DNA encodes the genetic information required for function and is packed into the nucleus by wrapping DNA around a protein complex comprised of histone proteins. Packing of the DNA into these complexes termed nucleosomes posses problems because DNA that requires expression must be easily accessible, while regions whose expression will be deleterious must remain transcriptionally inactive. To address this problem the DNA is further organized into large functional domains of heterochromatin and euchromatin, where the
former is transcriptionally repressed and the latter is transcriptionally active. To achieve this organization the nucleosomes within these domains are differentially post-transcriptionally modified to define chromatin domains.
The work of this thesis focuses on two distinct topics in heterochromatin function: how heterochromatin-induced transcriptional silencing is mediated in a sequence independent manner and how transcriptional silencing is maintained within the boundaries of the heterochromatin domain.
Chapter two presents work that examines the effect of the heterochromatin machinery on the chromatin structure of domains designated as heterochromatin. We find, through the creation of nucleosome occupancy maps of silencing-defective mutants, that nucleosome-free regions (NFRs), which associate with transcriptional start sites and regulatory regions in heterochromatin, had been eliminated upon the formation of heterochromatin. Additionally, the required heterochromatin machinery needed to eliminate NFRs within heterochromatin differs based on the type of sequence that is encountered. Through this work we propose that sequence independent transcriptional silencing is a result of the silencing factors acting in a sequence specific manner to eliminate NFRs.
Chapter 3 explores how the cell maintains and defines the transition between euchromatin and heterochromatin to prevent the encroachment of transcriptional silencing or activation into the neighboring chromatin domain. DNA sequence elements called boundary elements have been defined in many metazoans to be required for this activity. To understand how boundary elements function, we describe the development and potential uses of a genetic reporter gene system that displays boundary activity in Schizosaccharomyces pombe. Using this reporter, we show that two parallel mechanisms prevent heterochromatin spread from escaping its chromatin domain. Additionally, we report a novel function for a silencing factor, Swi6, in preventing heterochromatin spread at boundary elements.
