Sexual differentiation is a fundamental characteristic of all eukaryotes, dictating sex-specific morphology, physiology and behavior. Diploid organisms with heteromorphic sex chromosomes (XX or XY) require regulatory compensation of the X chromosome to maintain correct levels of genetic expression between the sexes, a process termed sex-specific dosage compensation (SSDC). The fruit fly, <i>Drosophila melanogaster</i> dosage compensates by upregulating transcription of most X-linked genes two-fold. Associated with this two-fold up regulation is the male-specific lethal (MSL) complex, a RNA-protein complex comprised of at least five known proteins; MSL1, MSL2, MSL3, males absent on the first (MOF), and maleless (MLE) and two non-translated RNA molecules; roX1 (RNA on the X chromosome) and roX2. The complex modulates the chromatin structure of the male X chromosome via acetylation of H4K16. MOF and MSL3 both exhibit an N-terminal chromodomain, whose function is unclear. The MSL3 chromodomain has been suggested to bind H3K36Me3. Chromodomains are a paradigm of how a single structural fold has evolved in diverse proteins to bind distinct targets. Chromodomains are common to nuclear regulators, and bind diverse targets including histones, DNA, and RNA. They function as recognition motifs of histone post-translational modifications and facilitate the translation of the histone code into a distinct local chromatin structure via recruiting the appropriate chromatin modulating machinery.<p> The goal of this research is to determine the structure of the <i>D. melanogaster</i> MOF and MSL3 chromodomains by X-ray crystallographic and/or nuclear magnetic resonance techniques, to advance our understanding of the structural characteristics of these diverse domains. Here we report the cloning and reproducible expression and purification of the <i>D. melanogaster</i> MOF and MSL3 chromodomains and their Homo sapiens orthologues. The <i>D. melanogaster</i> MOF chromodomain, whose NMR structure was published during this research, has been crystallized. Attempts to solve the crystal structure by molecular replacement, multiple-wavelength anomalous dispersion, and single-wavelength isomorphous replacement are reported.

Sexual differentiation is a fundamental characteristic of all eukaryotes, dictating sex-specific morphology, physiology and behavior. Diploid organisms with heteromorphic sex chromosomes (XX or XY) require regulatory compensation of the X chromosome to maintain correct levels of genetic expression between the sexes, a process termed sex-specific dosage compensation (SSDC). The fruit fly, <i>Drosophila melanogaster</i> dosage compensates by upregulating transcription of most X-linked genes two-fold. Associated with this two-fold up regulation is the male-specific lethal (MSL) complex, a RNA-protein complex comprised of at least five known proteins; MSL1, MSL2, MSL3, males absent on the first (MOF), and maleless (MLE) and two non-translated RNA molecules; roX1 (RNA on the X chromosome) and roX2. The complex modulates the chromatin structure of the male X chromosome via acetylation of H4K16. MOF and MSL3 both exhibit an N-terminal chromodomain, whose function is unclear. The MSL3 chromodomain has been suggested to bind H3K36Me3. Chromodomains are a paradigm of how a single structural fold has evolved in diverse proteins to bind distinct targets. Chromodomains are common to nuclear regulators, and bind diverse targets including histones, DNA, and RNA. They function as recognition motifs of histone post-translational modifications and facilitate the translation of the histone code into a distinct local chromatin structure via recruiting the appropriate chromatin modulating machinery.<p> The goal of this research is to determine the structure of the <i>D. melanogaster</i> MOF and MSL3 chromodomains by X-ray crystallographic and/or nuclear magnetic resonance techniques, to advance our understanding of the structural characteristics of these diverse domains. Here we report the cloning and reproducible expression and purification of the <i>D. melanogaster</i> MOF and MSL3 chromodomains and their Homo sapiens orthologues. The <i>D. melanogaster</i> MOF chromodomain, whose NMR structure was published during this research, has been crystallized. Attempts to solve the crystal structure by molecular replacement, multiple-wavelength anomalous dispersion, and single-wavelength isomorphous replacement are reported.

