Browsing by Subject "Heterochromatin"

Heterochromatin, or condensed chromatin, is a transcriptionally repressive form of chromatin that occurs in many eukaryotic organisms. At its natural locations, heterochromatin is thought to play important roles in genome organization as well as gene expression. Just as important is the restriction of this repressive form of chromatin to appropriate regions of the genome. In the budding yeast Saccaromyces cerevisiae, domains of condensed, transcriptionally silenced chromatin are found at telomeres and at the silent-mating type cassettes, HML and HMR. At these locations, a complex of Silent Information Regulator (SIR) proteins gets recruited to DNA through discrete silencer elements. Once recruited, the Sir protein complex then spreads along chromosomes in a step-wise manner. This process results in the silencing of gene expression. It is unclear whether silenced chromatin is established in the same manner at different genomic locations. Understanding how silenced chromatin is formed is important for determining how these chromatin structures are regulated.

To better understand how silenced chromatin is established in different genomic contexts, I used chromatin immuoprecipitation to follow the rate of silenced chromatin formation at different locations. The rates of Sir protein assembly were compared at two locations, telomere VI-R and HMR. I discovered that the silencers at these two locations were equally proficient at recruiting Sir proteins. However, the rate of Sir protein assembly onto nucleosomes was far more rapid at HMR than at the telomere VI-R. Furthermore, the rate of Sir protein assembly was more rapid on one side of the HMR-E silencer at HMR than the other. Moreover, insertion of the HMR-E silencer adjacent to the telomere VI-R significantly improved the rate of Sir protein assembly onto nucleosomes. Additionally, observations that the association of Sir protein occurs simultaneously across several kilobases at HMR and that silencing at HMR is insensitive to co-expression of wild-type and catalytically inactive Sir2 proteins suggest that HMR-E enables the assembly of silenced chromatin in a non-linear fashion. These results suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases.

In addition to the HMR-E silencer, HMR is also characterized by the presence of a second auxiliary HMR-I silencer and a tRNA gene that functions as a boundary element to restrict the spread of silenced chromatin. I used chromatin immunoprecipitation to determine how each of these regulatory elements contribute to the steady-state levels of Sir protein association with chromatin. Consistent with a role for HMR-E beyond recruitment, I discovered that the HMR-E silencer alone promoted higher levels of Sir proteins on nucleosomes compared to the telomere VI-R. The levels of Sir protein association with HMR were further elevated by the HMR-I silencer, even though this silencer does not recruit Sir proteins on its own and does not contribute to any of the known functions of silenced chromatin at HMR. Additionally, although the tRNA gene did block the spread Sir proteins, I discovered that the capacity for Sir proteins to spread beyond a few kilobases was severely limited even in the absence of the boundary.

The results of this thesis work provide new insights into the mechanisms of silenced chromatin establishment and regulation in budding yeast. I show here that the capacity of Sir proteins to assemble onto nucleosomes is inherently limited. Additionally, silencers vary in their ability to promote this assembly. I conclude that the silencer is a key factor in determining the relative size, efficiency, and location of silenced chromatin domains in the cell.

To ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability.

DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in Drosophila Menalogaster , is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4Ddb1 mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase.

In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.

In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.