Discovery of a Cellular Mechanism Regulating Transcriptional Noise

Stochastic fluctuations in gene expression (‘noise’) are often considered detrimental but, in other fields, fluctuations are harnessed for benefit (e.g., ‘dither’ or amplification of thermal fluctuations to accelerate chemical reactions). Here, we find that DNA base-excision repair amplifies transcriptional noise, generating increased cellular plasticity and facilitating reprogramming. The DNA-repair protein Apex1 recognizes modified nucleoside substrates to amplify expression noise—while homeostatically maintaining mean levels of expression—for virtually all genes across the transcriptome. This noise amplification occurs for both naturally occurring base modifications and unnatural base analogs. Single-molecule imaging shows amplified noise originates from shorter, but more intense, transcriptional bursts that occur via increased DNA supercoiling which first impedes and then accelerates transcription, thereby maintaining mean levels. Strikingly, homeostatic noise amplification potentiates fate-conversion signals during cellular reprogramming. These data suggest a functional role for the observed occurrence of modified bases within DNA in embryonic development and disease

Cross-phyla protein annotation by structural prediction and alignment

Background Protein annotation is a major goal in molecular biology, yet experimentally determined knowledge is typically limited to a few model organisms. In non-model species, the sequence-based prediction of gene orthology can be used to infer protein identity; however, this approach loses predictive power at longer evolutionary distances. Here we propose a workflow for protein annotation using structural similarity, exploiting the fact that similar protein structures often reflect homology and are more conserved than protein sequences. Results We propose a workflow of openly available tools for the functional annotation of proteins via structural similarity (MorF: MorphologFinder) and use it to annotate the complete proteome of a sponge. Sponges are highly relevant for inferring the early history of animals, yet their proteomes remain sparsely annotated. MorF accurately predicts the functions of proteins with known homology in >90% cases and annotates an additional 50% of the proteome beyond standard sequence-based methods. We uncover new functions for sponge cell types, including extensive FGF, TGF, and Ephrin signaling in sponge epithelia, and redox metabolism and control in myopeptidocytes. Notably, we also annotate genes specific to the enigmatic sponge mesocytes, proposing they function to digest cell walls. Conclusions Our work demonstrates that structural similarity is a powerful approach that complements and extends sequence similarity searches to identify homologous proteins over long evolutionary distances. We anticipate this will be a powerful approach that boosts discovery in numerous -omics datasets, especially for non-model organisms

The role of the EBNA2-EBF1 complex in EBV driven B cell transformation

The Epstein Barr virus (EBV) infection of B cells is associated with various malignancies, e.g. Burkitt’s lymphoma, Hodgkin’s lymphoma, lymphoproliferative diseases. Upon infection, EBV induces a complex viral and cellular gene expression program causing the transformation of naïve resting B cells to lymphoblastoid cell lines (LCLs) in vitro. These are continuously proliferating cultures and used to study molecular mechanisms of the transformation process. EBV nuclear antigen (EBNA) 2 is one of the first genes expressed after EBV infection and a key regulator of the transformation process. It preferentially binds to B cell specific enhancers and promotors via cellular adaptor proteins. CBF1 is the main DNA anchor but the knowledge about the contribution of B cell specific transcription factors (TFs) to EBNA2’s activity is limited. The early B cell factor (EBF) 1, a key TF during B cell development, has been identified as an EBNA2 co-factor. By identifying the α1-helix in the EBNA2 N-terminal dimerization (END) domain as a crucial region for the interaction with EBF1, I was able to generate mutant EBV lacking this region (EBVΔα1) in order to study the role of the EBNA2-EBF1 complexes during the transformation of infected B cells. Analysis of cellular processes and RNA expression revealed that EBVΔα1 was impaired in reprogramming cellular gene expression leading to a cell cycle arrest at the early S phase and impaired metabolism. Nevertheless, it was possible to establish long-term LCLΔα1 cultures on CD40 ligand expressing feeder cells. The analysis of primary EBVΔα1 infected B cells and LCLΔα1 revealed reduced expression of MYC and LMP1, which are important EBNA2 target genes and required for optimal cell proliferation and survival. Moreover, reverse genetics identified 3 classes of chromatin binding sites 1) EBF1 independent EBNA2 binding, 2) EBF1 dependent EBNA2 binding, 3) sites at which EBF1 and EBNA2 required the complex formation in order to bind. These data indicated that a functional EBNA2-EBF1 complex is required for optimal B cell transformation upon EBV infection. Subsequently, two CRISPR/Cas9 based genome editing approaches were explored to establish conditional EBF1 LCLs to further delineate the contribution of EBF1 to the EBNA2 induced transformation process of EBV infected B cells

