5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...

Indiana University-Purdue University Indianapolis (IUPUI) / The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.

There is a lack of low toxicity, specific anticancer therapies and in many cancer types there are limited effective treatments. Enhancer RNAs are noncoding RNA transcripts transcribed from enhancer regions. Increasing evidence of the function of eRNA in gene regulation suggests the possibility of eRNA involvement in cancer development. This report examines literature on enhancer RNA as a potent component in transcription control specifically in cancer development. Therefore, I conducted a systematic literature review to further clarify the involvement of eRNAs in cancer. There is strong evidence of eRNA upregulating oncogenes. For instance, the eRNA (CCAT1) upregulates the oncogene MYC in colorectal cancer. Other eRNAs were also found to be required for p53-dependent cell-cycle arrest and tumour inhibition. A study showed the interplay of a long noncoding RNA with eRNAs in p53-regulated enhancers, while another showed p53-bound enhancer regions transcribing an eRNA which mediates G1 arrest, DNA repair, and tumorigenesis through its interaction with the (BRCA2) gene. Finally, a study across numerous cancer patient samples revealed a cancer/lineage specificity of eRNAs and explored the clinical feasibility of eRNA-targeted therapy. These studies demonstrate how eRNAs can be a link in cancer signalling pathways both as a regulator of oncogenes and tumour suppressor genes, as well as suggest a promising future of eRNA-targeted cancer therapy.

Myocyte Enhancer Factor 2D (Mef2D) is a member of the Mef2 family. As a transcription factor, Mef2D regulates the expression of genes that impinge on cellular viability, tissue development, and fuel metabolism. Interestingly, Mef2D function appears to be tissue dependent. The function of Mef2D appears to be tissue-specific, with differences in activity in the immune system, neuronal tissue, and muscle. Here, we review the published literature describing the role of Mef2D across tissues. Little is known about the role of Mef2D in the beta-cell. To determine the function of Mef2D in the beta-cell, we built overexpression and knockdown cell lines. We determined the effect of Mef2D overexpression or knockdown on mitochondrial respiration, insulin secretion, cell survival, and gene expression in the INS-1 832/13 beta-cell line. Our data demonstrates that Mef2D knockdown enhances mitochondrial respiration, insulin secretion, and cell survival. Conversely, Mef2D overexpression inhibits mitochondrial respiration, insulin secretion, and cell survival. We demonstrate that some of this effect is due to modulated expression of the mitochondrial gene mtND6. These findings demonstrate that Mef2D overexpression is detrimental to beta-cell function and that Mef2D knockdown is beneficial. These data suggest that Mef2D may be a viable target to enhance functional beta-cell mass as a treatment for Type 1 and Type 2 Diabetes.

Genomweite Bemühungen haben eine große Anzahl langer nichtkodierender RNAs (lncRNAs) identifiziert, obwohl ihre möglichen Funktionen weitgehend rätselhaft bleiben. Hier verwendeten wir ein System zur synchronisierten Blüteninduktion in Arabidopsis, um 4106 blütenbezogene lange intergene RNAs (lincRNAs) zu identifizieren. Blütenbezogene lincRNAs sind typischerweise mit funktionellen Enhancern assoziiert, die bidirektional transkribiert werden und mit verschiedenen funktionellen Genmodulen assoziiert sind, die mit der Entwicklung von Blütenorganen zusammenhängen, die durch Koexpressionsnetzwerkanalyse aufgedeckt wurden. Die Master-regulatorischen Transkriptionsfaktoren (TFs) APETALA1 (AP1) und SEPALLATA3 (SEP3) binden an lincRNA-assoziierte Enhancer. Die Bindung dieser TFs korreliert mit der Zunahme der lincRNA-Transkription und fördert möglicherweise die Zugänglichkeit von Chromatin an Enhancern, gefolgt von der Aktivierung einer Untergruppe von Zielgenen. Darüber hinaus ist die Evolutionsdynamik von lincRNAs in Pflanzen, einschließlich nicht blühender Pflanzen, noch nicht bekannt, und das Expressionsmuster in verschiedenen Pflanzenarten war ziemlich unbekannt. Hier identifizierten wir Tausende von lincRNAs in 26 Pflanzenarten, einschließlich nicht blühender Pflanzen. Ein direkter Vergleich von lincRNAs zeigt, dass die meisten lincRNAs speziesspezifisch sind und das Expressionsmuster von lincRNAs einen hohen Transkriptionsumsatz nahe legt. Darüber hinaus zeigen konservierte lincRNAs eine aktive Regulation durch Transkriptionsfaktoren wie AP1 und SEP3. Konservierte lincRNAs zeigen eine konservierte blütenbezogene Funktionalität sowohl in der Brassicaceae- als auch in der Grasfamilie. Die Evolutionslandschaft von lincRNAs in Pflanzen liefert wichtige Einblicke in die Erhaltung und Funktionalität von lincRNAs. / Genome-wide efforts have identified a large number of long non-coding RNAs (lncRNAs), although their potential functions remain largely enigmatic. Here, we used a system for synchronized floral induction in Arabidopsis to identify 4106 flower-related long intergenic RNAs (lincRNAs). Flower-related lincRNAs are typically associated with functional enhancers which are bi-directionally transcribed and are associated with diverse functional gene modules related to floral organ development revealed by co-expression network analysis. The master regulatory transcription factors (TFs) APETALA1 (AP1) and SEPALLATA3 (SEP3) bind to lincRNA-associated enhancers. The binding of these TFs is correlated with the increase in lincRNA transcription and potentially promotes chromatin accessibility at enhancers, followed by activation of a subset of target genes. Furthermore, the evolutionary dynamics of lincRNAs in plants including non-flowering plants still remain to be elusive and the expression pattern in different plant species was quite unknown. Here, we identified thousands of lincRNAs in 26 plant species including non-flowering plants, and allow us to infer sequence conserved and synteny based homolog lincRNAs, and explore conserved characteristics of lincRNAs during plants evolution. Direct comparison of lincRNAs reveals most lincRNAs are species-specific and the expression pattern of lincRNAs suggests their high evolutionary gain and loss. Moreover, conserved lincRNAs show active regulation by transcriptional factors such as AP1 and SEP3. Conserved lincRNAs demonstrate conserved flower related functionality in both the Brassicaceae and grass family. The evolutionary landscape of lincRNAs in plants provide important insights into the conservation and functionality of lincRNAs.