Epidemiological studies have identified psychological stress as a significant risk factor in breast cancer. The stress response is regulated by the HPA axis in the brain and is mediated by glucocorticoid receptor (GR) signalling. It has been found that early life events can affect epigenetic programming of GR, and methylation of the GR promoter has been reported in colorectal tumourigenesis. Decreased GR expression has also been observed in breast cancer. In addition, it has been previously demonstrated that unliganded GR can serve as a direct activator of the BRCA1 promoter in mammary epithelial cells. We propose a model whereby methylation of the GR promoter in the breast significantly lowers GR expression, resulting in insufficient BRCA1 promoter activation and an increased risk of developing cancer. Antibody-based methylated DNA enrichment was followed by qPCR analysis (MeDIP-qPCR) in a novel assay developed to detect locus-specific methylation levels. It was found that 13% of primary breast tumours were hypermethylated at the GR proximal promoter whereas no methylation was detected in normal tissue. RT-PCR and 5’ RACE analysis identified exon 1B as the predominant alternative first exon in the breast. Tumours methylated near exon 1B had decreased GR expression compared to unmethylated samples, suggesting that this region is important for transcriptional regulation of GR. It was also determined that GR and BRCA1 expression was decreased in breast tumour compared to normal tissue. Furthermore, the relative expression of GR and BRCA1 measured by qRT-PCR was correlated in normal tissue but this association was not found in tumour tissue. From this, it appears that lower GR levels with associated decreased BRCA1 expression in tissues may be a predisposing factor for breast cancer. Based on these results we propose a role for GR as a potential tumour suppressor gene in the breast due to its association with BRCA1, also a tumour suppressor gene, as well as its consistently decreased expression in breast tumours and methylation of its proximal promoter in a subset of cancer patients. / Thesis (Master, Biochemistry) -- Queen's University, 2012-09-26 18:19:11.006

MeCP2 was identified by its ability to bind DNA in a methylation-specific manner. Yet, how it interprets the DNA methylome remains unclear. Several mechanisms have been proposed, including a role in transcriptional repression. MeCP2 is highly abundant in the brain, and loss-of-function mutations result in a neurological disorder called Rett syndrome (RTT). Strikingly, RTT-causing missense mutations are almost all located in either the methyl-CpG-binding domain (MBD) or a region that has been shown to bind the NCoR/SMRT co-repressor complex (NID). This suggests that the MBD and the NID are the key functional domains in MeCP2, and that the role of MeCP2 is to form a ‘bridge’ between chromatin and the co-repressor complex to regulate gene expression. To test this ‘bridge’ hypothesis, I have made an allelic series of knock-in mice with truncated forms of MeCP2 to determine whether the other regions are dispensable for protein function. The three other regions of MeCP2 (the N-terminus before the MBD, the Intervening region between the MBD and the NID, and the C-terminus after the NID) were deleted in a step-wise manner to produce progressively smaller truncated proteins. Knock-in mice which lack just the N-terminus or both the N- and C-termini are phenotypically normal. Therefore, these regions, which together make up 46% of the protein sequence, are dispensable for MeCP2 function in vivo. Additional deletion of the Intervening region, retaining only 34% of the original sequence, results in mild RTT-like symptoms in the knock in mice. This is likely to be caused by this protein’s decreased stability and reduced ability to bind the NCoR/SMRT complex in the brain. The most severely truncated protein is nevertheless able to reverse the Mecp2-null phenotype when reactivated after the onset of symptoms. Together, these findings strongly support the ‘bridge’ hypothesis.

