Evidence for a Stem Cell-Specific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells

Loh, Teck Peng; Sievert, L L; Scott, Richard W.

doi:10.1128/mcb.10.8.4045-4057.1990

Molecular and Cellular Biology

1990

DOI: 10.1128/mcb.10.8.4045-4057.1990

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Evidence for a Stem Cell-Specific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells

Teck Peng Loh

L L Sievert

Richard W. Scott

Abstract: A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear … Show more

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Cited by 11 publications

(2 citation statements)

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“…It is also abrogated by a mutation which creates an Spl binding site in the enhancer region (9). The 5' noncoding region of the viral genome functions as a negative element in EC cells (14,32). The host range mutants of Mo-MuLV which can replicate in EC cells carry mutations in this region (38).…”

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confidence: 99%

Embryonal Long Terminal Repeat-Binding Protein Is a Murine Homolog of FTZ-F1, a Member of the Steroid Receptor Superfamily

Tsukiyama

Ueda

Hirose

et al. 1992

Molecular and Cellular Biology

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The embryonal long terminal repeat-binding protein, ELP, is present in undifferentiated mouse embryonal carcinoma cells. It binds to and suppresses transcription of the Moloney leukemia virus long terminal repeat in undifferentiated murine embryonal carcinoma cells. We report here that ELP is a mouse homolog of Drosophila FTZ-F1, which positively regulates transcription of the fushi tarazu gene in blastoderm-stage embryos of the fly. As members of the steroid receptor superfamily, ELP and FTZ-F1 have both DNA binding and putative ligand binding domains which are well conserved between the two. ELP and FTZ-F1 function in cells in the extremely early stage of development. A high degree of conservation between the two transcription factors during the evolution of these species indicates the importance of their functions in early-stage embryogenesis. In addition, the sequence elements they recognize do not contain repeat units, in contrast to other steroid receptors, which usually bind to either palindromic or direct repeat sequences.

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confidence: 99%

Embryonal Long Terminal Repeat-Binding Protein Is a Murine Homolog of FTZ-F1, a Member of the Steroid Receptor Superfamily

Tsukiyama

Ueda

Hirose

et al. 1992

Molecular and Cellular Biology

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“…There are several logistical difficulties in performing such analyses on embryonic material, and we and others have therefore sought to exploit cultured cell systems which recapitulate differentiation events occurring during embryonic development (30,(37)(38)(39)48). The F9 line of embryonal carcinoma (EC) cells has a number of advantages for work of this type in that homogeneous populations of both stem cells and their differentiated derivatives can be obtained in quantities suitable for biochemical studies of transcription factor activity (20-22, 36, 47), and exogenous genes can be readily introduced into both cell types by conventional transfection procedures (10,(24)(25)(26)(27)38). F9 EC cells have a number of properties in common with the primitive endoderm cells of the blastocyst, and like the primitive endoderm, they will differentiate into both parietal and visceral endoderm, the differentiation pathway that is followed being dictated by the procedure used.…”

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Sequences and Factors Required for the F9 Embryonal Carcinoma Stem Cell E1a-Like Activity

Murray

Stott

Rigby

1991

Molecular and Cellular Biology

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F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

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TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells

Wolf

Hug²,

Goff³

2008

Proc. Natl. Acad. Sci. U.S.A.

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Murine leukemia viruses (MLVs) and related retroelements are potently restricted in embryonic cells by postintegration transcriptional silencing, likely to protect the germ line from insertional mutagenesis. This silencing is in large part attributable to the presence of a nuclear repression complex, which targets a sequence element of the proviral DNA, the repressor-binding site. The repressor-binding site closely overlaps the tRNA primer binding site, a highly conserved sequence essential for virus replication and defining the site of initiation of DNA synthesis during reverse transcription. We have recently demonstrated that the cellular corepressor TRIM28 is recruited to the proline tRNA primer-binding site used by many MLVs and is required to mediate this silencing. Here, we show that TRIM28 is also required for the restriction of retroviruses using a completely distinct tRNA for the priming of their DNA synthesis, namely Lys-1,2 tRNA. These results generalize the role of TRIM28 in retroviral restriction and suggest that this system has evolved to restrict multiple retroviruses.urine leukemia viruses (MLVs) are unable to replicate in embryonic cells (1-3). The block to replication occurs after the viral DNA has integrated into the host genome and is caused by transcriptional silencing of the proviruses (2, 4). This silencing is the result of several cumulative effects that include poor enhancer function of the 5Ј LTR (5-7), the binding of several cell-type specific transacting transcriptional repressors (6, 8), and de novo DNA methylation (4). A critical target of this repression is the DNA element known as the repressor binding site (RBS) (2, 5, 9, 10). The RBS element comprises 17 bp that overlap closely with the 18-bp primer binding site (PBS) of MLV (2), a sequence complementary to the cellular proline tRNA (tRNA Pro ) used to prime minus-strand DNA synthesis during reverse transcription (11). This silencing can be abrogated by a single base-pair substitution in the PBS known as the B2 mutation (2). Characterization of RBS-mediated restriction demonstrated that the 17-bp sequence was able to induce transcriptional silencing of reporter constructs in EC cells independently of position and orientation of the RBS sequence (10, 12). Furthermore, exonuclease III protection assay and EMSA experiments revealed the presence of a DNA-binding activity that is specifically enriched in stem cell nuclear extracts (10, 12). Based on these experiments, a trans-acting DNA-binding factor was postulated to exist; we have recently demonstrated that a high molecular weight complex binds the RBS sequence, and that a key component of this complex is the TRIM28 protein (13). TRIM28 (also known as Kap-1, or Tif1-beta) is a well characterized transcriptional corepressor that is recruited to its target genes by interactions with the Krüppel associated box (KRAB) zinc-finger DNA-binding proteins (14). This interaction is mediated via the KRAB box domain and leads to these DNA-binding proteins, serving as sequence-specific transcr...

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Evidence for a Stem Cell-Specific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells

Cited by 11 publications

References 51 publications

Embryonal Long Terminal Repeat-Binding Protein Is a Murine Homolog of FTZ-F1, a Member of the Steroid Receptor Superfamily

Embryonal Long Terminal Repeat-Binding Protein Is a Murine Homolog of FTZ-F1, a Member of the Steroid Receptor Superfamily

Sequences and Factors Required for the F9 Embryonal Carcinoma Stem Cell E1a-Like Activity

TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells

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