A truncated form of the human CAF-1 p150 subunit impairs the maintenance of transcriptional gene silencing in mammalian cells.

Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA. In S. cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacDelta mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin. Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner. This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state. Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies. We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5' truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells. Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes. These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin.

Members of the SRY family regulate the human LINE retrotransposons.

LINEs are endogenous mobile genetic elements which have dispersed and accumulated in the genomes of most higher eukaryotes via germline transposition, with up to 100 000 copies for the human LINE-1 (L1H) sequences. Although severely repressed in most normal tissues, L1H is still functional, with evidence for both germline and somatic-essentially in tumors-transpositions. Yet, no transcription factor that could regulate their transcription and be responsible for their transposition has hitherto been described. Here we show that factors belonging to the family of the testis-determining factor gene SRY (the SOX family) can modulate L1H promoter activity over a 10-fold range in a transient transfection assay using a luciferase reporter gene. These effects depend on two functional SRY binding sites which can be identified within the L1H promoter via mobility shift assays. Induction of endogenous L1Hs upon ectopic expression of the SOX11 transcription factor is further demonstrated, thus strengthening the physiological relevance of these new-and highly dispersed-target sites for the otherwise unclassical transcription factors of the SRY family.

mRNA retroposition in human cells: processed pseudogene formation.

Using a sensitive assay for detection of reverse transcription events, we demonstrate that human HeLa cells can 'retropose', i.e. reverse transcribe and integrate, the mRNA of a naive reporter gene, at a low but detectable frequency. Furthermore, we show that the retroposed copies have all the hallmarks of the processed pseudogenes naturally found in the mammalian genome: they lack intron and 5' promoter sequence, they have acquired a 3' poly(A) tail, and they are flanked by short repeats (< 15 bp) of target DNA sequence. These results demonstrate that human cells possess an endogenous reverse transcription activity, which is not restricted to transcripts of transposable elements, and which is likely to be involved in the formation, still ongoing, of a large fraction of the eukaryotic genome.

The dimerization/packaging sequence is dispensable for both the formation of high-molecular-weight RNA complexes within retroviral particles and the synthesis of proviruses of normal structure.

Retroviral particles contain a dimer of two genomic RNA molecules, linked by noncovalent intermolecular bonds. Studies by electron microscopy of viral RNA extracted from virions as well as in vitro studies have implicated a sequence, designated the dimer linkage sequence (DLS), in the dimerization process. The DLS has been localized within a short region encompassing the psi packaging sequence, between nucleotides 212 and 563 for the Moloney murine leukemia retrovirus (MoMLV) RNA. In this report, we show that viral RNAs lacking both the DLS and psi packaging sequences--and even an RNA lacking the first 6,537 nucleotides of MoMLV--can assemble within retroviral particles as high-molecular-weight, slow-migrating, heat-sensitive complexes closely related to those observed for wild-type viral RNAs. Furthermore, we show that proviruses of normal structure are generated upon infection of test cells with retroviral particles which contain the DLS/psi-deleted viral RNAs. These observations demonstrate that the DLS and psi packaging sequences are not essential in cis to form a functional RNA complex for reverse transcription and integration.

Generation of processed pseudogenes in murine cells.

Using as a reporter gene a non-coding proviral structure marked with an intron-containing indicator, we demonstrate the de novo formation, via a retrotransposition pathway, of canonical processed pseudogenes in cultured mammalian cells. Their structural features include endings corresponding to the start and termination of the RNA intermediate, intron loss, acquisition of a 3' poly(A) tail, and target site duplications of variable length. The absence of extracellular intermediates for these processes, and the elimination during retrotransposition of sequences in the reporter gene essential in cis for a retroviral cycle, further suggest that endogenous retroviruses or related elements are not involved. Pseudogene formation frequency is markedly increased (up to 10-fold) by several treatments including treatment with 5-azacytidine or tetradecanoyl phorbol acetate, or serum starvation, which do not act at the reporter gene transcription level, but rather on endogenous genes--including the LINE elements--necessarily involved in trans-complementation for retrotransposition.

High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.

Defective retroviruses can disperse in the human genome by intracellular transposition.

Using an assay for retrotransposition detection (T. Heidmann, O. Heidmann, and J. F. Nicolas, Proc. Natl. Acad. Sci. USA 85:2219-2223, 1988), we demonstrated that a defective retrovirus deleted for the gag, pol, and env open reading frames can disperse in the genome of human HeLa cells by intracellular transposition, at a frequency close to 10(-6) events per cell per generation. Transposition requires cooperation in trans for the gag and pol gene products and may be associated with the release of low amounts of noninfectious retroviruslike particles which are the hallmarks but not the intermediates of this transposition process. Similar events could account for the dispersion at high copy number of some of the human endogenous sequences related to retroviruses and for the occurrence of noninfectious retroviruslike particles in human placenta and several tumor cell lines (reviewed by E. Larsson, N. Kato, and M. Cohen, Curr. Top. Microbiol, Immunol, 148:115-132, 1989).