L’héritage épigénétique transgénérationnelle est un phénomène très controversé, selon lequel un phénotype non-génétiquement déterminé peut être transmis à la génération suivante. Jusqu'à présent, ce mode de transmission a été décrit dans quelques cas et il a été suggéré que les composants de la chromatine peuvent être impliqués, y compris des protéines du groupe Polycomb, qui agissent comme des répresseurs de gènes clés du développement et coordonnent la différenciation cellulaire et la prolifération. Les mécanismes moléculaires à la base du rôle de la répression génique Polycomb-dépendante à hérédité épigénétique transgénérationnelle sont loin d'être compris. Par conséquent, j’ai développé un système expérimental chez Drosophila melanogaster pour induire un héritage épigénétique transgénérationnelle stable, dans lequel des états d'expression génique alternatifs peuvent être transmis en présence de la même séquence d'ADN. A partir de ces « épilignes » stables, j’ai pu disséquer certaines des propriétés génétiques des épiallèles induits, tels que leur héritage quantitatif et leur capacité à communiquer à longue distance. En outre, les épiallèles montrent une synergie dans leur expression et transmission héréditaire. L'une des signatures moléculaires des épiallèles est une différence de répression médiée par les complexes Polycomb et par leur marque d’histone caractéristique. Cette distribution différente est indépendante de l’activité transcriptionnelles des gènes en aval, au moins dans un stade de développement précoce, et pourrait influer l'organisation tridimensionnelle du locus impliqué. Curieusement Ago2, un composant de la voie ARNi, a été montré interagir avec les épiallèles génétiquement et la protéine Ago2 se fixe directement à leur chromatine, ce qui indique un rôle possible pour le ncRNAs dans l'expression des épiallèles et éventuellement dans leur transmission. Ces résultats plaident en faveur e l’existence d’une hérédité épigénétique transgénérationnelle stable chez les métazoaires et fournissent un modèle qui se prête à une dissection moléculaire de ce phénomène. / Transgenerational epigenetic inheritance is a hotly debated phenomenon whereby a non-genetically determined phenotype can be transmitted to the next generation. So far, this mode of inheritance has been described in few cases and it was suggested that chromatin components might be involved, including Polycomb group proteins, which act as repressors of key developmental genes and coordinate cell differentiation and proliferation. The molecular mechanisms linking Polycomb-mediated silencing to transgenerational epigenetic inheritance are far from being understood. Therefore, I developed an experimental system in Drosophila melanogaster to induce stable transgenerational epigenetic inheritance, in which alternative gene expression states can be transmitted in the presence of the same DNA sequence. Starting from these highly stable “epilines”, I could dissect some of the genetic properties of the induced epialleles, such as their quantitative inheritance and their ability to trans-communicate. Moreover, the epialleles displayed synergy in their expression and transmission. One of the molecular signatures of the epialleles is the differential presence of the Polycomb repressive complexes and their related epigenetic marks. This different distribution is independent of the transcriptional activity of the downstream genes, at least in an early developmental stage, and could influence the three-dimensional organization of the locus involved. Intriguingly Ago2, an RNAi pathway component, has been found to genetically interact with the epialleles and to be directly bound on their chromatin, indicating a possible role for the ncRNAs in the expression of the epialleles and possibly in their transmission. These results make a case for strong and stable transgenerational epigenetic inheritance in metazoan and provide a model that is amenable for the molecular dissection of this phenomenon.