Research Symposium 2019, Health Disparities: Community Engagement

With the theme of “Health Disparities: Community Engagement,” this symposium aims to showcase the work done by researchers here in the Valley and beyond toward improving the health and well-being of the communities they serve and society as a whole

Study of crosstalk between G-protein coupled receptor-mediated signals and the Nuclear Factor-κB signal transduction cascade

Nuclear Factor-κB (NF-κB) is an ubiquitously expressed transcription factor that is activated in response to a broad spectrum of inflammatory stimuli, including the proinflammatory cytokine Tumour Necrosis Factor-α (TNF-α). Whereas NF-κB is pivotal for coordination of the immune/inflammatory response, its excessive activation is associated with the onset and propagation of multiple disease processes. NF-κB activity is mostly studied in cells subjected to proinflammatory stimuli, but in "real life" cells are simultaneously exposed to a plethora of signalling molecules that can modulate NF-κB activity. It has been known for many decades that sympathetic stress modulates immunity and inflammation, yet the molecular bases are not completely understood. Therefore, in this thesis, we focused on the activity of the β2- adrenergic receptor (β2-AR), one of the key mediators of the stress response, as a modulator of NF-κB function. In line with other reports describing the anti-inflammatory action of β2-AR agonists (β-agonists), we observed that cotreatment of human astrocytes with TNF-α and a β- agonist, inhibited the expression of several NF-κB-driven genes. However, we found that at the same time it potently enhanced the expression of other prototypical NF-κB target genes, including the proinflammatory cytokine Interleukin-6 (IL-6). We found that the IL-6 synergy, depended on the formation of an enhanceosome structure, and hypothesized that the IL-6 promoter acted as a "coincidence" detector, which requires input from multiple signalling cascades for maximal activation. Our previous research was limited to the study of β2-AR/NF-κB crosstalk in the central nervous system, using astrocytes as a cellular model system. In this thesis, we have extended our previous research to skeletal muscle cells. In addition, we have attempted to further unravel the molecular details of the very strong transcriptional synergy apparent at the IL-6 gene using a proteomics approach. Firstly, we have investigated signalling in response to TNF-α/β-agonist cotreatment in C2C12 cells, a murine skeletal muscle model, representing a physiologically relevant cell type to study β2-AR/NF-κB crosstalk. We observed many similarities in the outcome of β2-AR/NF-κB crosstalk in skeletal muscle cells as compared to astrocytes, although cell-type specific differences in the signalling cascades induced by β- agonists/TNF-α were also apparent. In particular, the very potent synergy at the IL-6 promoter was also detected in skeletal muscle cells. In addition, we found that the expression of several chemokines, influencing the migration potential of undifferentiated skeletal muscle cells, was upregulated upon TNF-α/β-agonist costimulation At the molecular level, we demonstrated that β-agonist-induced potentiation of NF-κB-dependent transcription of the IL-6 gene was associated with histone modifications, chromatin relaxation and formation of an enhanceosome structure. Secondly, using an unbiased proteomics approach, combining DNA-affinity purification and mass spectrometric analysis, we identified Transcription Enhancer Factor 1 (TEF-1) as a novel interactor of the IL-6 promoter. We found that TEF-1 recruitment to the IL-6 promoter was induced upon TNF-α/β-agonist costimulation and that it acted as a transcriptional repressor. Our results furthermore indicate that TEF-1 modulates the transcriptional activity of CREB, but not NF-κB, and that this is associated with altered accessibility of the IL-6 promoter to transcriptional regulators. Importantly, TEF-1 modulated NF-κB-dependent transcription in a gene selective manner. As the effects of β-agonists appear to be highly gene-selective, further elucidation of its molecular basis might lead to the identification of novel targets for the development of selective NF-κB inhibitors. In conclusion, these findings indicate that β2-AR/NF-κB crosstalk promotes potent transcriptional synergy for a subset of NF-κB target genes, including IL-6 and several chemokines. This synergy is apparent in multiple relevant cell types, suggesting it might have general significance. As IL-6 has been attributed with devastating properties in inflammatory disease, and as β-agonists are mainstream therapy for respiratory disease, our data warrant further investigation into the outcome of β2- AR/NF-κB crosstalk in vivo

