Opioid-linked overdose death rates have reached unprecedented levels in the United States. The growing incidence of Opioid Use Disorder (OUD) is concomitant with elevated rates of OUD in women during pregnancy and through parturition. Neonates born to mothers with active OUD can develop opioid dependence in utero and display various signs of postnatal withdrawal, a condition termed Neonatal Opioid Withdrawal Syndrome (NOWS). Common symptomatic features of NOWS include sleep disturbances, low birth weight, altered heart and respiratory rates, increased irritability, high-pitched crying, feeding difficulties. Many of these symptomatic presentations are driven by dysregulated function of the autonomic nervous system and hyperirritability of the sympathetic nervous system. Given the increasing incidence of NOWS, there is an alarming lack of knowledge regarding the long-term effects of perinatal opioid exposure on behavioral and neurodevelopmental outcomes. Murine models provide efficient means to understand the neurobiological adaptations impacted by opioid exposure during perinatal neurodevelopment that drive long-term effects on cognitive, social, affective, and reward-related behaviors. We describe a rodent model of third-trimester-equivalent opioid exposure which produces replicable, opioid withdrawal-related phenotypes including robust thermal hyperalgesia and altered ultrasonic vocalization (USV) profiles. We present results from two drug regimens of the model, differing in the schedule of opioid administration (once or twice daily injections of morphine from postnatal day (P) 1-14; 15.0 mg/kg). Beyond hyperalgesia and altered USV profiles, both drug regimens lead to weight loss. Furthermore, both models resulted in transcriptional adaptations within brain regions relevant to opioid dependence and withdrawal. Twice-daily exposure resulted in sex-specific changes in metabolic gene expression in the brainstem, while once-daily exposure resulted in down-regulation of genes related to myelin and dopaminergic circuitry development in the nucleus accumbens. We found minimal evidence for behavioral consequences associated with once-daily morphine exposure during adulthood; there were no significant effects of perinatal morphine on cognitive, reward-related, or fear learning tasks. This could potentially indicate compensatory mechanisms that mitigate the adverse effects of third trimester-equivalent morphine exposure over time. Lastly, we identified epigenetic mechanisms potentially driving dysregulation of normal development within the central nervous system following pre-natal opioid exposure in humans. We analyzed placental samples from pregnancies with opioid exposure and identified gene networks containing altered DNA methylation patterns. Notably, we found enrichment within the ‘integral component of the plasma membrane’ and ‘synapse assembly’ functional networks, indicating potential effects of prenatal opioid exposure on neural connectivity and transmission. Together, the transcriptional adaptations identified in rodent brain tissue and the epigenetic modifications identified in human placental tissue provide novel mechanistic insight as to how perinatal opioid exposure impacts neural and fetal development.

DNA methylation is an epigenetic mechanism with roles that range from the fine tuning of transcription to genome wide dynamic acclimation to changing environments and regulation of developmental processes. While recent work has confirmed the presence and regulatory functions of DNA methylation in non-bilaterians, its role and distribution in Porifera has never been addressed. In this study, we performed whole genome bisulfite sequencing of the demosponge Amphimedon queenslandica and show that DNA methylation occurs mostly in CpG dinucleotides of coding regions. While high levels of gene-body methylation correlate positively with high expression and co-occur with the histone modification H3K36me3, they are not associated with amelioration of spurious transcription as found in other metazoans; nonetheless, per-exon methylation levels are predictive for exon retention suggesting a role in mRNA splicing. Additionally, analyses of Amphimedon and other sponges genomic data consistently revealed biased dinucleotide frequencies that suggest a long history of methylation-driven CpG conversion. Despite a genome wide loss of CpG dinucleotides, these are positively selected in exons and in methylated genes. These results indicate DNA methylation as a component of early metazoans regulome and challenge hypothesis on CpG methylation acting as a means for codon usage optimization.