Recent studies have established that yolk hormones of maternal origin have significant effects on the physiology and behavior of offspring in birds. Herrington (2012) demonstrated that an elevation of progesterone in yolk elevates emotional reactivity in bobwhite quail neonates. Chicks that hatched from progesterone treated eggs displayed increased latency in tonic immobility and did not emerge as quickly from a covered location into an open field compared to control groups. For the present study, three experimental groups were formed: chicks hatched from eggs with artificially elevated progesterone (P), chicks hatched from an oil-vehicle control group (V), and chicks hatched from a non-manipulated control group (C). Experiment 1 examined levels of progesterone with High Performance Liquid Chromatography/tandem Mass Spectroscopy (HPLC/MS) from prenatal day 1 to prenatal day 17 in bobwhite quail egg yolk. In Experiment 2, bobwhite quail embryos were passively exposed to an individual maternal assembly call for 24 hours prior to hatching. Chicks were then tested individually for their preference between the familiarized call and a novel call at 24 and 48 hours following hatching. For Experiment 3, newly hatched chicks were exposed to an individual maternal assembly call for 24-hrs. Chicks were then tested for their preference for the familiarized call at 24 and 48-hrs after hatch. Results of Experiment 1 showed that yolk progesterone levels were significantly elevated in treated eggs and were present in the egg yolk longer into prenatal development than the two control groups. Results from Experiment 2 indicated that chicks from the P group failed to demonstrate a preference for the familiar bobwhite maternal assembly call at 24 or 48-hrs after hatch following 24-hrs of prenatal exposure. In contrast, chicks from the C and V groups demonstrated a significant preference for the familiarized call. In Experiment 3, chicks from the P group showed an enhanced preference for the familiarized bobwhite maternal call compared to chicks from the C and V groups at 24 and 48-hrs after hatch. The results of these experiments suggest that elevated maternal yolk hormone levels in pre-incubated bobwhite quail eggs can influence auditory perceptual learning in embryos and neonates.

Acute myeloid leukemia (AML) is a subset of leukemia that is characterized by the clonal expansion of cytogenetically and molecularly abnormal myeloid blasts. These blasts are highly proliferative accumulating in bone marrow and blood which leads to severe infections, anemia, and bone marrow failure. The poor prognosis of AML patients caused by the low tolerance to intensive chemotherapy has encouraged the pursuit of alternative therapeutic approaches. Differentiation therapy which involves the use of agents that can release the differentiation block in these leukemic blasts has emerged as a promising therapeutic approach. The use of All-trans retinoic acid (ATRA) represents a successful example of such an approach, nonetheless its efficacy is restricted to one subtype of AML. Efforts have been focused on finding differentiation agents which are effective for the other more common AML subtypes. Anti-CD44 targeted antibodies that activate the CD44 cell surface antigen are a promising candidate. Previous studies have shown that anti-CD44 treatment has been able to release the differentiation block in AML1 through AML5 subtypes. The exact mechanism by which anti-CD44 treatment is able to induce its effects has not been fully elucidated. Recent studies highlight the role that epigenetic mechanisms play during haematopoiesis and leukemogenesis and therefore, in this work we investigated the epigenetic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44-induced differentiation results in an extensive change of histone modification levels. We found that inhibiting enzymes responsible for the H3K9ac, H3K4me, H3K9me, and H3K27me modifications, attenuated the anti-proliferative and differentiation promoting effects of antic-CD44 treatment. Taken together, these data highlight the promising potential of using anti-CD44 as a therapeutic agent across multiple subtypes in AML

Living cells maintain stable transcriptional programs while exhibiting plasticity that allows them to respond to environmental stimuli. The Polycomb repressive complex 2 (PRC2) is a key regulator of chromatin structure that maintains gene silencing through the methylation of histone H3 on lysine 27 (H3K27me), establishing chromatin-based memory. Two variants of PRC2 are present in mammalian cells, PRC2-EZH2 which is predominantly present in differentiating cells, and PRC2-EZH1 that predominates in post-mitotic tissues. PRC2-EZH1α/β pathway is involved in the response of muscle cells to oxidative stress. Atrophied muscle cells respond to oxidative stress by enabling the nuclear translocation of EED and its assembly with EZH1α and SUZ12. Here we prove that the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) is required for the assembly of PRC2-EZH1 components. The absence of MALAT1 significantly decreased the association between EED and EZH1α proteins. Biochemical analysis shows that the presence of MALAT1 increases the enzymatic activity of PRC2-EZH1 in vitro. In addition, we show that the simultaneous expression of PRC2 core components is necessary for their solubility. The successful expression of PRC2 proteins enables the execution of several downstream experiments, which will further explain the nature of the interplay between MALAT1 and PRC2.

No description available.

