Derivation of species-specific hybridization-like knowledge out of cross-species hybridization results

BACKGROUND: One of the approaches for conducting genomics research in organisms without extant microarray platforms is to profile their expression patterns by using Cross-Species Hybridization (CSH). Several different studies using spotted microarray and CSH produced contradicting conclusions in the ability of CSH to reflect biological processes described by species-specific hybridization (SSH). RESULTS: We used a tomato-spotted cDNA microarray to examine the ability of CSH to reflect SSH data. Potato RNA was hybridized to spotted cDNA tomato and potato microarrays to generate CSH and SSH data, respectively. Difficulties arose in obtaining transcriptomic data from CSH that reflected those obtained from SSH. Nevertheless, once the data was filtered for those corresponding to matching probe sets, by restricting proper cutoffs of probe homology, the CSH transcriptome data showed improved reflection of those of the SSH. CONCLUSIONS: This study evaluated the relative performance of CSH compared to SSH, and proposes methods to ensure that CSH closely reflects the biological process analyzed by SSH

Regulated T cell pre-mRNA splicing as genetic marker of T cell suppression

Includes abstract. Includes bibliographical references

COMPUTATIONAL APPROACHES IN THE ESTIMATION AND ANALYSIS OF TRANSCRIPTS DIFFERENTIAL EXPRESSION AND SPLICING: APPLICATION TO SPINAL MUSCULAR ATROPHY

Spinal Muscular Atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. SMA is caused by deletion or mutations in the survival motor neuron 1 gene (SMN1), which are expected to generate alterations in RNA transcription, or splicing and most importantly reductions in mRNA transport within the axons of motor neurons (MNs). SMA ultimately results in the selective degeneration of MNs in spinal cord, but the underlying reason is still not clear entirely. The aim of this study is to investigate splicing abnormalities in SMA, and to identify genes presenting differential splicing possibly involved in the pathogenesis of SMA at genome-wide level. We performed RNA-Sequencing data analysis on 2 SMA patients and 2 controls, with 2 biological replicates each sample, derived from their induced Pluripotent Stem Cells-differentiated-MNs. Three types of analyses were executed. Firstly, differential expression analysis was performed to identify possibly mis-regulated genes using Cufflinks. Secondly, alternative splicing analysis was conducted to find differentially-used exons (DUEs; using DEXSeq) as splicing patterns are known to be altered in MNs by the suboptimal levels of SMN protein. Thirdly, we did RNA-binding protein (RBP) - motif discovery for the set of identified alternative cassette-DUEs, to pinpoint possible mechanisms of such alterations, specific to MNs. The gene ontology enrichment analysis of significant DEGs and alternative cassette-DUEs revealed various interesting terms including axon-guidance, muscle-contraction, microtubule-based transport, axon-cargo transport, synapse etc. which suggests their involvement in SMA. Further, promising results were obtained from motif analysis which has identified 22 RBPs out of which 7 RBPs namely, PABPC1, PABPC3, PABPC4, PABPC5, PABPN1, SART3 and KHDRBS1 are known for mRNAs stabilization and mRNA transport across MN-axon. Five RBPs from PABP family are known to interact directly with SMN protein that enhance mRNA transport in MNs. To validate our results specific wet-lab experiments are required, involving precise recognition of RNA-binding sites correspondent with our findings. Our work has provided a promising set of putative targets which might offer potential therapeutic role towards treating SMA. During the course of our study, we have observed that current methods for an effective understanding of differential splicing events within the transcriptomic landscape at high resolution are insufficient. To address this problem, we developed a computational model which has a potential to precisely estimate the \u201ctranscript expression levels\u201d within a given gene locus by disentangling mature and nascent transcription contributions for each transcript at per base resolution. We modeled exonic and intronic read coverages by applying a non-linear computational model and estimated expression for each transcript, which best approximated the observed expression in total RNA-Seq data. The performance of our model was good in terms of computational processing time and memory usage. The application of our model is in the detection of differential splicing events. At exon level, differences in the ratio of the sum of mature and the sum of nascent transcripts over all the transcripts in a gene locus gives an indication of differential splicing. We have implemented our model in R-statistical language

