Epigenetic mechanisms of retrovirus vector silencing
Subtitle: DNA methylation, silent chromatin and silencer blocking by insulators.
Published: 2004
Language: eng
Number of pages: 207
Description: Gene silencing of retrovirus vector expression in primitive cells has hampered the use of retrovirus vectors in gene therapy. This thesis seeks to understand retrovirus and Lentivirus vector silencing mechanisms, and the blocking of retrovirus silencers by insulators to design silencing resistant vectors for stem cell gene therapy. By studying single copy vector transduced ES cell clones, it was shown that retrovirus and Lentivirus vectors are silent, variegated expressing or low expressing. Variegated retrovirus and Lentivirus vectors are unstable in embryonic stem (ES) cells and extinguished during in vitro differentiation. Therefore, retrovirus and Lentivirus vector silencing is a heterogeneous phenomenon including silencing, variegation and extinction. Silent retrovirus and Lentivirus vectors are marked by silent chromatin characterized by histone H3 deacetylation and linker histone H1 binding. Variegated silent retrovirus vectors are marked by silent chromatin similar to silent vectors. Extinction of variegated expressing retrovirus vectors associates with increased MeCP2 binding. Both DNA methylation dependent and independent epigenetic silencing pathways induce silent chromatin on silent and variegated silent retrovirus vectors. DNA methylation plays a functional role but is dispensable in silencing, variegation and extinction. The establishment of a transient transgenic mouse silencer-blocking assay allowed the identification of retrovirus silencer-blocking activity in the cHS4 insulator that is distinguished from enhancer-blocking activity by independence of CTCF binding. It was also found that the gypsy insulator stimulates reporter gene expression when located in retrovirus vector LTRs but does not block retrovirus silencing. I propose that the decision for retrovirus silencing, variegation and extinction is made based on the balance of the integration site and viral silencers, and that silencing is controlled by dynamic interplay among multiple epigenetic pathways. My findings provide important new insight into retrovirus and Lentivirus stem cell silencing and the silencer blocking results provide new guidance for the use of insulators in retrovirus and Lentivirus vectors.
Notes: Adviser:  James Ellis.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 5004.