69 The Relationship Between Retroviral Integration and Host Cell Transcription 2 L. Maxfield*, C. Dow, J. Coffin Tufts Univ, Boston, MA
Background: Retroviral DNA integration occurs throughout the genome; however, local "hot spots" for integration exist where a strong preference for certain sites over others are seen. Our goal is to define properties of host cell DNA that influence sites of retroviral integration.
Method: Traditionally, integration was thought to prefer transcriptionally active over inactive DNA. However, previous data from our lab suggested that there are fewer integration events into a DNA template undergoing active transcription than when it is not. Since these data were generated using a stably transfected foreign gene that was only weakly inducible, we have extended this observation by comparing integration events into a highly inducible endogenous gene under both induced and uninduced transcriptional states. For this purpose, we have chosen the avian metallothionein (MT) gene because it has a low level of constitutive expression, yet can be induced to a very high level of expression by addition of 100 mM ZnSO4. To determine transcription levels, we quantitated unspliced RNA as a function of time after zinc induction, using real-time RT-PCR. MT RNA products were induced to a level more than 100-fold that seen in samples not induced by zinc. Transcription remained consistently high throughout the time that viral integration takes place.
Results: By mapping integration sites downstream of the MT gene transcription start site, and comparing the frequency of integration under both induced and uninduced conditions, we have confirmed that there are fewer integration events into a gene undergoing active transcription. We have mapped 38 retroviral integration events into uninduced MT DNA templates; while only 6 integration events have been mapped into MT gene templates that have been induced by zinc. Therefore, 100-fold activation of a DNA template reduces the number of integration events into that gene region by about 6-fold. These events have been confirmed by direct DNA sequencing to be unique integration events into the MT gene.
Conclusions: Several possible models for our observation are as follows. When a DNA template is undergoing active transcription, integration might be blocked by the RNA polymerase II complex due to steric hindrance, the integrase complex may require DNA to be in a double-stranded conformation, which would not be the case during transcription. Or finally, transcription might lead to remodeling of chromatin into a structure less favorable for integration.