Identification and Characterization of Two Novel Primate-specific Histone H3 Variants, H3.X and H3.Y

Chromatin is the packaged form of DNA in the eukaryotic nucleus, with the nucleosome as its basic unit. The nucleosome consists of DNA, wrapped around a histone octamer which is comprised of two copies of each of the four core histones H2A, H2B, H3 and H4. To allow DNA-related processes to occur, access to the DNA has to be regulated. One regulatory mechanism is the exchange of the canonical core histones by one of their replacement variants. Thus far, five members of the histone H3 family have been described: H3.1, H3.2, H3.3, testis-specific tH3 and centromere-specific CENP-A. Searching the database, we have recently identified two novel histone H3 variant genes on human chromosome 5, now termed H3.X and H3.Y. In my PhD thesis, I have analyzed their expression patterns, characterized their mRNA as well as their protein products and investigated their potential function(s). H3.X and H3.Y constitute primate-specific genes which have been found, in addition to humans, also in the chimpanzee and the macaque, but not in other mammals or even lower eukaryotes. H3.X and especially H3.Y mRNA is expressed at low but significant levels in the human osteosarcoma cell line U2OS and in some human bone, breast, lung and ovary tumor tissues, as well as in testis and certain areas of the brain. Tagged H3.X and H3.Y proteins behave similar to H3.1, H3.2 and H3.3 in their nuclear localization and stable incorporation into chromatin. However, in contrast to H3.1 and H3.3, novel H3 variants primarily form heterotypic nucleosomes. Endogenous H3.Y protein is expressed in a small number of U2OS cells (<0.1%), localizes to the nucleus and constitutes a stable chromatin component. The number of H3.Y-expressing U2OS cells can be increased by specific stress conditions, such as nutritional stress paired with high cell density. Matching its expression pattern in U2OS cells, H3.X and/or H3.Y protein(s) have also been observed in a small subpopulation of neurons in the human hippocampus. H3.Y has an influence on the expression of certain genes, most of them being implicated in regulating the cell cycle and chromatin structure. In line with this finding, knockdown of H3.X and H3.Y impairs cell growth. In conclusion, the results of this work show that H3.Y (and maybe H3.X) are novel primatespecific histone H3 variants which play a role in cell cycle regulation. Their expression is induced by certain stress stimuli and their presence in brain and testis suggests a potential role in primate-specific brain functions and speciation

Genetic Dissection of Therapeutic Intervention Targets in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer, accounting for 20% of all breast cancer diagnoses. Currently, there are no TNBC-specific targeted therapies in the clinic and therefore, broad-spectrum cytotoxic chemotherapeutic regimens remain the standard of care. Due to high rates of innate drug resistance, many TNBC patients do not respond to these regimens and, therefore, have no other therapeutic options. Our group employed a pan-genomic loss of function screen to systematically dissect the molecular architecture that functionally supports TNBC to uncover new therapeutic entry points. We further applied a paclitaxel-based synthetic lethal approach to increase our discovery space to also identify those molecular components that modulate chemoresponsiveness. To encompass the heterogeneity found within the TNBC patient population, we screened four triple negative tumor-derived breast cancer cell lines that represent the spectrum of TNBC oncogenic aberrations and chemoresponse profiles. We also accounted for the two molecular subtypes, claudin-low and basal-like that comprise the majority of TNBC cases. These screens revealed a number of core modulators of tumor cell viability and paclitaxel-induced cellular stress that have not been previously appreciated for supporting TNBC biology at the cell autonomous level. In particular, this strategy implicated the signaling supported by the cytokine receptor, CXCR3, and its ligand, CXCL9, to promote mitotic fidelity and tumor cell survival in the basal-like TNBC molecular subtype. In addition, we uncovered a requirement for the AMPK family member, SIK2, as a key nutrient sensor that may inhibit excessive autophagy. Inhibition of SIK2 enhanced autophagic flux and a loss of cell viability in a variety of TNBC genetic backgrounds. Finally, we discovered that cancer testes antigen transcription factor, ZNF165, directly repressed expression of negative TGFβ regulators thereby specifying a pro-tumorigenic TGFβ gene expression profile. Given that the expression of ZNF165 is otherwise restricted to the male testes, ZNF165 may represent a mechanism by which tumors engage anomaly-expressed proteins to promote survival. Taken together, our screening approach uncovered novel TNBC tumor cell vulnerabilities that identified cellular processes that could lead to new therapeutic approaches.Doctor of Philosoph

