Abstract
BackgroundRetroviral DNAs are profoundly silenced at the transcriptional level in embryonic cell types. The transcriptional profile of pluripotent stem cells has been demonstrated to be extremely heterogeneous from cell to cell, and how the silencing of retroviral DNAs is achieved is not yet well characterized.ResultsIn the current study, we investigated the transcriptional silencing dynamics in stem cells by independently monitoring the expression of two Moloney murine leukemia virus (MMLV) retroviral vectors newly introduced into embryonic carcinoma (EC) cells. Although MMLV is efficiently silenced by epigenetic mechanisms in most such cells, a small number of the doubly-transduced EC cells transiently show double-positive proviral expression. These cells were sorted and their expression patterns were studied over time as silencing is established.ConclusionsOur data suggest that retroviral silencing occurs stochastically, in an individual locus-specific fashion, and often without synchronous silencing of both viruses in the same cells. Surprisingly, the chromatin modifications that mark the silenced proviruses are unchanged even in cells that temporarily escape silencing. This local silencing effect is a feature of stem cell epigenomic regulation that has not previously been revealed.
Highlights
Retroviral DNAs are profoundly silenced at the transcriptional level in embryonic cell types
Infection by the MLVs typically results in high-level constitutive expression of the provirus, while in mouse embryonic stem cells, embryonic carcinoma cell lines, and other primitive cell types, the viral DNA is heavily silenced at the transcriptional level [9,10,11]
F9 cells infected with two reporter viruses rapidly and efficiently silence both proviruses To monitor the stability of retroviral restriction in embryonic cells, two Moloney murine leukemia virus (MMLV) viral vectors, one containing a GFP gene and one an mCherry reporter gene, were separately packaged into virus particles, and the viruses were mixed and used to co-infect F9 embryonic carcinoma (EC) cells at a multiplicity of infection (MOI) of approximately 2
Summary
Retroviral DNAs are profoundly silenced at the transcriptional level in embryonic cell types. Retroviral vectors utilizing alternative PBS sequences that are not recognized by this silencing machinery escape the rapid silencing but are still subject to some transcriptional repression [16,17] The machinery mediating this silencing is used to repress or regulate expression of many of the endogenous proviruses resident in the mouse genome, most strikingly the so-called IAP elements [18], through the DNA binding factor YY1 [19]. That not all endogenous proviruses are or even regulated in this pattern, and that some retroelements show the inverse behavior, expressing well in ES cells and not in differentiated cells; these include the HERV-H proviruses in human cells [20] and the virus-related L1td1/Ecat gene in mouse [21] For these elements other regulatory factors must be in play
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