MicroRNAs (miRNAs) sind endogenene kurze RNA-Moleküle, die zentrale Aufgaben bei der Regulation der eukaryotischen Zellhomöostase erfüllen. MiRNAs wurden bereits als potente Immunregulatoren beschrieben. Trotz dieser Erkenntnisse blieb die Rolle dieser kurzen RNA Moleküle in Infektionen mit Mycobacterium tuberculosis weitgehend unerforscht. Im Rahmen dieser Arbeit wurde ein miRNA-Expressionsprofil von Makrophagen generiert, die mit Mycobacterium tuberculosis infiziert waren. Dies ermöglichte die Identifizierung von miRNAs, welche bei der Infektion differenziell reguliert waren. Anhand eines ex-vivo-Modells von Todesrezeptor-induzierter Apoptose konnte gezeigt werden, dass miRNA-133b apoptoseresitente Zellen empfindlich gegen Tumornekrosefaktor-alpha (TNFalpha), TNF-related apoptosis-inducing ligand (TRAIL) oder CD95 ligand (Fas/APO1 ligand) induzierte Zytotoxizität machte. Eine umfassende Studie führte zur Identifizierung der anti-apoptotischen Proteine Fas apoptosis inhibitory molecule (FAIM) und glutathione-S-transferase pi (GSTP1) als direkte Zielgene für miRNA-133b. Desweiteren zeigte sich die Expression von Osteoprotegerin (OPG) und Fettsäuresynthase (FASN), als miRNA-133b abhängig. Dies unterstrich die pleiotrope Art der pro-apoptotischen Aktivität dieser miRNA. Die Expression von miRNA-133b wurde durch Mitglieder der Toll-like Rezeptor (TLR)-Familie aktiviert. MiRNA-133b Transfektion führte zu einer verstärkten Aktivierung des Transkriptionsfaktors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). Diese resultierte in erhöhten Mengen an Interleukinen 6 und 8 (IL6/8). Diese Ergebnisse stellen die erste detaillierte Charakterisierung von miRNA-133b im Zusammenhang der Todesrezeptor-vermittelten Apoptose und der angeborenen Immunität dar. Die erforschten molekularen Wechselwirkungen ergänzen und bereichern das Verständnis über die regulatorischen molekularen Mechanismen, die mit der Tumorentstehung und Entzündung verbunden sind. / MicroRNAs (miRNAs) are endogenous short RNA molecules which perform essential tasks in the regulation of eukaryotic cell homeostasis. During the past few years miRNAs have emerged as very potent immune regulators. Despite the consequences of this discovery for our understanding of immune response regulation hitherto virtually nothing is known about miRNA function during innate immunity to Mycobacterium tuberculosis. Herein, a miRNA expression profile of human macrophages infected with Mycobacterium tuberculosis was generated. This led to the identification of miRNAs being differentially regulated during infection. By using an experimental ex-vivo model of death receptor (DR)-induced apoptosis it could be demonstrated that miRNA-133b rendered apoptosis-resistant cells sensitive to tumor necrosis factor-alpha (TNFalpha)-, TNF-related apoptosis-inducing ligand (TRAIL)- or CD95 ligand (Fas/APO1 ligand)-activated cytotoxicity. Comprehensive analysis led to the discovery of the anti-apoptotic proteins Fas apoptosis inhibitory molecule (FAIM) and glutathione-S-transferase pi (GSTP1) as direct miRNA-133b targets. Moreover, underlining the pleiotropic and synergistic nature of miRNA activity, the expression of osteoprotegerin (OPG) and fatty acid synthase (FASN) could be further proven as miRNA-133b dependent. Expression of miRNA-133b increased following innate immune activation by members of the Toll-like receptor (TLR) family. MiRNA-133b enhanced the activity of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). This translated into increased levels of the pro-inflammatory interleukins 6 and 8 (IL6/8). The results presented in this work represent the first detailed characterization of miRNA-133b in the context of DR-mediated apoptosis and innate immunity. The molecular interactions dissected herein improve the understanding of the regulatory processes associated with tumorigenesis and the immune response.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.

Background: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). Methods: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. Results: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. Conclusions: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.

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