Le spermatozoïde des animaux à reproduction sexuée est une cellule extrêmement spécialisée, dont la chromatine très particulière est le siège de nombreux remodelages tant lors de la gamétogenèse que lors de la formation du zygote. Chez D. melanogaster comme chez les mammifères, lors de la spermiogenèse, les histones qui condensent l'ADN sont remplacées par des petites protéines basiques spécifiques du noyau spermatique : les protamines. Cette architecture est stabilisée par des liaisons disulfures. Lors de la fécondation, ces protéines sont éliminées du noyau paternel, qui réincorporent des histones pour former une chromatine fonctionnelle. Toutefois, les mécanismes régissant la mise en place et l'enlèvement des ponts disulfures et des protamines sont inconnus chez la Drosophile.Au cours de ma thèse, j'ai démontré l'importance de deux thiorédoxines sexuelles pour la reproduction.D'une part, j'ai pu montrer que DHD, qui est une thiorédoxine strictement maternelle, est essentielle à l'éviction des protamines de la chromatine paternelle lors de la fécondation. Sans cette protéine essentielle, la décondensation du noyau mâle n'a pas lieu, les protamines ne sont pas enlevées et le développement zygotique ne peut pas avoir lieu. Cette thiorédoxine est directement responsable de la réduction des liaisons disulfures qui stabilisent la chromatine spermatique.D'autre part, j'ai démontré que TrxT, une thiorédoxine exclusivement testiculaire, est nécessaire au bon déroulement de la spermiogenèse. Sans cette protéine, les spermatides subissent des dommages à l'ADN et sont éliminées.Ce travail met en évidence les rôles essentiels des thiorédoxines sexuelles pour la reproduction / In animal sexual reproduction, spermatozoon is a very specialized cell. Its very peculiar chromatin is remodeled both during spermiogenesis and fertilization. During mammalian and drosophilian spermiogenesis, histones involved in DNA condensation are replaced with sperm specific small nuclear basic proteins : the protamines. This sperm specific architecture is stabilized by disulfide bonds. At fertilization,protamines are removed from the male nucleus and maternally-provided histones are incorporated to form a functional paternal chromatin. However, the mecanisms involved in the incorporation and the removal of protamines of their disulfide bonds are unknown in Drosophila.During my PhD, I demonstrated that two sexual thioredoxins are important for spermiogenesis and fertilization in D. melanogaster. In one hand, I showed that DHD, a female specific thioredoxin, is essential for protamine eviction at fertilization. Without this major protein, male nucleus does not decondense, protamines are not removed from sperm chromatin and zygotic development does not occur. Besides, I demonstrated that DHD is directly responsible for the reduction of the disufide bonds which stabilize sperm chromatin.On the other hand, I showed that TrxT, a testis-specific thioredoxin, is needed for spermiogenesis. Without this protein, DNA damages appear on spermatid nuclei, and those spermatozoon are then eliminated during spermatogenesis.This work highlights that drosophilian sex-specific thioredoxins are essential for sexual reproduction success

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-13T16:00:10Z No. of bitstreams: 2 Dissertação - Rogério Orfaly Addad da Silva - 2006.pdf: 843452 bytes, checksum: 27a7d9be950071d5241ff583004bbe06 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-13T16:00:31Z (GMT) No. of bitstreams: 2 Dissertação - Rogério Orfaly Addad da Silva - 2006.pdf: 843452 bytes, checksum: 27a7d9be950071d5241ff583004bbe06 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-13T16:00:31Z (GMT). No. of bitstreams: 2 Dissertação - Rogério Orfaly Addad da Silva - 2006.pdf: 843452 bytes, checksum: 27a7d9be950071d5241ff583004bbe06 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2006-12-08 / Thirty six Nelore bulls with age between 30 and 120 months were used to evaluation of the seminal quality and the reproductive aptitude. The animals had been kept in extensive conditions, with feeding the grass, receiving mineral supplementation and serving a herd with 3,782 females of some categories (heifers and cows). Data enclosed measured of scrotal circumference and evaluation of the semen collected by eletroejaculation and evaluated the physical characteristics (volume, mass activity, motility, strong), morphologic (major, minor and total defects) and spermatic structure integrity (integrity of plasmatic membrane, acrosome and chromatin) in this period had been carried through that preceded three consecutive mating season (2003, 2004 and 2005). The data had been submitted analyze of variance, the averages compared for test SNK and the coefficient of correlation of Pearson was calculated using the SAS. The bulls presented scrotal circumference of 33.8 ± 2.8cm, 15.6 ± 8.5% of total spermatic defects, 40.1 ± 23.1% of gametes with normal membrane and acrosome and 8.8 ± 3.8% of fragmented chromatin. The relations between scrotal circumference, physical and morphologic characteristics, spermatic integrity and the reproductive performance of the herd had been low and not significant (P< 0.05). In sexually mature bulls the integrity of plasmatic membrane, acrosome and nuclear chromatin had not been the most frequent trouble of the spermatic morphology. / Foram utilizados 36 touros da raça Nelore com idade entre 30 e 120 meses, para avaliação da qualidade seminal e da aptidão reprodutiva. Os animais foram mantidos em condições extensivas, com alimentação a pasto, recebendo suplementação mineral e servindo um rebanho com 3782 fêmeas de várias categorias (novilhas, vacas primíparas e pluríparas). Foram realizadas colheitas de dados que abrangiam medidas de circunferência escrotal e avaliação do sêmen colhido por eletroejaculação e avaliadas as características físicas (volume, turbilhonamento, motilidade, vigor), morfológicas (defeitos maiores, menores e totais) e de integridade espermática (integridade de membrana plasmática, de acrossoma e de cromatina) no período que antecedeu três estações de monta consecutivas (2003, 2004 e 2005). Os dados foram submetidos a analise de variância, as médias comparadas pelo teste SNK e o coeficiente de correlação de Pearson foi calculado utilizando o SAS. Os touros apresentaram circunferência escrotal de 33,8 ± 2,8cm, 15,6 ± 8,5% de defeitos espermáticos totais, 40,1 ± 23,1% de gametas com membrana e acrossoma íntegros e 8,8 ± 3,8% de cromatina fragmentada. As relações entre circunferência escrotal, as características físicas, morfológicas e de integridade espermática e o desempenho reprodutivo do rebanho foram baixas e não significativas (P>0,05). Em touros sexualmente maduros a integridade de membrana plasmática, acrossoma e fragmentação de cromatina nuclear não foram os distúrbios mais freqüentes da morfologia espermática.

