells by apoptosis. FITC Annexin V staining and nuclear morphology showed that significant proportions of the 1 μM (p=0.010 and p=0.027) and 5 μM (p=0.002 and p=0.018) 5-aza treated DMS 79 cells were apoptosing, with little apoptosis seen in HEK cells. 5-Aza induced negligible HEK cell death, as determined by microscopic analyses. The effect of dexamethasone (Dex; a synthetic Gc) on HEK and DMS 79 cells was examined to determine whether Gc treatment could enhance apoptosis. Treatment with Dex alone, and in combination with 5-aza, resulted in significant HEK cell death (p=0.046 and p=0.005 respectively), but not apoptosis. This was unexpected as HEK cells express very little unmethylated GR, and may be due to excessive drug exposure or combined drug toxicity. The same effect was observed with DMS 79 cells (p=0.003 and p<0.0005 respectively), with 5-aza appearing to enhance cell death induced by Dex. No effects on apoptosis were seen confirming earlier reports that GR-mediated apoptosis is ligand-independent. As 5-aza does not selectively demethylate the GR, cells were exposed to the GR antagonist, RU486, to establish whether apoptosis associated with 5-aza treatment is specifically due to demethylation and subsequent expression of the GR. Treatment with RU486 in conjunction with 5-aza induced cell death (p=0.014), but not apoptosis, of HEK cells. Again, this may have been due to excessive drug exposure or combined drug toxicity. Flow cytometric data showed that DMS 79 cell death was induced by both RU486 (p=0.004), and RU486 in combination with 5-aza (p=0.003). Furthermore, although not significant, RU486 treatment appeared to inhibit apoptosis induced by 5-aza in the DMS 79 cells. The data suggest that re-expression of the GR may be responsible for apoptotic induction. Our findings, although not significant, hint that endogenous re-expression of the GR leads to apoptosis. Unlike mutations, epigenetic marks are reversible and clinical trials with DNMTis have shown promising results. The identification of a novel endogenous mechanism that specifically induces apoptosis of SCLC cells offers great promise for the development of targeted therapeutics for the treatment of this deadly disease.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Project objectives: 1) Characterization of variation for yield heterosis in melon using Half-Diallele (HDA) design. 2) Development and implementation of image-based yield phenotyping in melon. 3) Characterization of genetic, epigenetic and transcriptional variation across 25 founder lines and selected hybrids. The epigentic part of this objective was modified during the course of the project: instead of characterization of chromatin structure in a single melon line through genome-wide mapping of nucleosomes using MNase-seq approach, we took advantage of rapid advancements in single-molecule sequencing and shifted the focus to Nanoporelong-read sequencing of all 25 founder lines. This analysis provides invaluable information on genome-wide structural variation across our diversity 4) Integrated analyses and development of prediction models Agricultural heterosis relates to hybrids that outperform their inbred parents for yield. First generation (F1) hybrids are produced in many crop species and it is estimated that heterosis increases yield by 15-30% globally. Melon (Cucumismelo) is an economically important species of The Cucurbitaceae family and is among the most important fleshy fruits for fresh consumption Worldwide. The major goal of this project was to explore the patterns and magnitude of yield heterosis in melon and link it to whole genome sequence variation. A core subset of 25 diverse lines was selected from the Newe-Yaar melon diversity panel for whole-genome re-sequencing (WGS) and test-crosses, to produce structured half-diallele design of 300 F1 hybrids (MelHDA25). Yield variation was measured in replicated yield trials at the whole-plant and at the rootstock levels (through a common-scion grafted experiments), across the F1s and parental lines. As part of this project we also developed an algorithmic pipeline for detection and yield estimation of melons from aerial-images, towards future implementation of such high throughput, cost-effective method for remote yield evaluation in open-field melons. We found extensive, highly heritable root-derived yield variation across the diallele population that was characterized by prominent best-parent heterosis (BPH), where hybrids rootstocks outperformed their parents by 38% and 56 % under optimal irrigation and drought- stress, respectively. Through integration of the genotypic data (~4,000,000 SNPs) and yield analyses we show that root-derived hybrids yield is independent of parental genetic distance. However, we mapped novel root-derived yield QTLs through genome-wide association (GWA) analysis and a multi-QTLs model explained more than 45% of the hybrids yield variation, providing a potential route for marker-assisted hybrid rootstock breeding. Four selected hybrid rootstocks are further studied under multiple scion varieties and their validated positive effect on yield performance is now leading to ongoing evaluation of their commercial potential. On the genomic level, this project resulted in 3 layers of data: 1) whole-genome short-read Illumina sequencing (30X) of the 25 founder lines provided us with 25 genome alignments and high-density melon HapMap that is already shown to be an effective resource for QTL annotation and candidate gene analysis in melon. 2) fast advancements in long-read single-molecule sequencing allowed us to shift focus towards this technology and generate ~50X Nanoporesequencing of the 25 founders which in combination with the short-read data now enable de novo assembly of the 25 genomes that will soon lead to construction of the first melon pan-genome. 3) Transcriptomic (3' RNA-Seq) analysis of several selected hybrids and their parents provide preliminary information on differentially expressed genes that can be further used to explain the root-derived yield variation. Taken together, this project expanded our view on yield heterosis in melon with novel specific insights on root-derived yield heterosis. To our knowledge, thus far this is the largest systematic genetic analysis of rootstock effects on yield heterosis in cucurbits or any other crop plant, and our results are now translated into potential breeding applications. The genomic resources that were developed as part of this project are putting melon in the forefront of genomic research and will continue to be useful tool for the cucurbits community in years to come.