Über Long-Tails, Mikroarrays, und Markersets: Integrative wissenschaftliche Ansätze in funktioneller Genomik, Populationsgenetik und genetischer Epidemiologie

The work in the three presented articles provides several demonstrations of how an integrative approach to scientific research has led to a better understand of biological phenomena. The first article incorporates research from the overlapping fields of biotechnology, functional genomics, and bioinformatics. The study's objective is to describe the nature of the distribution of gene expression levels measured with microarrays with the aim of developing an inter-array normalization method. The normalization method is compared to other existing normalization methods and is found to be especially suited to so-called boutique microarrays. The second article uses genotyping data generated by microarrays with the goal of examining the population genetic structure of the European human population. This study combines aspects of the fields of biotechnology, bioinformatics, and population genetics and sheds light on the genetic differences between Europeans by characterizing a strong correlation between geographic and genetic distance. In the final article, focus switches from genetic differences to genetic similarities in the same European individuals by examining the relationship structure of genetic nearest neighbors. Observations about these relationships lead to the proposal of a genetic matched-pair study design that contributes a methodological improvement to the field of genetic epidemiology. The proposed study design has the potential to increase the power of analysis of genome-wide association studies which are used to discover disease-causing genes. A presentation of previously unpublished research which was generated during the course of the work is also included. Finally, a discussion of long-tail data distributions initially observed in the first article leads to conclusions on the fundamental properties of the informational content of genetic marker sets ascertained in the last two articles.Die Arbeiten, die in den drei vorliegenden Artikeln präsentiert werden, zeigen, wie ein integrativer wissenschaftlicher Ansatz zu einem besseren Verständnis biologischer Phänomene führt. Der erste Artikel verknüpft Forschung aus den sich überlappenden Fachgebieten Biotechnologie, funktionelle Genomik und Bioinformatik. Das Ziel der Studie war es, mittels Mikroarrays die Verteilungsform der Genexpressionsniveaus zu bestimmen, um eine Normalisierungsmethode zu entwickeln. Diese Normalisierungsmethode wurde mit anderen bereits bekannten Normalisierungsmethoden verglichen und sie erwies sich als besonders geeignet für sogenannte Boutique-Mikroarrays. Der zweite Artikel verfolgt das Ziel, mit Hilfe von humanen Genotypisierungsdaten aus Mikroarrays die populationsgenetische Struktur der europäischen Population zu charakterisieren. Diese Studie verbindet Aspekte der Forschungsgebiete Biotechnologie, Bioinformatik und Populationsgenetik und gibt damit Aufschluss über die Muster genetischer Unterschiede zwischen Europäern: Es konnte eine hohe Korrelation zwischen geographischen und genetischen Distanzen gezeigt werden. Der letzte Artikel richtet den Blick auf die genetischen Gemeinsamkeiten der selben europäischen Individuen, indem er die Verwandtschaftsstruktur mittels eines genetischen "nearest neighbors"-Algorithmus untersucht. Die beobachteten Verwandtschaftsstrukturen führen zum Vorschlag eines genetischen Matched-Pair-Studiendesigns, das auf dem Gebiet der genetischen Epidemiologie eine erhebliche methodische Verbesserung darstellt. Das vorgeschlagene Studiendesign kann die Aussagekraft der statistischen Analysen bei Genom-weiten Assoziationsstudien erhöhen, also bei Studien, die durchgeführt werden, um krankheitsverursachende Gene zu identifizieren. Darüber hinaus werden bisher unveröffentlichte Forschungsergebnisse vorgestellt, die im Zusammenhang mit den obigen Studien gewonnen wurden. Eine abschließende Diskussion der Long-Tailed-Verteilung der Daten, die zunächst in der ersten Studie beobachtet wurde, führt zu Schlussfolgerungen über die grundlegenden Eigenschaften des Informationsgehaltes genetischer Markersets, welche nachfolgend in den letzten beiden Studien bestätigt wurden