Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor

Abstract

Replication of the murine leukemia viruses is strongly suppressed in mouse embryonic stem (ES) cells. Proviral DNAs are formed normally but are then silenced by a large complex bound to DNA by the ES cell-specific zinc-finger protein ZFP809. We show here that ZFP809 expression is not regulated by transcription but rather by protein turnover: ZFP809 protein is stable in embryonic cells but highly unstable in differentiated cells. The protein is heavily modified by the accumulation of polyubiquitin chains in differentiated cells and stabilized by the proteasome inhibitor MG132. A short sequence of amino acids at the C terminus of ZFP809, including a single lysine residue (K391), is required for the rapid turnover of the protein. The silencing cofactor TRIM28 was found to promote the degradation of ZFP809 in differentiated cells. These findings suggest that the stem cell state is established not only by an unusual transcriptional profile but also by unusual regulation of protein levels through the proteasomal degradation pathway. Infection of these cells by MLVs results in normal virus entry, reverse transcription of the viral RNA, and integration of viral DNA, but the proviral DNA is then transcriptionally silenced. A specific DNA sequence used to prime viral DNA synthesis, the primer binding site complementary to the 3′ portion of proline tRNA (PBSpro), is present on those viruses that are most strongly and rapidly silenced (7-10). A large protein complex, specifically expressed in ES cells, binds to the PBSpro sequence to mediate the transcriptional repression of proviral DNA (5,9,11,12). This silencing complex contains the transcriptional repressor TRIM28, also known as KAP1 or TIF1β (6), and is tethered to the PBSpro DNA by ZFP809, a zinc-finger protein with sequence-specific DNA-binding activity (13). TRIM28 acts as a scaffold for a number of factors that mediate the silencing of the nearby DNA, including the NuRD histone deacetylase complex, histone H3K9 methyltransferase ESET, and heterochromatin protein 1 (14-17). TRIM28, a RING-domain protein, also plays an important role in the protein modification pathway. Like other members of the RING domain family, TRIM28 is an E3 ligase that mediates transfer of ubiquitin or the small ubiquitin-like modifier (SUMO) to target substrates (18,19). ZFP809 contains two domains, a KRAB box at the N terminus that is responsible for the interaction with TRIM28 and a zincfinger domain containing seven zinc fingers that provide its sequence-specific DNA-binding activity (Fig. 1A). Whereas most of the components of the silencing complex are expressed ubiquitously, ZFP809 is expressed specifically in embryonic cells and not in differentiated cells (13,20). ZFP809 is the key ES cellspecific component of the silencing complex. Ectopic expression of suitable forms of ZFP809 in differentiated cells is sufficient to induce the formation of the DNA-binding silencing complex and to establish the silencing in those cells (13), suggesting that the lack of restriction of MLVs in differen...

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KRAB-Zinc Finger Protein ZNF268a Deficiency Attenuates the Virus-Induced Pro-Inflammatory Response by Preventing IKK Complex Assembly

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