Foi estudado a influência das estações não reprodutiva e reprodutiva, dos crioprotetores glicerol e etilenoglicol e do processo de congelação e descongelação sobre a motilidade, o vigor, a morfologia e o complexo DNA-proteína de espermatozóides em garanhões, comparando o sêmen fresco, o exposto a congelação e descongelação sem crioprotetores, o exposto aos crioprotetores sem congelação e o exposto aos crioprotetores com congelação e descongelação. Foram utilizados seis garanhões da raça Mangalarga Paulista, colhendo 12 ejaculados de cada animal na estação não reprodutiva (1) e 12 ejaculados na estação reprodutiva (2), analisando a motilidade, o vigor, a concentração, a morfologia e a patologia do complexo DNA-Proteína. A patologia do complexo DNA-Proteína foi avaliada em sêmen fixado com etanol-ácido-acético glacial 3:1 (v/v), tratado com HCL 4N a 25oC e corado com azul de toluidina a 0,025% em tampão Mcllvaine, empregando microscopia óptica com aumento de 1000x. Os resultados mostraram que a motilidade, o vigor, a morfologia e a anomalia do complexo DNA-Proteína dos espermatozóides diferem entre os grupos congelado e não congelado (P<0,05). Para o grupo fresco, a taxa de patologia do complexo DNA-Proteína apresentou diferença significativa (P<0,05) entre as estações 1 e 2. O processo de congelação e descongelação exerce influência negativa sobre o complexo DNA-Proteína de espermatozóides em garanhões. / To study the effects of freezing and thawing on DNA-protein complexes in spermatozoa, 12 ejaculate were collected from each of 6 stallions (Mangalarga Paulista breed) in each of two consecutive breeding seasons. Motility, vigor, concentration, morphology and incidence of the DNA-protein complex pathologies of spermatozoa were evaluated and compared among fresh semen, semen expose to freezing and thawing without cryoprotectants and semen exposed to the cryoprotectors glycerol and ethylene glycol left at room temperature or frozen. Incidence of the DNA-protein complex pathologies was evaluated in semen smear fixed in na ethanol-acetic-acid mixture (3:1, v/v) for 1 minute, washed in 70% ethanol for 3 minutes and air dried. To induce nuclear metachromasy, smears were treated with 4N HCL at 25ot for 20 minutes followed by rinsing in distilled water. Preparation were stained with a 0.025% toluidine blue solution in Mailvaine buffer for 15 minutes. Frequency of spermatozoa exhibiting nuclear metachromasy was determined in 1000 cells/animal by light microscopy at 1000X magnification. Motility, vigor, morphology and DNA-protein complex pathologies of spermatozoa were different for semen frozen compared to not frozen (P<0.05). For fresh semen, there were effects of seasons on the incidence of DNA-protein complex pathologies (P<0.05) For semen frozen without cryoprotectants there were significant effects seasons (P<0.05) The process of cryopreservation and thawing influences negatively the DNA-protein complexes in stallion spermatozoa.

