Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

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Following fertilization, the embryo undergoes a sequence of precisely timed cleavage cycles to produce a blastocyst. The timing of these cycles likely depends in part on appropriate levels of gene activity. I have investigated whether high mobility group (HMG) proteins 14 and 17, which are associated with chromatin containing transcribed genes, are expressed in mouse embryos and are required to maintain normal early developmental timing. As assayed using RT-PCR, mRNAs encoding both HMG-14 and HMG-17 were present throughout preimplantation development to the blastocyst stage. By immunofluorescence, both proteins were detected in the nuclei of prophase I-arrested oocytes and embryos beginning at the 2-cell stage. To investigate their function, antisense oligonucleotides targeting the 5' end of each mRNA species were injected into 1-cell stage embryos which were then cultured to develop to the blastocyst stage. At the 2- and 4-cell stages, only weak nuclear immunofluorescence was observed; however, by the 8-cell stage, the staining pattern of injected embryos was indistinguishable from controls. Thus, the injected antisense oligonucleotides transiently depleted the cellular supply of HMG-14 and HMG-17. Furthermore, the embryos in which both HMG-14 and MAG-17 had been depleted progressed significantly more slowly through successive stages of preimplantation development, as compared with embryos in which the proteins were individually depleted or injected with nonsense oligonucleotides. Therefore, it can be concluded that depletion of HMG-14 and HMG-17 from embryonic chromatin transiently delays preimplantation development, demonstrating a crucial role for these proteins in maintaining the normal temporal coordination of development.

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Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.