Nuclear architecture and structural dynamics - molecular basis of chromatin remodeling induced by human ISWI machines -

One striking observation made when revealing the genetic code of humans was the relative small number of protein encoding genes that were found. The increased complexity of this biological system was not reflected by an increased number of genes. Nevertheless for higher vertebrates an expansion in genome sizes was observed [Yoo and Crabtree, 2009]. Only a small fraction of the genetic code is translated into proteins (approximately 2 %), although over 90% of the genome is transcribed. Therefore, not the number of coding sequences grew, but the complexity of the machinery helping to interpret and functionally use the information increased during evolution [International Human Genome Sequencing Consortium, 2004; Carninci et al., 2008]. A complex interaction network generates a specific spatial and temporal pattern of cellular activities. This allows formation of complex organisms based on a limited number of genes. The highly dynamic chromatin structure is a prerequisite for the flexible interpretation of the genetic information. One important class of enzymes directly changes the structure of chromatin by assembling and repositioning nucleosomes. These chromatin remodeling complexes nicely reflect the complexity of the DNA organization and interpretation machinery. Assembly of chromatin remodeling complexes from several ATP-dependent DNA translocases in combination with numerous regulatory subunits generates a large combinatorial diversity [Rippe et al., 2007; Ho and Crabtree, 2010]. Especially during development of the nervous system, activity of diverse remodeling complexes is crucial [Yoo and Crabtree, 2009]. ATPases of the ISWI class are often found in complexes playing an important role during neural development. The genome of the fruit fly Drosophila melanogaster encodes only one ISWI ATPase. In mammalians like human and mouse it is more complex, they have two different ISWI class remodeler named SNF2H and SNF2L. Although having a highly similar protein structure, both enzymes show a distinct spatial and temporal expression pattern [Lazzaro and Picketts, 2001]. Nevertheless, the detailed differences and molecular basis of this specialization remained unclear. The data presented in this thesis reveal that human ISWI remodeler are highly cooperative nucleosome binder. It is shown that cooperative binding of these chromatin modifiers is predominately seen for nucleosomal substrates. DNA is bound much less cooperative, but with a high affinity. In addition, both enzymes form higher molecular weight complexes, mainly di- and tetramers, also in absence of DNA or nucleosomal substrates. They both differ qualitatively and quantitatively in the way they bind to nucleosomes. For mono-nucleosomal substrates, SNF2H shows a more cooperative binding than SNF2L does, while affinity to nucleosomes is higher for SNF2L. These differences were also observed for dynamic binding in presence of ATP. Under competitive conditions, SNF2H preferentially binds nucleosomes with longer linker DNA, while substrate binding of SNF2L is not affected by linker length. In contrast to substrate binding, ATP-hydrolysis activity of SNF2L is dependent of nucleosomal linker DNA length and increases with longer linkers, whereas hydrolysis activity of SNF2H is already maximal stimulated by nucleosome core particles. As also published by other groups SNF2H is unable to translocate such linker less nucleosomes [He et al., 2008]. Strikingly, SNF2L pushes the histone octamere over the end of the nucleosomal DNA. In summary, activity of both paralogs is regulated with respect to the substrate structure, but on contrary levels. In order to analyze chromatin structure and dynamical changes in vivo, induced e. g. by chromatin remodeling machines, an effective method to differentially characterize accessible, active chromatin regions and inactive heterochromatin is crucial. As shown in this thesis, limiting amounts of micrococcal nuclease allow a specific characterization of accessible euchromatin regions. The isolated chromatin fragments displayed a specific nuclear distribution within distinct decondensed regions as determined by fluorescence in situ hybridization. Comparative microarray hybridization to an ENCODE chip revealed a correlation of these open chromatin fractions with active and therefore euchromatic marks, like histone H3K4 methylation, RNA polymerase II association and transcriptional activity. Furthermore, differences in methylation of mono-, di- and tri-nucleosomal DNA also revealed a specific global pattern of 5-methyl-cytosine modifications within human chromatin. DNA methylation in vivo is mainly restricted to nucleosomal linker regions. Therefore, the established method can be used for further characterisation of active chromatin domains in a relative high resolution. As a next step, the dynamical changes of chromatin structure can be analysed. Direct manipulation by knock-down or overexpression of distinct remodeling enzymes, e. g. SNF2H and SNF2L, within HeLa cells will address the question how chromatin structure and status of these initially charaterized regions changes. Furthermore the question whether DNA methyltransferases gain access to their substrate by structural dynamics, or if their activity is limited to linker DNA regions in general, can be dissected by this approach in a more detailed way.