No description available.

In multicellular organisms, cell growth and differentiation is controlled in part by apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Resistance to Fas-mediated apoptosis is regulated through the production of a soluble Fas isoform (sFas), created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔ6) that can bind FasL and block apoptosis. Long noncoding RNAs (lncRNAs) are >200-nucleotide sequences that are important regulators of cellular programs. However, their role in erythropoiesis is just beginning to be appreciated with studies limited to murine systems. I studied the potential role of lncRNAs during human red blood cell development using RNA from cultured CD34+ purified from fetal liver (FL), cord blood (CB), or adult bone marrow (BM) to screen 82 documented lncRNAs. This screen revealed that the Fas Anti-sense (Saf) was consistently increased during maturation and levels high for BM compared to FL or CB. Next, I characterized the regulatory sequence of Saf and by In silico analysis identified canonical binding sites for the erythroid-specific transcription factors KLF1 and GATA1. Chromatin immunoprecipitation (ChIP) assays confirmed binding of both factors to their target sequences and luciferase reporter constructs revealed synergistic activity evidenced by increase in luciferase expression relative to controls. Genome wide expression analysis using cells with overexpression of Saf showed no effect on global gene transcription, suggesting Saf function at post transcriptional levels. Saf was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. Using a combination of biochemical assays, overexpression and knockdown studies, I showed how this occurs. Cell fractionation and RT-PCR demonstrated that Saf is localized in the nucleus. I found that Saf directly interacted with Fas pre-mRNA to form RNaseA-resistant double-stranded RNA intermediates at regions that flank Exon6. Post-transcriptional function of Saf was confirmed by qRT-PCR demonstrating significantly increased levels of sFas for Saf overexpressing cells. Enrichment for sFas RNA coincided with reduced Fas on the cell surface and increased sFas protein levels when conditioned supernatants were assayed by ELISA. Conversely, siRNA-mediated knockdown of Saf significantly reduced sFas production compared to non-targeting siRNA controls. Saf-interacting proteins were identified by mixing in vitro transcribed and biotin-labeled Saf RNA with nuclear lysates followed by mass spectrometry analysis. This screen identified human splicing factor 45 (SPF45) which has a known role in Fas pre-mRNA alternative splicing. Specific SPF45/Saf interaction was confirmed by RNA pulldown and western blot with SPF45-specific antibodies and the ability to detect sequences for Saf, Fas and sFas by RT-PCR of RNA that immunoprecipitated with SPF45. SPF45 knockdown decreased sFas transcripts and this reduction corresponded to limited production of sFas and increased sensitivity to Fas-mediated apoptosis when cells were exposed to the Fas-activating antibody CH11. Importantly, overexpression of Saf in SPF45 knockdown cells failed to rescue production of sFas supporting the hypothesis that Saf and SPF45 co-participate in modulating Fas pre-mRNA splicing. Protein phosphorylation modulates the interaction of the splicing factors with RNA. The effect of phosphorylation on the Saf-SPF45 interaction was evaluated using stable cell lines expressing a myc-tagged SPF45 protein or versions modified to introduce alanine in place of threonine 71 (T-71-A) or serine 222 (S-222-A) to prevent phosphorylation. Mutation in S-222-A reduced the interaction of SPF45 with Saf. I conclude that Saf interacts with Fas pre-mRNA at sequences that flank exon 6 and recruits phosphorylated SPF45 as mechanism to recognize exon 6 and allow for alternative splicing of Fas Pre-mRNA. Collectively, these studies reveal a novel mechanism to regulate apoptosis that may be responsible for cell proliferation and drug resistance.