Protein arginine methylation is an important post-translational modification that is vital in regulating various cellular processes such as gene transcription, cell signaling, and RNA processing. Protein arginine methyltransferases (PRMTs) are responsible for performing this important modification. PRMT1 (protein arginine methyltransferase 1) and Hmt1 (hnRNP methyltransferase 1) are the predominant PRMTs in humans and yeast, respectively. Despite growing momentum in this field, relatively little is understood about PRMT regulation. Further work discovering how PRMTs are regulated will greatly advance our understanding of diseases where PRMTs have been implicated, such as heart disease, viral pathogenesis, and cancer. It has been discovered that a human protein called hCaf1 (human Ccr4-associated factor 1) is a regulator of PRMT1 with respect to certain substrates, and also colocalizes with PRMT1. We present data that suggest the yeast homolog of hCaf1, Pop2, may also perform a similar function on Hmt1. We provide data on the expression and purification of a truncation of Pop2 from S. cerevisiae, including the temperature sensitivity of one construct of Pop2 and its susceptibility to precipitation. We also demonstrated concentration-dependent inhibition of Hmt1-catalyzed methylation of histone H4 by Pop2 in vitro. Yeast cell lysates also showed altered patterns of methylation in the presence and absence of Pop2 in vivo. In an effort to understand the mechanism employed by Pop2 to accomplish this regulatory function, pull-downs were performed suggesting that Pop2 directly interacts with histone H4, a substrate of Hmt1. Mutagenic studies with Pop2 suggested a region that may be responsible for this interaction. Given these data, we hypothesized that Pop2 is able to inhibit the methylation of histone H4 via a substrate-sequestering mechanism. Further experimentation will determine the precise interaction surfaces of Pop2 and substrate, and continue to define the details of methylation inhibition by Pop2, including the scope of its influence in the cell.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19626号 / 医博第4133号 / 新制||医||1016(附属図書館) / 32662 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 清水 章, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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A research report submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the Degree of Master of Science in Medicine. Johannesburg October, 1997 / Fragile X syndrome is, second to Down syndrome, the commonest form of genetic mental retardation. The aim of this research project was to investigate the impact of having a child with this syndrome on the family relationships. The subjects were 21 mothers and 9 fathers of affected children. The data were collected by means of specially constructed questionnaires in interviews with 19 mothers and 8 fathers and completed by post in three cases. A control group of parents with a normal child, matched for sex and age of the affected child, family size and ethnic groups, was interviewed. The data were computerised and analyzed. The results showed that more experimental parents than controls enjoyed their child’s nature, but disliked the behavioural problems. About half of the experimental parents tended not to reward good behaviour physically. However, although most of the affected children were accepted by their siblings, they had fewer friends and more problems with their peers. Some parents thought that their relationship with their spouse had improved and others thought that it had deteriorated after the affected child’s birth. Most parents in both study groups would request prenatal diagnosis in subsequent pregnancies and significantly more experimental parents than controls would request a termination of pregnancy for an affected fetus. Most parents were satisfied with the health service they received. These results show that family dynamics are disturbed by the presence of a child with FMR. Counsellors and therapists working with these families should be aware of the effects of the syndrome on the family / IT2017

Deformed Epidermal Autoregulatory Factor-1 (DEAF-1) is a transcription factor that binds TTCG motifs and has roles in fetal development, clinical depression and cancer. In order to further our understanding of the DEAF-1 protein, this study characterizes previously unidentified DEAF-1 interacting proteins and post-translational modifications of DEAF-1. A region encompassing the DNA binding domain of DEAF-1 interacts with the C-terminal Bax interacting domain of the Ku70 subunit of the DNA-PK holoenzyme. Ku70 acts as an anti-apoptotic protein through C-terminal domain and so DEAF-1 was assessed for its ability to influence apoptosis after various stimuli. DEAF-1 acted as a pro-apoptotic protein after intrinsic stimuli. Apoptotic activities occurred through a nuclear, DNA independent mechanism and a mutation that eliminated Ku70 interactions also inhibited DEAF-1 pro-apoptotic activities. Analysis of mammalian purified DEAF-1 indicated a number of phosphorylation sites and also a methylated arginine residue. Various assays were performed on mutated forms of DEAF-1 to determine the significance of the modified sites on DEAF-1 functions and properties. Lysine mutation of the methylated arginine site appeared to augment protein-protein interactions with itself and also Ku70. Alanine mutations at three of the identified phosphorylation sites increased DEAF-1 pro-apoptotic activities. In vitro kinase assays identified CDK5 as potential kinase that can phosphorylate DEAF-1. These studies provide new insight into potential functions, properties, and regulation of DEAF-1.

Genomic DNA methylation is an epigenetic marker that reflects influences of the environment, aging, and diseases. Although causal mechanisms of these alterations are understudied, the first step to addressing changes in DNA methylation is to map alterations appearing in a particular context. For example, human populations have diverse situational exposures. As an extreme example, the isolated populations of nomadic hunter/gatherer individuals in the Kalahari Desert lack access to most of the conveniences of modern lifestyles. Due to climate and behavioral adaptations of the lifestyle of these isolated populations (acoustic sensitivity to predators, irregular water and food availability), I predicted and demonstrated altered methylation landscapes compared with a non-Khoesan group of southern Africans with more industrialized lifestyles. The sites of differential methylation were assessed for potential functional impact at several example loci. A related project addressed nucleotide variants at interrogated methylation loci. For instance, C to T polymorphisms occurring in some individuals cannot initially be discriminated from loci that have differential cytosine methylation (following bisulfite treatment) in an array-based assay. My solution to this problem was the development of a computational approach to detect loci in methylation array data, which show tiered patterns created by SNP alleles rather than the usual continuum of differential methylation values. This approach was applied to the Kalahari populations and HapMap groups to show the utility of the approach. In the Kalahari populations, post-infancy ages are not recorded. We used functions that utilize DNA methylation to calculate estimates of aging and compared these results with predictions reported by the sample collectors, which were based primarily on interactions with non-nomadic neighbors. I compared the same aging estimates to known ages in the non-Khoesan samples and found correspondence. Although DNA methylation is a good predictor of cellular age, another method is telomere length measurement. To assess a relationship between predictors, I assessed associations in 300 samples between age, DNA methylation, and telomere length. Initial results indicated multiple correlated loci when accounting for gender and ethnicity using a linear model approach. / 2020-07-31T00:00:00Z

