UCSF

In eukaryotic cells, genomic DNA is packaged with histones to form chromatin. While euchromatin is found in gene-rich regions of the genome, heterochromatin is associated with centromeres and telomeres that are gene-poor, transcriptionally less active and contain high densities of repetitive non-coding DNA sequences. The structural component of heterochromatin consists of unique proteins that bind to distinct histone tail modifications. This structure serves important cellular functions that include regulating gene expression and maintaining chromosomal integrity. Despite extensive research, there remain many unanswered questions about how heterochromatic structures are assembled.

The overall goal of this study is to gain a better understanding of how heterochromatin assembles in fission yeast. More specifically, we are interested in the basic question: what determines where heterochromatin forms? We hypothesized that non-coding DNA sequences embedded in heterochromatin play crucial roles in specifying where heterochromatin forms. To test this hypothesis, we dissected these non-coding sequences and we investigated the roles of some protein factors bound to them. The findings of this study are divided into 3 chapters.

Chapter II of this dissertation describes a systematic study to identify DNA sequences that are sufficient to nucleate heterochromatin. In this study, we developed an ectopic heterochromatic reporter construct to dissect heterochromatic sequences and to test their capability in inducing heterochromatin formation at a given locus. Using this construct, we found that several large DNA fragments from pericentromeric regions are able to induce ectopic heterochromatin formation. Furthermore, we found a unique hexameric DNA motif that is necessary but not sufficient for heterochromatin formation.

In chapter III, we describe a novel protein player in heterochromatin assembly. This protein, Seb1, binds specifically to non-coding RNAs that are transcribed from heterochromatic regions. Using genetic and biochemical tools, we demonstrate that Seb1 directs heterochromatin formation by recruiting several chromatin-modifying enzymes to heterochromatic regions.

Finally, in chapter IV we describe the role of heterochromatic transcript polyadenylation in heterochromatin assembly. By investigating the functions of a poly(A)-binding protein and a putative deadenylase, we found that polyadenylation of heterochromatic transcripts inhibits heterochromatin formation. Together, the results of this study shed new light on mechanisms of heterochromatin assembly.