Understanding the factors that affect c-Myc in prostate cancer is critical to developing an effective means of treatment for aggressive, castration-resistant forms of the disease. Myc is an oncogene known to be overexpressed and stabilized in many types of cancer, prostate included. Recent insights into breast cancer have revealed that Myc protein retains a longer half-life in cancer cells, but the cause for this has yet to be deduced. Due to its close proximity and proven interaction with Myc, I propose that the lncRNA PVT1 is stabilizing Myc and facilitating its activation of target genes in castration-resistant prostate cancer. To explore this hypothesis, metastatic prostate cancer DU145 cells were transfected with both siRNAs and ASOs targeting PVT1. Cells were analyzed for changes in different protein levels, as well as binding partners, through the use of immunoprecipitation and western blot. These results were verified with RT-qPCR data to confirm knockdown levels of PVT1. Proliferation assays were also conducted to explore the proliferative abilities of cells when PVT1 levels were decreased. Transfection of PVT1 with siRNA yielded about a 50% knockdown, while ASO targeting brought PVT1 levels down 80%. PVT1 inhibition had different effects on the level of c-Myc in cells depending on the method of transfection used--while transfection with anti-PVT1 siRNAs slightly decrease the amount of c-Myc protein in the cell, transfection using ASOs significantly increases c-Myc. Most importantly, proliferation of DU145 cells decreased with PVT1 knockdown by ASOs. Removal of this lncRNA, therefore, hinders that oncogenic potential for growth of prostate cancer cells. Since Myc poses a difficult target for cancer therapy, any new method to mitigate its oncogenic signaling would be invaluable. Targeting lncRNA PVT1 may be a successful method of doing just that, but more work needs to be done to explore the effects of different knockdown strategies. It is clear, however, that the relationship between Myc and PVT1 is a convoluted interaction that warrants further research. A breakthrough in this area could lead to huge improvements in the way prostate cancer is diagnosed and treated.

In my thesis, I focused on several underexplored areas of RNA splicing regulation. In the first part, I analyzed how chromatin and transcription regulatory elements change pre-mRNA splicing. In the second part, I studied why long non-coding RNAs (lncRNAs) are spliced less efficiently than protein-coding mRNAs. Finally, I was testing the importance of intron for the activating function of lncRNAs. It has been shown that chromatin and promoter identity modulate alternative splicing decisions. Here, I tested whether local chromatin and distant genomic elements that influence transcription can also modulate splicing. Using the chromatin modifying enzymes directly targeted to FOSL1 gene by TALE technology, I showed that changes in histone H3K9 methylation affect constitutive splicing. Furthermore, I provide evidence that deletion of transcription enhancer located several kilobases upstream of an alternative exons changes splicing pattern of the alternative exon. Many nascent lncRNAs undergo the same maturation steps as pre-mRNAs of protein- coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences. Genome-wide analysis of intergenic lncRNAs (lincRNAs) revealed that, in general, they do not...

