AbstractsBiology & Animal Science

Exploring the role of structured DNA elements and their specific binders in genome regulation

by Michael Filarsky

Institution: Universität Regensburg
Department: Biologie und Vorklinische Medizin
Degree: PhD
Year: 2013
Record ID: 1099441
Full text PDF: http://epub.uni-regensburg.de/28670


After the description of the double helix structure by Watson and Crick, the function of DNA in the cell was seen as rather static, a long stretched out database of information, which was read and interpreted by the protein machinery doing all the interesting work. That notion however, quickly changed with the discovery of the DNA’s potential to adopt various alternative conformations, like Z-DNA, quadruplex DNA or triplexes. Although initially characterized in vitro, these structures were soon implicated in the development of certain diseases and suggested to have possible roles in gene regulation and genome organization. The major focus thereby lay on the triplexes, three stranded structures comprising the DNA duplex and a single stranded RNA or DNA that binds in the major groove of the double helix. Strikingly, the recent advancements in high-throughput sequencing technology revealed the enrichment of putative triplex forming sequences within the regulatory regions of the genome. These observations combined with the discovery of an ever-growing number of functional ncRNAs, led to the assumption that triplex structures could present an interface between these ncRNAs and the chromatin. The data in this thesis reveal that the intergenic spacer region of the mammalian rRNA genes contains a cluster of possible triplex forming sequences in its regulatory enhancer and terminator elements. The triplex forming potential of these motifs was predicted by in-silico analysis of the rDNA sequence and validated using EMSA experiments. Microscale thermophoresis (MST) assays were established to investigate the formation of triplex structures in various conditions in vitro. These experiments presented new insights on the characteristics of the triplex formation process and suggest the complex formation to be readily possible in physiological conditions. Thus the results provide the basis for further investigations on a possible role for enhancer-derived triplex forming ncRNAs in the regulation of the rRNA genes. Besides their presence in regulatory gene regions, sequences prone to form non-canonical DNA structures were also shown to be part of matrix attachments regions (MARs) throughout the genome. These MAR sites are tethered to the nuclear matrix and mediate the anchoring of chromatin loops on this putative protein scaffold within the nucleus. The MAR elements are implicated to play various roles in transcriptional regulation and chromatin organization. Interestingly, MARs have also been shown to interact with specific MAR binding proteins and were implicated to form triplex structures with ncRNAs at the nuclear matrix. However, the exact nature of these processes and their implications for gene regulation and chromatin organization are so far poorly understood. In the second part of this thesis Tip5, a subunit of NoRC, the key player in epigenetic rRNA gene repression, is revealed as nuclear matrix binding protein. The AT-hook and TAM domains of the protein are identified as motifs mediating the localization of Tip5 to the…