Molecular mechanisms of silencing virally transduced genes.

摘要

Transcriptional silencing has been an interesting topic of cellular and molecular biology for many years, and the mechanism for this phenomenon has been a mystery. Now, silencing of virally transduced genes has become a major block for gene therapy. Understanding the molecular mechanism of silencing is the first step towards developing appropriate strategies to overcome this problem.; The data presented in this dissertation demonstrate that histone deacetylase inhibitors sodium butyrate and trichostatin A (TSA) strongly reactivate silenced, virally transduced gene (adeno-associated viral vector $rAAV/CMVlacZ$ expression. The reactivation by butyrate and TSA is independent of transgene chromosomal integration sites in stably transduced HeLa cells, and is not promoter or cell-type specific because they also reactivated a silenced globin vector $rAAV/HS2abAS3$ in hematopoietic K562 cell clones. The specificity of TSA to histone deacetylase suggests that histone deacetylation is integrally involved in silencing virally transduced genes.; Through DNaseI analysis and nuclear run-on experiments, it is now demonstrated that viral transgene chromatin domain opening precedes transcriptional activation upon TSA treatment. Chromatin immunoprecipitation-PCR analysis reveals that acetylation of histone H4 N-terminal lysine residues K5, K12, and K16 initiates chromatin domain opening, while acetylation of lysine K8 maintains open chromatin structure and high level transcription. Removing TSA results in rapid histone lysine deacetylation, chromatin domain closing, and transcriptional silencing. A model is proposed that histone deacetylase is recruited to the transgene locus by a host protein or protein complex recognizing viral terminal repeat sequences. Local deacetylation causes chromatin condensation and restricts the accessibility of transcriptional factors to the promoter. Treatment with TSA inhibits histone deacetylase, induces local histone hyperacetylation and chromatin decondensation, and activates transcription. Removing TSA results in rapid conversion of viral transgene chromatin to a condensed state, and rapid transcriptional silencing occurs due to stably recruited histone deacetylases.; This study reveals for the first time the molecular mechanisms involved in silencing virally transduced genes and essential roles for specific histone lysine residues in transcriptional regulation of chromosomal genes in mammalian cells. This study provides new insights into chromatin regulatory functions and suggests new directions for viral vector development. This work also has implications for gene therapy. Efficient viral gene transfer followed by drug treatment to relieve suppression may provide a powerful combination for treatment of genetic and infectious diseases.