L'empreinte génomique, une régulation épigénétique unique entraînant une expression génique spécifique aux parents d'origine, est essentielle au développement et à la croissance des mammifères. H19 est un ARN long non codant exprimé en milieu maternel qui est un régulateur central du réseau de gènes à empreinte contrôlant le développement. H19 est exprimé pendant le développement embryonnaire dans de nombreux tissus, y compris toutes les cellules hématopoïétiques. Le rôle de H19 au cours du développement embryonnaire n'a été documenté que pour le placenta où il contrôle la croissance. Le rôle de H19 dans la lymphopoïèse n'a pas été étudié. Notre laboratoire a précédemment trouvé H19 comme principal transcrit exprimé sélectivement par les proB du foie foetal (FF), et exprimé de façon différentielle par des immigrants thymiques précoces et tardives. Cependant, le rôle du gèneH19 dans celui du développement des cellules B, ou même dans le système immunitaire, reste méconnu. Ici, nous avons réalisé une caractérisation complète des perturbations du développement et de la fonction du système immunitaire des souris pour lesquelles un grand segment du locus H19 a été supprimé. Dans cette étude, nous avons constaté que le mutant H19 avait un impact spécifique sur le développement des cellules du FF induisant une augmentation sélective du nombre des cellules proB BP1+ présentant des perturbations importantes du réarrangement du locus IgH.On observe également chez les animaux H19-/- adultes une expansion anormale du compartiment B mature. Bien que H19 ne soit plus exprimé après la naissance, les lymphocytes B des souris adultes mutantes présentent un phénotype altéré. On observe en effet des perturbations importantes du profil d'expression du marqueur B220. Les souris H19-/-présentent un défaut d'expansion des lymphocytes B du centre germinatif, ainsi qu'une chute de la production des IgM spécifiques dans le sérum après immunisation. Indiquant une réponse défectueuse des cellules B. De manière cohérente, nous avons trouvé une réactivité réduite au BCR des cellules B naïves H19-/-, associée les expériences de reconstitution compétitive ont mis en évidence un altération cellule-intrinsèque de la réponse humorale chez les animaux mutants. Un défaut d'induction de l'expression des molécules du CMHII, CD40,et CD86, qui pourrait être à l'origine des perturbations de la réponse humorale observée chez les souris H19-/-. Les analyse de transcriptome réalisées sur les lymphocytes B du centre germinal des animaux mutants ont mis en évidence une expression différentielle des gènes impliqués dans la régulation de l'intensité du signal émanant du récepteur B à l'antigène. Au total ce travail nous a permis de démontrer l'activité régulatrice exercée par l'ARN non codant H19 sur le développement et la fonction du système immunitaire. / Genomic imprinting, a unique epigenetic regulation resulting in a parent-of-origin specificgene expression, is essential for normal mammalian development and growth. H19 is amaternally expressed long non-coding RNA that is a central regulator of the imprinting gene network controlling development and growth. H19 is expressed throughout embryonic development in multiple tissues including all hematopoietic cells. The role of H19 during embryonic development has only been documented for the placenta where it controls growthand the role of H19 in lymphopoiesis has not been investigated. The laboratory has previouslyfound H19 as the major differentially expressed transcript in two microarrays comparing fetalliver (FL) and bone marrow (BM) derived pro-B cells, as well as between early and latethymic settling progenitors. However, a role for imprinting gene H19 in B cell development,or even in immune system remains elusive. Here we sought to analyze mice where a large segment of the H19 locus has been deleted. In our work, we found that loss of H19 have specific impact on the FL B cell development byproducing increased numbers of BP1+ proB cell. Although BP1+ proB cells from H19-/- FLshowed impaired Ig heavy chain V-D-J rearrangement, that increase resulted in a net enlarged B cell compartment in the adult periphery of H19 mutant. In adult mice, although H19 is notexpressed in B lymphocytes after birth, B cells from H19-/- mice exhibited altered B cellsurface phenotype, represented by an upregulated B220 expression on all B cell subsets. After immunization with different T cell dependent antigens, H19-/- exhibits reduced GC B cells, and impaired specific IgM titer in the serum, indicating a defected B cell response in H19-/-mice. Competitive reconstitution analysis showed a B cell autonomous impairment in the Bcell response. Consistently we found a reduced BCR responsiveness of H19-/- naïve B cells that together with less efficient upregulation of MHCII and CD40 expression after immunization might be responsible for the impaired immune response in H19-/- mice. Genome-wide transcription analysis revealed differential expression of genes involved inregulating the intensity of B cell receptor signaling. This work brings new insights on the regulation role of long non-coding RNA H19 in the early B cell development and immune system