La méthylation des histones constitue un niveau important de contrôle épigénétique chez les eucaryotes. Mes études portent sur la caractérisation des facteurs potentiellement intervenant dans la mise en place et la lecture de la méthylation pour mieux apprécier son rôle et des mécanismes sous-jacents dans la régulation de la transcription et du développement des plantes chez l’Arabidopsis thaliana. Ainsi, la première partie de mes travaux de thèse a contribué à l’étude d’une protéine à domaine SET (SET DOMAIN GROUP7, SDG7) et à montrer que SDG7 est nécessaire au bon déroulement de l'induction de VIN3 et du processus de vernalisation pour la floraison. Nos résultats suggèrent que SDG7 pourrait méthyler une protéine non-histone encore inconnue dans la régulation de la transcription et le contrôle de la durée de vernalisation. La deuxième partie de ma thèse porte sur l’étude de SDG8 et les H2B-UBIQUITIN-ligases HUB1/HUB2 pour examiner un cross-talk éventuel entre la triméthylation de H3K36 (H3K36me3) et la monoubiquitination d’H2B (H2Bub1). Nous avons montré que H3K36me3 et H2Bub1 sont déposés largement indépendamment, qui diffère d’une dépendance hiérarchique de déposition préalablement observée chez la levure. La dernière partie de ma thèse a permis l’identification des protéines HUA2/HULK2 à domaine PWWP comme lecteurs éventuels de H3K36me3 dans la régulation de la floraison et du développement des plantes. / Histone methylation is one of the keys epigenetic marks evolutionarily conserved in eukaryotes. My study focuses on the characterization of factors potentially involved in the deposition and reading of lysine (K) methylation to appreciate its role and underlying mechanisms in the regulation of transcription and plant development, using Arabidopsis thaliana as a model organism. In the first part of my thesis, I report on our study of SET DOMAIN GROUP7 (SDG7), a protein containing the evolutionarily conserved SET domain, which is generally recognized as a signature of K-methyltransferases. We found that SDG7 plays an important role in the regulation of VIN3 induction associated with cold duration measure during vernalization treatment. Intriguingly, levels of several different histone methylations were found unchanged in the sdg7 mutant plants and the recombinant SDG7 protein failed to show a histone-methyltransferase activity in vitro. We thus conclude that SDG7 might methylate a yet unknown non-histone protein to regulate transcription and proper measurement of the duration of cold exposure in the vernalization process. In the second part, I studied interaction between SDG8 and HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2. My results unravel that H3K36me3 and H2Bub1 are deposited largely independently in Arabidopsis, which is in contrast to the dependent crosstalk of these two different epigenetic marks previously reported in yeast. In the last part of my thesis, I report on the identification of the PWWP-domain proteins HUA2/HULK2 as readers of H3K36me3 and demonstrate that sdg8 and hua2 genetically interacts in the regulation of flowering time.

Asf1 est une protéine chaperon d’histone, qui participe à l’assemblage et au désassemblage des histones H3/H4 sur l’ADN. Asf1 n’est pas essentiel pour la viabilité cellulaire chez S. cerevisiae, mais les voies de surveillance des dommages à l’ADN sont activées de façon constitutive dans les cellules dépourvues d’Asf1 et celles-ci sont hypersensibles à plusieurs types de stress génotoxiques. Chez S. cerevisiae, Asf1 forme un complexe stable avec Rad53 en absence de stress génotoxique. Nos résultats suggèrent qu’au moins trois surfaces d’interaction sont impliquées dans le complexe Asf1-Rad53. Le domaine FHA1 de Rad53 fixe Asf1 phosphorylé sur T270, l’extrémité C-terminale de Rad53 fixe la même surface d’Asf1 impliquée dans la fixation des co-chaperones HirA/CAF-1, et un troisième site putative est constituée de la surface d’Asf1 impliquée dans la fixation de l’histone H3 avec le domaine kinase de Rad53. Lors des stress génotoxiques, Rad53 est phosphorylée et activée. Mes résultats montrent une dissociation totale du complexe Rad53-Asf1 après traitement HU, mais la préservation du complexe après traitement des cellules avec une gamme de concentration de MMS. Nous pensons que la régulation du complexe traduisent des réponses cellulaires distinctes adaptées à des stress génotoxiques spécifiques. Par ailleurs, grâce à la structure du complexe formé par un peptide C-terminal de Rad53 et le domaine N-terminal d’Asf1, nous avons isolé une mutation rad53_A806R-L808R. Nous avons constaté que cette mutation déstabilise l’interaction entre Asf1 et Rad53 et augmente la viabilité des mutants rad9 et rad24 aux stress génotoxiquex. Ce mutant rad53_A806R-L808R semble retourne plus vite dans le cycle cellulaire et/ou traverse plus vite la phase S par rapport à