Advances in Functional Decomposition: Theory and Applications

Functional decomposition aims at finding efficient representations for Boolean functions. It is used in many applications, including multi-level logic synthesis, formal verification, and testing. This dissertation presents novel heuristic algorithms for functional decomposition. These algorithms take advantage of suitable representations of the Boolean functions in order to be efficient. The first two algorithms compute simple-disjoint and disjoint-support decompositions. They are based on representing the target function by a Reduced Ordered Binary Decision Diagram (BDD). Unlike other BDD-based algorithms, the presented ones can deal with larger target functions and produce more decompositions without requiring expensive manipulations of the representation, particularly BDD reordering. The third algorithm also finds disjoint-support decompositions, but it is based on a technique which integrates circuit graph analysis and BDD-based decomposition. The combination of the two approaches results in an algorithm which is more robust than a purely BDD-based one, and that improves both the quality of the results and the running time. The fourth algorithm uses circuit graph analysis to obtain non-disjoint decompositions. We show that the problem of computing non-disjoint decompositions can be reduced to the problem of computing multiple-vertex dominators. We also prove that multiple-vertex dominators can be found in polynomial time. This result is important because there is no known polynomial time algorithm for computing all non-disjoint decompositions of a Boolean function. The fifth algorithm provides an efficient means to decompose a function at the circuit graph level, by using information derived from a BDD representation. This is done without the expensive circuit re-synthesis normally associated with BDD-based decomposition approaches. Finally we present two publications that resulted from the many detours we have taken along the winding path of our research

DIS3 and its Role in CircRNA Degradation

Circular RNAs (circRNAs) are covalently closed, single-stranded endogenous RNAs lacking 5′ end caps and 3′ poly(A) tails. Although the low abundance, these molecules show cell type-, tissue- or developmental stage-specific expression. For decades, circRNAs were considered as byproducts of aberrant splicing. Recent findings unrevealed their cellular functions such as microRNA (miRNAs) or RNA binding proteins (RBPs) sponges, scaffolds and decoys. Although circRNA biogenesis is considerably well understood, it remains intriguing how circRNAs are ultimately degraded, as they are stable and resistant to RNA exonucleolytic decay. Using a biochemical approach, we aim to identify the cellular degradation pathways of circRNAs and the endonucleases involved. To achieve this purpose, using an enzymatic ligation method, we performed in vitro synthesis of selected circRNAs and confirmed their circularity. Then, we measured the stability of the synthetic circRNAs and their linear counterpart in cell lysates of different purification approaches. Our data confirmed the high stability of circRNAs compared to the linear counterparts and showed high sensitivity of degradation for circular RNAs in cytoplasmic extracts suggesting cytoplasmic decay pathways. Next, we used mass spectrometry analysis to identify the endonucleases involved in circRNAs degradation and validated them with in vitro degradation assays. Furthermore, we characterized the function of DIS3 and its PIN domain using in vitro and in vivo experiments. In vitro experiments show that DIS3 can degrade synthetic circRNAs alone or associated with the exosome and that the PIN domain is responsible for its endoribonuclease activity. RNA-seq analysis from CRISPR/Cas9-mediated DIS3 knockout cells confirmed the potential role of DIS3 in degrading a subset of circRNAs. Among them, three candidates such as circOXCT1, circRERE and circFAM208, show upregulation in DIS3 knockout while their linear RNA counterparts do not change. Finally, proteomic studies of DIS3 function in the nucleus and cytoplasm elucidate the molecular mechanisms behind the regulation of DIS3, which might affect circRNA metabolism. Altogether our study adds a new aspect to the function of DIS3 in regulating circRNA degradation pathway

CRISPR-based screening & functional characterization of long non-coding RNAs in melanoma

Melanoma, the most lethal form of skin cancer, is increasingly prevalent in Western populations and is characterized by its high metastatic potential. This cancer type is distinguished by a significant accumulation of somatic mutations, primarily due to UV radiation, leading to a mutation rate that exceeds that of most other solid tumors. The progression of melanoma involves the uncontrolled proliferation and spread of malignant melanocytes, with notable disruptions in the MAPK-ERK and PI3K-AKT-mTOR signalling pathways contributing to the development of advanced therapeutic strategies. Recent research has highlighted the importance of the non-coding regions of the genome, previously considered "junk DNA", for their regulatory functions. Long non-coding RNAs (lncRNAs), which are typically longer than 200 nucleotides, have been identified as key players in cellular development, differentiation, and cancer progression. Numerous studies have established a link between lncRNAs and the growth and progression of melanoma, with elevated lncRNA expression levels observed in melanoma cases. Herein, the present study attempted to elucidate the involvement of a specific set of lncRNAs in mechanisms underlying cell growth and proliferation. These lncRNAs, which had been observed to exhibit increased expression in melanoma cell lines and in short-term cultures derived from brain and lymph node metastasis, are examined by employing CRISPRi screening. Furthermore, functional characterization analyses were conducted on the most promising lncRNA candidates, including BDNF-AS, GMDS-AS1, and a novel, non-annotated lncRNA named XLOC030781 based on preliminary data that validate their importance in melanoma. Suppression of these lncRNAs led to apoptosis and inhibited cell cycle progression, with XLOC030781 playing a notable role in melanoma migration, broadening its functional scope in relation to this malignancy. Finally, the implementation of fluorescence in situ hybridization technique provided data on their subcellular localization, offering complementary information on their functionality