Thesis advisor: Marc A.T. Muskavitch / The protozoan Plasmodium falciparum, the mosquito-borne pathogen that causes human malaria, remains one of the most difficult infectious parasites to combat and control. Campaigns against malaria eradication have succeeded, in most instances, at the level of vector control, rather than from initiatives that have attempted to decrease malaria burden by targeting parasites. The rapid evolution and spread of insecticide-resistant mosquitoes is threatening our ability to combat vectors and control malaria. Therefore, the development, procurement and distribution of new methods of vector control are paramount. Two aspects of vector biology that can be exploited toward these ends are vector behaviors and vector-specific insecticide targets. In this thesis, I describe three aspects of vector biology with potential for the development of improved means of vector control: photopreference behavior, long non-coding RNA (lncRNA) targets and epigenetic gene ensemble targets. My studies of photopreference have revealed that specific mosquito species within the genus Anopheles, An. gambiae and An. stephensi, exhibit different photopreference behaviors, and that each gender of mosquito in these species exhibits distinct light-dependent resting behaviors. These inter-specific behavioral differences may be affected by differing numbers of long-wavelength sensing Opsin genes in each species, and my findings regarding species-specific photopreferences suggest that some behavioral interventions may need to be tailored for specific vector mosquito species. Based on the advancement of next-generation sequencing technologies and the generation by others of assembled genomes of many anopheline mosquito species, I have identified a comprehensive set of approximately 3,000 lncRNAs and find that RNA secondary structures are notably conserved within the gambiae species complex. As lncRNAs and epigenetic modifiers cooperate to modulate epigenetic regulation, I have also analyzed the conservation of epigenetic gene ensembles across a number of anopheline species, based on identification of homologous epigenetic ensemble genes in An. gambiae compared to Drosophila melanogaster. Further analyses of these ensembles illustrate that these epigenetic genes are highly stable among many anopheline species, in that I detect only eight gene family expansion or contraction events among 169 epigenetic ensemble genes within a set of 12 anopheline species. My hope is that my findings will enable deeper investigations of many behavioral and epigenetic processes in Anopheles gambiae and other anopheline vector mosquitoes and thereby enable the development of new, more effective means of vector and malaria control. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events, during which a differentiated oocyte gives rise to a totipotent zygote. During OET a transcriptionally silent oocyte undergoes massive reprogramming of gene expression, which transforms it into a transcriptionally active zygote. Although numerous studies have contributed to understanding the mechanism of OET, many genes involved in OET are yet to be identified. A whole new level of possible regulation of OET came with the discovery of long non-coding RNAs (lncRNA). LncRNAs are pol II transcripts longer than 200 nucleotides, that are typically spliced and polyadenylated but do not encode proteins. While lncRNAs have been studied in many model systems including embryonic stem cells, their expression in oocytes and early embryos and contribution to OET were largely unexplored at the beginning of this project. In my PhD project, I aimed to identify, annotate, and analyze lncRNAs expressed during OET. First, using RNA-Seq, 1600 highly reliable lncRNAs were identified and annotated in mouse oocytes and early embryos. Majority of lncRNAs were novel with expression exclusively at OET stages. A significant fraction of these lncRNAs was found associated with LTR retrotransposons, contributing to their novelty and...

No description available.

High throughput RNA-sequencing data has revealed a cellular transcriptome that represents a much larger part of the genome than was thought even 10 years ago. The newfound transcripts are not translated to protein and a few are known to have essential functions regulating gene expression. However, most non-coding RNAs in the transcriptome have no identified function or mechanism of action. H19 is a long non-coding RNA (lncRNA) that looks much like an mRNA with a 5’ methyl cap and a polyA tail, but it is not translated. H19 is associated with cell proliferation and most human cancers. The mechanisms governing H19’s role in these processes is unknown, but we wondered how translation avoidance is involved. Exciting preliminary results from the Weinberg lab show that H19 co-sediments with the 40S ribosomal subunit, suggesting the RNA is associated with cytoplasmic proteins. We hypothesized that RNA binding proteins may be mediating H19’s ability to avoid translation despite its having the characteristics of mRNA. Therefore, we are working to identify these RNA binding proteins associated with H19 by adapting a recently reported RNA antisense purification method.

Myoclonus dystonia (MD) is a rare autosomal-dominant combined dystonia movement disorder characterised by quick, involuntary muscle jerks (myoclonus) paired with sustained muscular contraction (dystonia). Although known to be genetically heterogeneous, the most common genetic factor is mutations within SGCE, the gene encoding ε-sarcoglycan, accounting for approximately 45% of cases. Previous linkage analyses conducted on a family displaying inherited MD without SGCE mutations lead to the identification of another critical region, DYT15. Preliminary data suggested that mutations within the long non-coding RNA (lncRNA) CD108131, found within the DYT15 locus, resulted in decreased expression of both the SGCE transcript, as well as the SGCE protein. Validation of the remaining variants of interest yielded no new candidate genes. A low coverage area coinciding with the entire sequence of TMEM200C was discovered, however subsequent sequencing data revealed no potential disease-causing variants. Therefore, to further characterise the relationship between CD108131 and SGCE suggested by the preliminary data, a CRISPR-Cas9 knockout was developed in HEK293 cells using a double-cut strategy that allowed for complete excision of the CD108131 gene. Stable CD108131 knockout mutant cell lines were examined for differences in gene expression. QRT-PCR analysis was conducted and revealed a significant decrease in SGCE expression in the absence of CD108131. Additionally, expression also trended towards a decrease for ZBTB14, however ARHGAP28 and RPPH1 were not significantly altered. This data demonstrates that the lncRNA CD108131 is likely to have a regulatory effect on SGCE, and perhaps ZBTB14, transcription.