DNA-dependent RNA polymerases from the fungus Aspergillus nidulans

The aim of the work presented here was the isolation and characterization of the DNA-dependent RNA polymerases from the fungus Aspergillus nidulans, which was a part of a project concerning the regulation of gene expression in this lower eukaryote.The transcription of a genome and the regulation mechanisms involved are basic steps in the development and differentiation of an organism. The regulation mechanisms necessary for the development of a single fertilized egg cell into an organism like men, must be very precise and complicated, if one considers that the organism consists of dozens of different cell types, each having a specific function as part of the whole. The signals, which trigger a cell to develop into a highly specialized blood or brain cell, are largely unknown at the moment. Because the developmental and differentiation process in higher eukaryotes is so complex, relatively simple differentiating organisms, like Aspergillus nidulans may be more suitable for the study of the molecular mechanisms underlying the developmental regulation of gene expression. The limited knowledge of the biochemical organization of Aspergillus, as compared to a lower eukaryote like yeast, is certainly a disadvantage, but at the same time a challenge for the investigator. On the other hand, the genetics of Aspergillus has been extensively studied and this can be of great use in biochemical and developmental studies of this organism.The transcription of coding sequences of the DNA into RNA, is one of the first processes of a complex chain of events underlying the expression of genetic information. The specific mechanisms involved in the regulation of transcription are largely unknown, but they must directly or indirectly affect the activity of the DNA-dependent RNA polymerases, responsible for the differential transcription of genetic information. Apart from the regulation at the cellular level of the enzyme, other mechanisms must be responsible for the differential transcription of specific classes of genes, that are transcribed by a common enzyme. Structural modification of the chromatin could influence the accessibility of specific genes and hence their ability to be transcribed by an RNA polymerase. Transcription may also be controlled directly by regulators, altering the interaction between the enzyme and a specific gene or genes. It is therefore important to purify eukaryotic nuclear RNA polymerases and to study their structure and function. For understanding the actual transcription mechanism, the development and study of cell-free systems, supplemented with purified RNA polymerases and well characterized templates, will be required.This thesis describes how the DNA-dependent RNA polymerases I and II from Aspergillus nidulans can be successfully purified and subsequently characterized with respect to their catalytic properties and subunit composition (Chapters 2 and 3). Preparation of protoplasts from Aspergillus (Chapter 4) was initally thought to be necessary for the isolation of the RNA polymerases, because desintegration of the rigid cell wall of Aspergillus was the first difficulty encountered. Although large amounts of protoplasts could be prepared, the procedure appeared to be rather timeconsuming and impractical as a standard, large-scale procedure for the isolation of the RNA polymerases. Protoplasts, however, can be very useful when micro-assays or a gentle treatment to break open the cell wall are required. This is demonstrated in Chapter 5, where the effect of inhibitors of RNA synthesis has been studied in vivo in metabolically active protoplasts. The isolation and characterization of RNA polymerase III could not be achieved within the limited time available for the project

RIP1-HAT1-SirT complex identification and targeting in treatment and prevention of cancer

Purpose: Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes.Experimental Design: We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and in vivo studies with different mice models.Results: Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3-SIRT1/2-HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential in vitro, in leukemic blasts ex vivo, and in vivo in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8-mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene-induced mammary gland hyperproliferation in vivoConclusions: These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme

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Partially methylated domains are hypervariable in breast cancer and fuel widespread CpG island hypermethylation

Global loss of DNA methylation and CpG island (CGI) hypermethylation are key epigenomic aberrations in cancer. Global loss manifests itself in partially methylated domains (PMDs) which extend up to megabases. However, the distribution of PMDs within and between tumor types, and their effects on key functional genomic elements including CGIs are poorly defined. We comprehensively show that loss of methylation in PMDs occurs in a large fraction of the genome and represents the prime source of DNA methylation variation. PMDs are hypervariable in methylation level, size and distribution, and display elevated mutation rates. They impose intermediate DNA methylation levels incognizant of functional genomic elements including CGIs, underpinning a CGI methylator phenotype (CIMP). Repression effects on tumor suppressor genes are negligible as they are generally excluded from PMDs. The genomic distribution of PMDs reports tissue-of-origin and may represent tissue-specific silent regions which tolerate instability at the epigenetic, transcriptomic and genetic level

Differential transcription of the orphan receptor ROR beta in nuclear extracts derived from Neuro2A and HeLa cells

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BLUEPRINT: mapping human blood cell epigenomes

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Genome-wide epigenomic profiling for biomarker discovery

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Towards cracking the epigenetic code using a combination of high-throughput epigenomics and quantitative mass spectrometry-based proteomics

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Differential binding and transcriptional behaviour of two highly related orphan receptors, ROR alpha(4) and ROR beta(1)

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Brd4-independence in ground state pluripotency.

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