Transcriptional regulation at a glance

Considering that 80 genomes have been sequenced, providing us with the static information of the genome, it is still a long way to reveal the relationship between complex genotypes and phenotypes. The transcriptional regulation process is one of the obstacles that need to be understood to bridge our current information gap. It describes the first step from the genomic sequence information to RNA templates used for protein production or as direct functional units, like non-coding RNAs (e.g. micro RNAs). This introduction aims to highlight the key aspects of the transcriptional process from our current understanding

Regulationseinheiten in Evolution, Entwicklung und humaner Krankheit

Misregulation of gene expression can result in broad types of diseases and abnormalities. For elucidating the molecular mechanisms underlying evolution, development and disease, the 2% protein-coding regions in the human genome have been studied for decades, but the the 98% non-coding regions remain less understood. Many studies have revealed that evolutionary conserved non-coding elements (CNEs) act as cis-regulatory elements (CREs). Nevertheless, not all CREs are evolutionary conserved and, hence, the identification of CREs has proved challenging. Some CREs work cooperatively to regulate gene expression. Together with their target gene(s), these CREs form regulatory units. For instance, topologically associating domains (TADs) are large regulatory units in the genome, controlling and restricting the interactions between genes and CREs. Many studies have found the disruption of TADs can lead to gene expression misregulation. This thesis aimed at characterizing regulatory units. In particular, we explored the mechanisms by which regulatory unit disruption can lead to changes in gene expression in evolution, development, and disease. The first part of this thesis focused on the identification and characterization of small regulatory units consisting of two CREs. We identified 5,500 pairs of adjacent CNEs (to which we further refer as “CNE-CNE pairs”) in the human genome with either expanded, conserved or contracted inter-CNE sequences compared to the common mammalian ancestor. Particularly, the CNEs in CNE-CNE pairs with conserved and mildly contracted inter-CNEs sequences were most likely to perform as active or poised enhancers; in addition, both CNEs of the pairs exhibited similar epigenetic profiles, suggesting that the CNE-CNE pairs tend to act as small regulatory units. Furthermore, transposon deletions and insertions were associated with the contraction or expansion of inter-CNE sequences, indicating that transposon activity might disrupt the link between the two CNEs in the CNE-CNE pairs, leading to a loss of cis-regulatory function. Our study identified novel regulatory units, and highlighted the existence of cooperative interaction between adjacent CREs in the regulatory units that are distance-sensitive and can be disrupted by transposon activity. Our findings contribute to understanding the mechanism by which selective forces act on CREs in the context of evolution and human genetic diseases. The second part of this thesis investigated the mechanisms by which regulatory units can be disrupted, leading to gene expression misregulation in disease. To address this, we first constructed 1,467 consensus TADs in the “normal genome” and 1,622 consensus TADs in the “cancer genome”. To evaluate the prognosis value of knowing the location of structural variants such as copy number variants (CNVs) within the TADs in cancer patient survival outcome, we applied Cox regression analysis. In this manner, we identified 35 prognostic TADs; 54% of these TADs did not contain any genes with a known association to cancer causality, indicating that a large fraction of the TADs have prognostic value independently of coding variants. Furthermore, 34% of the 35 prognostic TADs underwent strong structural perturbations in the cancer genome. Hence, the prognostic value of at least a fraction of the 35 TADs appeared to be associated with the disruption of normal CRE interactions. Our study emphasized the importance of identifying perturbed regulatory units in monitoring cancer development and progression. Understanding the molecular mechanisms regulating gene expression will help us to understand the formation and evolution