Rad53-WT, et augmente la réparation de l’ADN ou l’adaptation aux dommages du simple mutant rad24Δ. / Asf1 is a histone chaperone, which participates in the assembly and disassembly of histones H3/H4 on DNA. Asf1 is not essential for cell viability in yeast, but the DNA damage checkpoints are constitutively activated in cells lacking Asf1 and they are hypersensitive to several types of genotoxic stress. In yeast, Asf1 forms a stable complex with Rad53 in the absence of genotoxic stress. Our results suggest that this complex involves at Ieast three interaction surfaces. One site involves the H3-binding surface of Asf1 with an as yet undefined surface of Rad53, probably reside in the kinase domain of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T270. The third site involves the C-terminal 21 aa of Rad53 bound to the conserved Asf1 N-terminal domain, where Rad53 competes with histone H3/H4 and co-chaperones HirA/CAF-1 for binding to the same surface of Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl methane sulfonate, suggesting a regulation of the complex as a function of the stress.In addition to these results, we also found that the rad53-A806R+L808R mutation at the C-terminus of Rad53 destabilized the Asf1-Rad53 interaction and increased the viability of rad9 and rad24 mutants to genotoxic stress. The rad53-ALRR mutant also appeared to re-enter the cell cycle and/or traverse S-phase more rapidly than wild type and increased repair or adaptation when combined with the rad24 mutant.

abstract: Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin. I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions. SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug. Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019

abstract: The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access of target DNA to Cas9. The Haynes lab has constructed fusions of enzymatic chromatin-modifying domains designed to remodel chromatin and increase Cas9 editing efficiency. With a library of PFs available, this research focuses on analyzing the behavior of Cas9 in chromatin that has been artificially opened by PFs. The types and frequency of INDELs (insertions & deletions) were determined after non-homologous end joining (NHEJ) in PF and Cas9-treated cells using quantitative Sanger sequencing and Synthego’s ICE software. Furthermore, NOME-seq analysis was carried out to map nucleosome position in PF and Cas9 treated cells. Although this experiment was unsuccessful, the heat map generated with data obtained from Synthego ICE predicts a possible presence of nucleosome in the vicinity suggesting that perhaps a fully open chromatin state was not achieved. Linear Regression analysis with certain assumptions confirms that with the increase in distance downstream of cut-site, the editing frequency decreases exponentially. Nevertheless, further experimental work should be carried out to investigate this hypothesis. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019

Transcriptional enhancers represent the primary basis for differential gene expression. These elements regulate cell type specificity, development, and evolution, with many human diseases resulting from altered enhancer activity. To date, a key gap in our knowledge is how enhancers select specific promoters for activation. To fill this gap, in this thesis, I first developed an Integrated Method for Predicting Enhancer Targets (IM-PET). Leveraging abundant “omics” data, I devised and characterized multiple genomic features for distinguishing true enhancer-promoter (EP) pairs from non-interacting pairs. I integrated these features into a probabilistic predictor for EP interactions. Multiple validation experiments demonstrated a significant improvement over extent state-of-the-art approaches. Systematic analyses of EP interactions across twelve human cell types reveals global features of EP interactions. Second, we used a well-established viral infection model to map the dynamic changes of enhancers and super-enhancers during the CD8+ T cell responses. Our analysis illustrated the complexity and dynamics of the underlying EP interactome during cell differentiation. Taking advantage of the predicted EP interactions, we constructed stage-specific transcriptional regulatory networks, which is critical for understanding the regulatory mechanism during CD8+ T cell differentiation. Third, recent progress in mapping technologies for chromatin interactions has led to a rapid increase in this type of interaction data. However, there is a lack of a comprehensive depository for chromatin interactions identified by all major technologies. To address this problem, we have developed the 4DGenome database through comprehensive literature curation of experimentally derived interactions. We envision a wide range of investigations will benefit from this carefully curated database.

