Histone Modifications in Trypanosoma Brucei

Trypanosoma brucei maintains an infection in its mammalian host by switching its surface antigen, thus evading host antibodies, in a process known as antigenic variation. Variant surface glycoproteins (VSG), the surface antigen, are expressed from genes located at as many as 20 Expression Sites (ES), which are present in subtelomeric regions of several chromosomes. How trypanosomes maintain monoallelic expression of VSG is a major question in trypanosome biology. Several theories have been proposed, including the presence of an Expression Site Body (a dedicated ‘transcription factory’), regulation of RNA elongation, and some kind of transcriptionally restrictive chromatin structure at inactive ES. In other organisms, the role of histone posttranslational modifications (PTMs) in influencing transcriptional states has been well-documented. To begin to understand the role of chromatin structure in the regulation of gene expression in trypanosomes, I developed a protocol for the purification of histones from mammalian-infective trypanosomes and identified PTMs using Edman degradation and mass spectrometry. I found that the N-termini of H4 and, possibly, H3 have a number of posttranslational modifications (PTMs), while H2A and H2B, in contrast, have relatively few. I also found a series of acetylated lysines at the C-terminus of H2A. Interestingly, I observed that alanine 1 of H2A, H2B, and H4 is monomethylated, a novel modification found in trypanosomatids. Next, I attempted to identify the targets of the putative histone deacetylase TbSIR2RP-1, a SIR2-related protein. Yeast SIR2 plays a key role in establishing heterochromatin at subtelomeric loci. TbSIR2RP-1 was chosen as a possible mediator of ES silencing, although this was later shown not to be the case. I showed that, unlike its yeast counterpart, TbSIR2RP-1 may not be an H4 deacetylase. Finally, I began to characterize H3 K4 methylation, which, in other organisms, is traditionally associated with transcriptionally active chromatin. I showed that nucleosomes containing the histone variant H2BV are enriched for H3 K4 methylation. I initially hypothesized that this is due to ubiquitination at a conserved lysine at the H2BV C-terminus, but find that this is not the case. Instead, I propose that replacement of major histones H2A/H2B with H2AZ/H2BV acts as a trigger to stimulate H3 K4 methylation, which represents an alternative pathway leading to H3 methylation, compared to what is observed in yeast

Current and future treatment strategies for rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, and can be subcategorized histologically and/or based on PAX-FOXO1 fusion gene status. Over the last four decades, there have been no significant improvements in clinical outcomes for advanced and metastatic RMS patients, underscoring a need for new treatment options for these groups. Despite significant advancements in our understanding of the genomic landscape and underlying biological mechanisms governing RMS that have informed the identification of novel therapeutic targets, development of these therapies in clinical trials has lagged far behind. In this review, we summarize the current frontline multi-modality therapy for RMS according to pediatric protocols, highlight emerging targeted therapies and immunotherapies identified by preclinical studies, and discuss early clinical trial data and the implications they hold for future clinical development

Regulation of Stability and Copy Number of Tandem Repeats in <i>Saccharomyces cerevisiae</i>

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, I used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. I developed powerful, highly sensitive assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating rDNA copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of the CUP1 array, I showed that instability at both tandem arrays can be induced by DNA replication stress and altered transcription of the locus. Importantly, while changes in instability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and, importantly, it restricts transcription-induced amplification of the CUP1 array. My work suggests that the modulation of replication and transcription is a simple, reversible strategy to alter instability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the ubiquity of DNA replication, transcription, and chromatin marks like histone acetylation, the mechanisms I have characterized could significantly advance our understanding of the behavior of other tandem repeats and their role in shaping eukaryotic genomes

Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain cancer-related death in children. These aggressive high-grade gliomas cannot be effectively treated and are associated with dismal prognosis. Whilst radiation therapy (RT) prolongs survival, it is a palliative therapy, as half of children with DIPG die within 1 year of diagnosis and almost all are dead by 2 years. These statistics have not changed for decades, despite a multitude of clinical trials. No chemotherapeutic regimen has been shown to improve survival, emphasizing the need to find novel and effective treatments. One of the principal reasons for this poor outcome was our limited knowledge of the biology of DIPG’s. Due to their location in brainstem, surgical resection is not feasible and up until recently, even performing a limited biopsy was considered too dangerous. In the last decade, DIPG tumor tissue has become available through autopsies and biopsies. This combined with the genome revolution has resulted in a transformation in our understanding of the underlying biology of this disease. Moreover, viable DIPG cells can now be grown in the laboratory which have allowed development of in-vitro (neurospheres) and in-vivo models (allograft and xenograft). This chapter summarizes recent advances in DIPG and potential novel therapies

Functional Classification of Immune Regulatory Proteins

SummaryThe members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. Guided by the Brotherhood approach, we present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative

Characterising the Role of the Calcium-dependent Citrullinating Enzyme Peptidyl Arginine Deiminase 2 in Ovarian Cancer

Epithelial ovarian cancer (EOC) represents the fifth most common cause of cancer mortality among women worldwide and accounts for the highest fatalities amongst gynaecological malignancies. The dysregulation of calcium-dependent peptidyl arginine deiminase 2 (PADI2) plays a key role in the tumorigenesis of several cancers; however, the role of PADI2 in the pathogenesis of EOC is yet to be investigated. Using RNA-seq and microarray data from primary serous ovarian cancers (The Cancer Genome Atlas data set) combined with survival data, the expression of PADI2 was assessed using each platform (n=262 and n=564). Kaplan-Meier analysis and Log-rank tests showed an association of PADI2 mRNA expression with overall survival using both platforms (RNA-seq cohort p=0.008 and microarray cohort p=0.0112). Low expression of PADI2 was associated with improved survival. Expression studies were used to examine the overexpression and knockout (CRISPR/Cas9 editing) of PADI2 expression, respectively, in the human-derived high-grade serous OVCAR-4 and mouse-derived EOC ID8-Luc2 cell lines. In OVCAR-4 cells, PADI2 overexpression reduced proliferation (22%) and cellular aggregation (94%), while increasing apoptosis (34%) and autophagy (38%), in a Ca2+- and citrullination-dependent manner, at 72 hours. PADI2 overexpression also induced cisplatin cytotoxicity by 10% at 72 hours. In ID8-Luc2 cells, PADI2 overexpression significantly decreased cisplatin cytotoxicity by 24%, independently of exogenous Ca2+ supplementation or induced citrullination, at 72 hours. In addition, qRT-PCR validation of TCGA gene co-expression data indicated that PADI2 overexpression correlated with expression of a number of genes. This was confirmed in functional studies where there was a 1.69-fold increase in ARHGEF10L and 0.75-fold decrease in FZD5 expression upon PADI2 overexpression. In this thesis, The Cancer Genome Atlas expression studies of PADI2 showed that higher PADI2 confers decreased survival of EOC patients. Conversely, PADI2 overexpression in vitro, induced apoptosis/autophagy and decreased proliferation/cellular aggregation possibly via deregulating FZD5 and ARHGEF10L. Collectively, this work suggests that PADI2 may serve as a potential therapeutic target in human EOC. Codon based selection analyses were used to test for evidence of positive selection in PADI2. The phylogenetic maximum likelihood analysis of PADI2 orthologues retrieved from 31 species indicated that there was no evidence of positive selection in PADI2 codons, but numerous residues were under evolutionary constraint