of life and to find cures for diseases.Eine fehlerhafte Regulation der Genexpression kann zu einem breiten Spektrum an Krankheiten und Abnormitäten führen. Zur Aufklärung der molekularen Mechanismen, die Evolution, Entwicklung und Krankheit zugrunde liegen, werden die 2% protein-kodierenden Regionen im menschlichen Genom seit Jahrzehnten untersucht, die 98% nichtkodierenden Regionen sind bisher deutlich weniger gut verstanden. Viele Studien haben gezeigt, dass evolutionär konservierte nicht-kodierende Elemente (KNEs) als cis-regulatorische Elemente (CREs) fungieren. Jedoch sind nicht alle CREs evolutionär konserviert, weswegen sich die Identifizierung von CREs als schwierig erwiesen hat. Einige CREs regulieren die Genexpression kooperativ. Zusammen mit ihrem Zielgen oder ihren Zielgenen bilden diese CREs Regulationseinheiten. Zum Beispiel sind topologisch assoziierende Domänen (TADs) große Regulationseinheiten im Genom, die die Interaktionen zwischen Genen und CREs kontrollieren und einschränken. Viele Studien haben bestätigt, dass die Störung der TAD-Struktur zu einer fehlerhaften Genexpression führen kann. Diese Arbeit zielte auf die Charakterisierung von Regulationseinheiten ab. Insbesondere hat sie die Mechanismen erforscht, durch die eine Störung von Regulationseinheiten zu Veränderungen der Genexpression führt, im Kontext von Evolution, Entwicklung und Krankheit. Der erste Teil dieser Arbeit hat sich mit der Identifizierung und Charakterisierung kleiner Regulationseinheiten, bestehend aus zwei CREs, befasst. Wir haben 5500 Paare benachbarter KNEs im menschlichen Genom identifiziert (im Folgenden als ”KNE-KNE-Paare” bezeichnet), bei denen die Inter-KNE-Sequenzen im Vergleich zu dem gemeinsamen Vorfahren der Säugetiere entweder expandiert, konserviert, oder kontrahiert sind. Insbesondere die KNEs in KNE-KNE-Paaren mit konservierten oder leicht kontrahierten Inter-KNE-Sequenzen wiesen die größte Wahrscheinlichkeit auf, als aktive Enhancer oder ”poised” Enhancer (d.h., in Bereitschaftsstellung) zu fungieren. Des Weiteren zeigten die zu einem Paar gehörenden KNEs ähnliche epigenetische Profile, was darauf hindeutet, dass die KNE-KNE-Paare als kleine Regulationseinheiten fungieren. Darüber hinaus waren Transposon-Deletionen (respektive Insertionen) mit einer Kontraktion (respektive Expansion) der Inter-KNE-Sequenzen assoziiert. Dies deutet an, dass die Transposonaktivität möglicherweise den funktionellen Zusammenhang zwis chen den beiden KNEs in den KNE-KNE-Paaren stört und so zu einem Verlust ihrer cis-regulatorischen Funktion führt. Unsere Studie hat neue Regulationseinheiten identifiziert und die Existenz kooperativer Interaktionen zwischen benachbarten CREs, die sich in distanzsensitiven und durch Transposonaktivität zerstörbaren Regulationseinheiten befinden, hervorgehoben. Unsere Ergebnisse tragen zum Verständnis des Mechanismus bei, durch den selektive Kräfte auf CREs im Kontext von Evolution und genetisch bedingten Erkrankungen des Menschen wirken. Der zweite Teil dieser Arbeit hat die Mechanismen untersucht, durch die Regulationseinheiten gestört werden können, was zu einer Fehlregulation der Genexpression bei Krankheiten führt. Hierzu haben wir zunächst 1467 Konsensus-TADs im „normalen Genom“ und 1622 Konsensus-TADs im „Krebsgenom“ konstruiert. Um den prognostischen Wert der Kenntnis der Position von Strukturvarianten wie Kopienzahlvarianten (KZVs) innerhalb der TADs bezüglich der Überlebensdauer von Krebspatienten zu ermitteln, haben wir eine Cox-Regressionsanalyse durchgeführt. Auf diese Weise haben wir 35 prognostische TADs identifiziert; 54% dieser TADs enthielten keine Gene mit einem bekannten Zusammenhang mit der Krebsursache, was darauf hinweist, dass ein großer Teil der TADs unabhängig von kodierenden Varianten einen prognostischen Wert hat. Darüber hinaus zeigten 34% der 35 prognostischen TADs starke strukturelle Störungen im Krebsgenom. Daher schien der prognostische Wert von mindestens einem Bruchteil der 35 TADs mit der Störung normaler CRE-Interaktionen verbunden zu sein. Unsere Studie zeigt, wie wichtig es ist, gestörte Regulationseinheiten zur Überwachung der Krebsentwicklung und -progression zu identifizieren. Das Verständnis der molekularen Mechanismen, die die Genexpression regulieren, wird uns helfen die Entstehung und Evolution des Lebens zu verstehen und Heilmittel für Krankheiten zu finden