During spermiogenesis, somatic chromatin is remodeled and a vast majority (> 90%) of DNA histones are replaced by short arginine-rich peptides called protamines. This compaction is immense, with protamine-DNA self-assembly in sperm chromatin resulting in a final volume roughly 1/6th of a somatic nucleus. This near crystalline organization of the DNA in sperm is thought crucial both for the transport of the paternal genes as well as for the protection of genetic information as sperm chromatin is transcriptionally inactive and all DNA repair mechanisms are shut down. Chapter 1 will include an overview of the topics discussed in this document, including: sperm chromatin, Sperm chromatin remodeling, DNA damage, and the effect of DNA damage to sperm DNA. Chapter 2 will contain a brief overview of the techniques used within this study. This includes: Small-angle X-ray Scattering, gel electrophoresis, DNA precipitation assays, and ethidium bromide dissociation assays. In chapter 3, we will discuss the effect of DNA packaging on the accessibility of free radicals to damage condensed DNA. A variety of polycations were used to condense plasmid DNA in reconstituted samples. After condensation, the DNA-polycation condensates were exposed to 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) for 1 hour, decondensed, and the plasmid DNA examined by gel electrophoresis. By comparing the intensities of the supercoiled, open coiled and linear bands, we were able to identify the presence of single-strand nicks and double-strand breaks in DNA. DNA packaging densities for all polycation-DNA systems were determined by small-angle X-Ray scattering (SAXS). Our results show that for similar length polycations, the amount of oxidative damage scales directly with the DNA packaging with more tightly condensed DNA being damaged less. However, our results also show that DNA damage is also dependent on polycation length, with DNA condensed by shorter polycations being damaged more than DNA condensed with longer polycations even at similar packaging densities. Protamine has long been thought to play a role in protecting spermatic DNA from damaging agents in vivo. However, the relationship between the hypercondensation of sperm chromatin, the DNA integrity, and the transfer of epigenetic information from sperm to oocyte and potential to alter gene expression in the early embryo are poorly understood. In Chapter 4, we examine how underprotamination affects free radical accessibility and DNA stability in reconstituted sperm chromatin. Specifically, reconstituted salmon protamine- plasmid DNA condensates (polyplexes) were formed at precise protamine/DNA ratios and subsequently subjected to exposure to AAPH free radicals. Agarose gel electrophoresis was then used to assess DNA damage by observing topology alternations in the decondensed polyplexes. FPG-DNA glycosylase has also been used to more accurately determine oxidative damage beyond just nicks and double-strand breaks in the various condensed states. We show that higher levels of protamination correlate to greater levels of protection to the DNA from oxidative damage up until full charge compensation. Furthermore, we also demonstrate that poorly compacted chromatin could be recovered by the introduction of small cationic peptides in underprotaminated condensates as well as actual sperm nuclei. SAXS studies were performed to show that the introduction of cationic peptides resulted in tighter DNA packaging densities in the underprotaminated sperm chromatin. In Chapter 5, we examine the role of disulfide bonds on DNA packaging in mammalian sperm chromatin. Mammalian protamine, unlike fish, are known to have cysteine residues capable of forming inter- and intra-protamine disulfide bonds. In bull, prior work had shown evidence for the formation of a unique hairpin secondary structure due to the folding of the ends of the protamine molecule by intramolecular disulfide linkages. Between folds is an arginine-rich region known as the DNA binding region. The DNA binding region has a local arginine fraction (~60-75%) that is much higher than the arginine fraction within the full bull protamine sequence (~50%). Previous work by the DeRouchey lab has shown that the percent arginine was crucial for DNA condensation in small arginine-rich peptides. We hypothesize that the fraction of arginine is also critical to DNA remodeling in sperm chromatin. SAXS studies showed that disulfide bond reduction resulted in complete decondensation of bull sperm nuclei. Here, we have used cysteine alkylation chemistry to add neutral or charged functional groups to the protamine cysteine, thereby inhibiting the formation of these disulfide bonds. This chemistry both prevents the formation of the hairpin as well as modifies the overall charge of the protamine. Through ethidium bromide exclusion assays, we measured binding of these altered protamines to calf thymus DNA and determined that a percent cationic charge of above 50% is necessary for the protamine to effectively condense DNA. In addition, we show that DNA condensation of bull protamine with the hairpin is nearly identical to piscine protamines which have no disulfide linkages but a net arginine fraction of 60-75%. Upon disruption of the hairpin, however, complete condensation does not occur despite a net charge on the protamine of +26.