Multiple studies have implicated the POZ-ZF and methyl-DNA-binding transcription factor, Kaiso, in the regulation of genes and pathways that are important for development and tumorigenesis. In Xenopus embryos and mammalian cultured cells, Kaiso has been implicated as a negative regulator of the canonical Wnt signaling pathway. Paradoxically however, Kaiso depletion extends lifespan and delays polyp onset in the ApcMin/+ mouse model of intestinal tumorigenesis, where aberrant activation of Wnt signaling results in the development of neoplasias. These findings call into question Kaiso’s role as a negative regulator of canonical Wnt signaling and led us to hypothesize that Kaiso promotes intestinal tumorigenesis by a mechanism independent of its role in canonical Wnt signaling. To further delineate Kaiso’s role in intestinal tumorigenesis and to determine Kaiso’s role in regulating canonical Wnt signaling in the murine intestine, we generated a Kaiso transgenic mouse model expressing an intestine-specific murine Kaiso transgene. We then crossed our Kaiso transgenic mice with ApcMin/+ mice and analyzed the resultant progeny. Unexpectedly, Kaiso transgenic mice exhibited intestinal inflammation, increased expression of Wnt target genes and deregulated progenitor cell differentiation, although ectopic expression of Kaiso was not sufficient to drive tumorigenesis in the intestine. In agreement with previous studies, ectopic Kaiso expression in ApcMin/+ mice resulted in a significantly shortened lifespan and increased tumour burden. While we were unable to determine the precise mechanism by which Kaiso promotes intestinal tumour development, we found that Kaiso-induced inflammation is enhanced in the ApcMin/+ background and ectopic Kaiso expression further intensifies Wnt target gene expression in this model. Collectively, these studies have identified novel roles for Kaiso in regulating inflammation and cell-fate determination in the intestine. Furthermore, our findings suggest that Kaiso may contribute to intestinal tumorigenesis by promoting inflammation, which has been shown to be a predisposing factor for colorectal cancer development. Lastly, we have demonstrated distinct tissue and organism-specific roles for Kaiso in regulating canonical Wnt signaling. While, the aforementioned studies were the primary focus of this thesis, we also examined Kaiso’s role in DNA methylation-dependent repression of two tumour-associated genes, cyclinD1 and HIF1A. Our studies revealed that Kaiso binds and regulates the cyclinD1 locus via both sequence-specific and methylation-dependent DNA binding, suggesting that these alternate modes by which Kaiso binds to DNA may not be mutually exclusive. Furthermore, we identified a previously unexplored role for Kaiso in regulating the expression of the master regulator of hypoxia, HIF1A, which implicates Kaiso in modulating hypoxia-driven tumorigenic processes. / Thesis / Doctor of Philosophy (PhD)

The fundamental unit of chromatin is the nucleosome, which consists of a core of eight proteins wrapped by DNA. This core is composed of four pairs of histone proteins: H2A, H2B, H3, and H4. The DNA wraps around the protein core ~1.7 times, facilitating compaction of DNA length in the cell. Further, the location of nucleosomes makes genomic elements encoded in the DNA, such as promoters or enhancers, accessible or inaccessible to RNA polymerase and transcription factors. Thus, where nucleosomes are located (or positioned), can play a major role in transcription or other cellular processes. Additionally, histone proteins are frequently post-translationally modified, and these modifications further play a role in cellular processes, and in some cases are even required for specific protein function. What positions nucleosomes, and the downstream results of positioning or post-translational modifications (PTMs) is a topic of prolific study. Nucleosome formation is not random. In vivo it is believed that chromatin remodelers are the primary determinant of where nucleosomes form, while in vitro the DNA itself is the primary determinant. Formation of nucleosomes in vitro is a potent tool to elucidate fundamentals of chromatin. Considering that in vitro nucleosome formation is dependent on free energy, morphology and base composition of the DNA influence the free energy of formation. We found that the ends of linear DNA fragments were much more likely to have in vitro nucleosomes form on them. While this has the potential to bias results, based on our observations we could not find any significant alteration of the overall underlying DNA sequence composition due to the end preference observed. Histone proteins frequently receive the PTMs of methylation or acetylation. Histone methylation is typically indicative of repressed genes, while histone acetylation is typically indicative of active genes. In vivo the addition and removal of methylation and acetylation is highly dynamic. We hypothesized that the histone PTMs of methylation and acetylation also played a role in where nucleosomes formed. Comparing both in vivo and in vitro datasets, we observed strikingly similar patterns of nucleosomes for several histone methylations and acetylations, suggesting that these PTMs do indeed direct nucleosome formation. Upon further investigation, the underlying DNA sequence preferences change when compared to unmodified nucleosomes. This suggests that the genome is encoded to position these marks in locations where they are likely to be needed.