Alternative splicing: transcriptional regulatory network in agroforestry

Alternative splicing (AS) in plants plays a key role in regulating the expression of numerous transcripts from a single gene in a regulatory pathway. Variable concentrations of growth regulatory hormones and external stimuli trigger alternative splicing to switch among different growth stages and adapt to environmental stresses. In the AS phenomenon, a spliceosome causes differential transcriptional modifications in messenger RNA (mRNAs), resulting in partial or complete retention of one or more introns as compared to fully spliced mRNA. Differentially expressed proteins translated from intron-retaining messenger RNA (mRNAir) perform vital functions in the feedback mechanism. At the post-transcriptional level, AS causes the remodeling of transcription factors (TFs) by the addition or deletion of binding domains to activate and/or repress transcription. In this study, we have summarized the specific role of AS in the regulation of gene expression through repression and activation of the transcriptional regulatory network under external stimuli and switch among developmental stages

Screening for PPAR Responsive Regulatory Modules in Cancer

Peroxisome proliferator-activated receptors (PPARs) have via their large set of target genes a critical impact on numerous diseases including cancer. Cancer development involves numerous regulatory cascades that drive the progression of the malignancy of the cells. On a genomic level, these pathways converge on regulatory modules, some of which contain colocalizing PPAR binding sites (PPREs). We developed an in silico screening method that incorporates experiment- and informatics-derived evidence for a more reliable prediction of PPREs and PPAR target genes. This method is based on DNA-binding data of PPAR subtypes to a panel of DR1-type PPREs and tracking the enrichment of binding sites from multiple species. The ability of PPARγ to induce cellular differentiation and the existence of FDA-approved PPARγ agonists encourage the exploration of possibilities to activate or inactivate PPRE containing modules to arrest cancer progression. Recent advances in genomic techniques combined with computational analysis of binding modules are discussed in the review with the example of our recent screen for PPREs on human chromosome 19

Forkhead box R1-mediated stress response linked to a case of human microcephaly and progressive brain atrophy

Forkhead box (Fox) family transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. This family was named after the ectopic head structures observed in mutants of the Drosophila gene forkhead (fkh). Since the discovery of fkh, hundreds of Fox genes have been identified in organisms ranging from yeasts to humans, making it one of the largest but least explored families of higher eukaryotic transcription factors. The NIH Undiagnosed Diseases Program (NIH UDP), a clinical site of the NIH Undiagnosed Diseases Network (UDN), identified a variant (p.M280L) in a single allele of the FOXR1 gene in an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy, and global developmental delay. The de novo missense variant in FOXR1 converts a highly conserved methionine residue at amino acid 280 to leucine and was predicted to contribute to the individual’s disease. The goal of this research is to investigate the biological role of FOXR1 and to determine how the M280L mutant leads to disease pathogenesis. At the protein level, the M280L mutant impaired FOXR1 expression and induced a nuclear aggregate phenotype when overexpressed in HEK 293T and COS7 cells due to protein misfolding and proteolysis. A FOXR1 C-terminal truncation mutant mimicked the M280L phenotype, suggesting that the C-terminal sequences of FOXR1 are important for FOXR1 protein stability. RNAseq and pathway analysis in HEK 293T cells indicated that FOXR1 acts as both transcriptional activator and repressor, playing central roles in heat shock response, chaperone cofactor-dependent protein refolding, and cellular response to stress. Indeed, FOXR1 expression is increased in HEK 293T in response to cellular stress, a process in which FOXRI directly controls HSPA6, HSPA1A and DHRS2 transcription. In contrast, the ability of the M280L mutant to respond to stress is compromised, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Combined, these results suggest that FOXR1 plays a role in cellular stress and that impairment of these functions may contribute to the disease phenotypes seen in the individual with the FOXRI M280L variant

Novel Bioinformatics Approaches for MicroRNA Detection and Target Prediction.

