Tissue-specific and reversible RNA interference in transgenic mice

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.     

A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells.

Chromatin packaging in mammalian spermatozoa requires an ordered replacement of the somatic histones by two classes of spermatid-specific basic proteins, the transition proteins and the protamines. Temporal expression of transition proteins and protamines during spermatid differentiation is under translational control, and premature translation of protamine 1 leads to precocious nuclear condensation and sterility. We have previously suggested that the double-stranded (ds) RNA binding protein Prbp (encoded by the gene Tarbp2) functions as a translational regulator during mouse spermatogenesis. Here we show that Prbp is required for proper translational activation of the mRNAs encoding the protamines. We generated mice that carry a targeted disruption of Tarbp2 and determined that they were sterile and severely oligospermic. Using immunohistological analysis, we determined that the endogenous Prm2 mRNA and a reporter mRNA carrying protamine 1 translational-control elements were translated in a mosaic pattern. We showed that failure to synthesize the protamines resulted in delayed replacement of the transition proteins and subsequent failure of spermiation. The timing of Prbp expression suggests that it may function as a chaperone in the assembly of specific translationally regulated ribonucleoprotein particles.     

Chromatin profiling using targeted DNA adenine methyltransferase

Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins. Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci. Knowledge of the identity of these in vivo target loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins. We report here large-scale mapping of in vivo binding sites of chromatin proteins, using a novel approach based on a combination of targeted DNA methylation and microarray technology. We show that three distinct chromatin proteins in Drosophila melanogaster cells each associate with specific sets of genes. HP1 binds predominantly to pericentric genes and transposable elements. GAGA factor associates with euchromatic genes that are enriched in (GA)n motifs. A Drosophila homolog of Saccharomyces cerevisiae Sir2p is associated with several active genes and is excluded from heterochromatin. High-resolution, genome-wide maps of target loci of chromatin proteins ('chromatin profiles') provide new insights into chromatin structure and gene regulation.     

The study of macromolecular complexes by quantitative proteomics

We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure. In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.