INTRODUCTION: Bladder cancer (BC) is highly prevalent. It presents as either non-muscle invasive or muscle-invasive disease. The prognosis of muscle invasive disease is poor, with a 5-year survival rate of less than 50%. Treatment approaches for both types of BC have not advanced much in the last few years and new therapies are needed to overcome the large burden of BC. Recently, a large effort has been undertaken to classify BC into molecular subtypes. These analyses have revealed significant alterations in epigenetic modifiers in BC. A previous study from our group revealed that SH3GL2, a negative regulator of receptor tyrosine kinase (RTK) signaling, was lost with high frequency in BC, leading to increased growth of tumor cells in-vitro and in-vivo. Conversely, forced expression of SH3GL2 in BC cell lines attenuated oncogenic behaviors including growth and migration. In addition to genomic deletion, SH3GL2 is subject to methylation-induced silencing, a key epigenetic mechanism. OBJECTIVE: Epigenetic mechanisms of gene regulation are known to be perturbed in BC. The objectives of this study were to investigate methylation of the SH3GL2 promoter and to test whether agents that promote Deoxyribonucleic acid (DNA) demethylation could be used to re-express SH3GL2 thereby restoring regulation of RTK signaling. METHODS: Methylation of a specific CpG island in the SH3GL2 promoter was analyzed using methylation-specific Polymerase Chain Reaction (PCR) in a panel of BC cell lines with known SH3GL2 messenger Ribonucleic Acid (mRNA) status. Selected BC cell lines were treated with a variety of demethylating agents at different doses and for different times to evoke the re-expression of silenced SH3GL2. Demethylation inhibitors were combined with the histone deacetylase inhibitor, trichostatin A (TSA), to determine whether further re-expression could be achieved. RESULTS: The SH3GL2 promoter displayed differing extents of promoter methylation among cell lines examined. In RT4 cells, the only cell line with detectable expression of SH3GL2 mRNA and protein, the promoter was completely unmethylated. In contrast, T24 and 253J cells displayed significant promoter methylation with little to no SH3GL2 mRNA expressed, consistent with methylation-induced silencing. Treatment of T24 and 253J with 5-Aza-2’-deoxycytidine (5-Aza-dC, 20 M), a DNA methyltransferase (DNMT) inhibitor increased gene expression but this was not dose- or time-dependent. Two additional DNMT inhibitors, Zebularine and RG-108 were also tested. A much higher dosage of Zebularine was required to trigger activation (500 M) while RG-108 was unable to trigger gene reactivation at all. Combination treatment with 5-Aza-dC and TSA further increased SH3GL2 expression compared to either agent alone. These results suggest that DNA methyltransferase inhibition is an effective treatment to re-express SH3GL2 in cells with SH3GL2 promoter silencing. CONCLUSION: The present study shows silencing of SH3GL2 in a variety of BC cell lines as a consequence of DNA promoter hypermethylation. Treatment with demethylating agents was able to increase gene expression. Based on prior findings showing attenuation of tumor cell growth and migration with forced expression of SH3GL2, DNA methyltransferase inhibition represents an effective strategy to re-express SH3GL2 in BC and normalize tumor cell behavior. / 2020-07-03T00:00:00Z

Cloning through somatic cell nuclear transfer (SCNT) remains highly inefficient twenty years after the first demonstration of the technology with the birth of Dolly. By increasing efficiency by selecting the embryos early in development that are most likely to succeed following transfer into a surrogate mother, the technology could be more routinely utilized to enhance animal agriculture production. SCNT is believed to be highly inefficient as a result of incorrect DNA methylation and gene expression that are accumulated because of the SCNT technique. We proposed the use of a non-toxic, non-invasive detector of cell death, to quantitatively assess embryo competency prior to embryo transfer. We believed we could use SR-FLICA to identify the embryos with low levels of cell death as a result of proper DNA methylation and gene expression. By analyzing the whole embryo, differences in gene expression and DNA methylation were identified in embryos with high and low levels of cell death. However, the level of cell death did not prove to be a reliable indicator of embryo quality in predicting pregnancy outcome. This data supports the commonly held hypothesis that DNA methylation and gene expression after SCNT have random defects as a result of the random nature of resetting the DNA for embryo development. More research is required to identify the embryos which will prove to be successful following SCNT and embryo transfer.