MicroRNAs (miRNAs) are regulators of gene expression at the post-transcriptional level. Scientists have not been able to fully unlock their therapeutic potential because their functions and mechanisms of action have not been fully characterized. In this thesis we address shortcomings and provide solutions for detecting miRNAs in a high-throughput manner and for predicting miRNA targets - areas key to understanding miRNA function. Profiling expression of miRNAs using microarrays has its limitations owing to diverse melting temperatures and high sequence similarities, which affects sensitivity and specificity. A simple yet effective strategy that we employ involves base changes to probes complementary to mature miRNAs. Using nearest-neighbour thermodynamic principles we determine the best probes for all mature miRNAs that serve to eliminate cross-hybridization and create a uniform melting temperature profile. We present a set of probes that are designed for the human let-7 family and demonstrate their power to resolve these similar sequences in a microarray experiment using both spiked-in and true biological samples. The second problem that is tackled in this thesis involves improving miRNA target prediction, a key to understanding miRNA function in various biological processes. We use a combination of thermodynamic and sequence-based searches to identify endogenous sites on 5′-UTRs. There are two aspects that make our approach unique compared to other target prediction methodologies. First, we not only consider seed-matches on the 3′-UTR but also 5′-UTR matches with 3′-ends of miRNAs. Second, we show that non-conserved sites on the 5′-UTR can possibly contribute to species-specific targeting. We verify our claims through in vitro experiments using two predicted miRNA-target pairs: hsa-miR-34a and its target AXIN2, and cel-lin-4 and its target lin28. Extending results from the target prediction study, we show that upstream AUGs (uAUGs), which are known to post-transcriptionally regulate gene expression, are probable binding sites for miRNAs. We show that the cell- or tissue-specific repression of genes that harbour uAUGs can be explained by the expression of targeting miRNAs in those cells. The approaches suggested here will help further our understanding of how these tiny RNAs regulate gene expression.Ph.D.BioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62207/1/sasubram_1.pd

Charakterizace protein-proteinových interakcí mezi transkripčními faktory Forkhead box O (FOXO) a p53

Transkripční faktor p53 hraje klíčovou roli v regulaci buněčného cyklu, opravě DNA, apoptóze, obraně před nádorovými onemocněními a udržování buněčné homeostázy. Při buněčném stresu p53 přímo interaguje s transkripčním faktorem Forkhead box O (FOXO) 4, čímž zvyšuje expresi proteinu p21, což vede k indukci buněčné senescence. Molekulární mechanismus interakce těchto dvou klíčových transkripčních faktorů však zůstává nejasný kvůli absenci strukturních dat. Hlavním cílem této dizertační práce byla strukturní charakterizace interakce mezi FOXO4 a p53 prostřednictvím několika biofyzikálních technik včetně metody sedimentační rychlosti analytické ultracentrifugace (SV AUC), nukleární magnetické rezonance (NMR), chemického zesítění spojeného s hmotnostní spektrometrií. Dále byla studována DNA vazebná afinita obou proteinů k jejich příslušným konsenzuálním DNA sekvencím v přítomnosti nebo nepřítomnosti jejich vazebných partnerů a byl porovnán p53-vazebný povrch Forkhead domény tří různých FOXO proteinů. Připravené proteiny byly také použity pro optimalizaci nízkomolekulárních sloučenin umožňujících inhibici interakce mezi FOXO3 a DNA. Získané výsledky ukázaly, že p53 interaguje s FOXO4 prostřednictvím komplexních přechodných interakcí zahrnujících strukturované i neuspořádané oblasti obou proteinů. NMR...The transcription factor p53 plays a key role in cell cycle arrest, DNA repair, apoptosis, tumor suppression, and maintaining cellular homeostasis. Under cellular stress, p53 directly interacts with the Forkhead box O (FOXO) 4 transcription factor, thereby upregulating the expression of the p21 gene, resulting in the induction of cellular senescence. However, the detailed molecular mechanism behind FOXO4-p53 interaction remains unclear due to the unavailability of structural data. Therefore, main goal of this doctoral thesis was the characterization of the interaction between FOXO4 and p53 using several biophysical techniques including sedimentation velocity analytical ultracentrifugation (SV AUC), nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled to mass spectrometry. Furthermore, we also investigated the DNA binding properties of both proteins with their respective consensus DNA sequences in the presence or absence of their binding partners by fluorescence anisotropy measurements along with the comparison of p53-binding surfaces of the Forkhead domain of three different FOXO proteins by NMR spectroscopy. In addition, we also optimized small molecule compounds for the inhibition of FOXO3-DNA interaction. Our results revealed that the p53 interacts with FOXO4 through...Katedra fyzikální a makromol. chemieDepartment of Physical and Macromolecular ChemistryPřírodovědecká fakultaFaculty of Scienc

