Development of a novel site-specific targeting strategy for the study of the Nuclear Lamina

David Shapira

Research output: ThesisMaster's Thesis

Abstract

The Nuclear Lamina, a network of filamentous proteins lying underneath the inner nuclear membrane, defines the periphery of the nucleus as a discrete biochemical compartment. The organization of the genome is based around several such functional compartments, and the genomic regions which reside around the nuclear lamina compartment display distinct features such as low transcriptional activity and replication during late S-phase. Some regions show a stable association to the lamina while other regions have a more dynamic association to it.
Rif1 is a regulator of late replication timing that coats large chromatin domains (Rif1-associated domains or RADs), which predominantly overlap with chromatin domains found at the nuclear lamina. Upon Rif1’s deletion, the subset of RADs that is bound weakly or not at all with the lamina both repositions towards the interior of the nucleus and switches replication timing from late to early S-phase. In contrast, RADs which are simultaneously bound by Rif1 and the lamina, are refractory to Rif1 deletion and remain both peripheral and late replicating. This observation has led to the hypothesis that Rif1 may function in keeping chromatin loosely attached to the nuclear lamina, and that this localization is what gives the chromatin a late replication timing.
A lack of methods capable of targeting specific regions of the genome back to the NL have made it difficult to determine whether the detachment from the nuclear lamina is enough to result in a loss of late-replication timing. Here I develop a two-step method that first inserts short, targeted recombination sites into the genome at specific locations, enabling the later targeting and integration of larger constructs such as an array of Tet-response elements, into otherwise hard to manipulate regions.
Using such an approach will enable the site-specific targeting of chromatin to the nuclear lamina for the first time and will help further unravel the molecular mechanism of Rif1 as well as answer whether the nuclear periphery is able to impart a late replication timing upon chromatin targeted there.
Original languageEnglish
Awarding Institution
  • University of Edinburgh
Supervisors/Advisors
  • Buonomo, Sara, Supervisor
Publication statusPublished - 2018

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