Analysis of dysbindin interacting genes in the pathogenesis of schizophrenia

Dysbindin (dystrobrevin-binding protein 1 DTNBP1) has been implicated as a schizophrenia candidate gene. However while numerous positive associations have been reported, no non-synonymous alleles have been found which account for the association. A number of recent studies suggest that altered dysbindin expression may be the mechanism by which DTNBP1 variants confer susceptibility to schizophrenia. Therefore one objective of this study was to identify putative DTNBP1 cis-acting variants and perform association analyses of these variants with schizophrenia and allelic expression differences observed at the DTNBP1 locus. While four variants were associated with schizophrenia, logistic regression suggested that the signal observed at these polymorphisms was not independent of the most associated SNP rs4715984. Comparison with the results of a DTNBP1 allelic expression assay revealed that seven SNPs were associated with differential expression. Post hoc analysis revealed that the majority of the expression differences were accounted for by variation at two loci (rs2619538 and rsl3198512), one of which (rsl3198512) was subsequently shown to directly affect transcription in vitro using a luciferase reporter gene assay. As rs4715984 was not correlated with allelic expression differences it implies that a reduction in dysbindin expression through cis-acting variation may not be the primary aetiological factor in schizophrenia pathogenesis. This was supported by further analysis of a schizophrenia risk haplotype previously reported to be associated with differential expression as the refined haplotype was no longer correlated. A second objective of this thesis was to investigate the hypothesis that DTNBP1 could cause susceptibility to schizophrenia through its role within the BLOC-1 complex. Association analysis was performed on BLOC-1 genes which displayed evidence of being under the influence of cis-acting regulation. MUTED, a BLOC-1 gene previously reported as associated to schizophrenia was also investigated. However association results provided no compelling support for the hypothesis that DTNBP1 contributes to susceptibility to schizophrenia through the BLOC-1 complex.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Analysis of dysbindin interacting genes in the pathogenesis of schizophrenia

Dysbindin (dystrobrevin-binding protein 1 DTNBP1) has been implicated as a schizophrenia candidate gene. However while numerous positive associations have been reported, no non-synonymous alleles have been found which account for the association. A number of recent studies suggest that altered dysbindin expression may be the mechanism by which DTNBP1 variants confer susceptibility to schizophrenia. Therefore one objective of this study was to identify putative DTNBP1 cis-acting variants and perform association analyses of these variants with schizophrenia and allelic expression differences observed at the DTNBP1 locus. While four variants were associated with schizophrenia, logistic regression suggested that the signal observed at these polymorphisms was not independent of the most associated SNP rs4715984. Comparison with the results of a DTNBP1 allelic expression assay revealed that seven SNPs were associated with differential expression. Post hoc analysis revealed that the majority of the expression differences were accounted for by variation at two loci (rs2619538 and rsl3198512), one of which (rsl3198512) was subsequently shown to directly affect transcription in vitro using a luciferase reporter gene assay. As rs4715984 was not correlated with allelic expression differences it implies that a reduction in dysbindin expression through cis-acting variation may not be the primary aetiological factor in schizophrenia pathogenesis. This was supported by further analysis of a schizophrenia risk haplotype previously reported to be associated with differential expression as the refined haplotype was no longer correlated. A second objective of this thesis was to investigate the hypothesis that DTNBP1 could cause susceptibility to schizophrenia through its role within the BLOC-1 complex. Association analysis was performed on BLOC-1 genes which displayed evidence of being under the influence of cis-acting regulation. MUTED, a BLOC-1 gene previously reported as associated to schizophrenia was also investigated. However association results provided no compelling support for the hypothesis that DTNBP1 contributes to susceptibility to schizophrenia through the BLOC-1 complex.EThOS - Electronic Theses Online ServiceGBUnited Kingdo