Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots.

The Saccharomyces cerevisiae genome contains about 35 copies of dispersed retrotransposons called Ty1 elements. Ty1 elements target regions upstream of tRNA genes and other Pol III-transcribed genes when retrotransposing to new sites. We used deep sequencing of Ty1-flanking sequence amplicons to characterize Ty1 integration. Surprisingly, some insertions were found in mitochondrial DNA sequences, presumably reflecting insertion into mitochondrial DNA segments that had migrated to the nucleus. The overwhelming majority of insertions were associated with the 5' regions of Pol III transcribed genes; alignment of Ty1 insertion sites revealed a strong sequence motif centered on but extending beyond the target site duplication. A strong sequence-independent preference for nucleosomal integration sites was observed, in distinction to the preferences of the Hermes DNA transposon engineered to jump in yeast and the Tf1 retrotransposon of Schizosaccharomyces pombe, both of which prefer nucleosome free regions. Remarkably, an exquisitely specific relationship between Ty1 integration and nucleosomal position was revealed by alignment of hotspot Ty1 insertion position regions to peak nucleosome positions, geographically implicating nucleosomal DNA segments at specific positions on the nucleosome lateral surface as targets, near the "bottom" of the nucleosome. The specificity is observed in the three tRNA 5'-proximal nucleosomes, with insertion frequency dropping off sharply 5' of the tRNA gene. The sites are disposed asymmetrically on the nucleosome relative to its dyad axis, ruling out several simple molecular models for Ty1 targeting, and instead suggesting association with a dynamic or directional process such as nucleosome remodeling associated with these regions.

DNA polymerase epsilon, acetylases and remodellers cooperate to form a specialized chromatin structure at a tRNA insulator.

Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA. In contrast, DNA polymerase epsilon, functioned with the chromatin remodeller, Rsc, and the histone acetylase, Rtt109, to generate a histone-depleted region at the tRNA insulator. Rsc and Rtt109 were required for efficient binding of TFIIIB to the tRNA insulator, and the bound transcription factor and Rtt109 in turn were required for the binding of Rsc to tRNA. Robust insulation during growth and cell division involves the formation of a hypersensitive site at the insulator during chromatin maturation together with competition between acetylases and deacetylases.

DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo.

Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions.

Analyses of SUM1-1-mediated long-range repression.

In Saccharomyces cerevisiae, local repression is promoter specific and localized to a small region on the DNA, while silencing is promoter nonspecific, encompasses large domains of chromatin, and is stably inherited for multiple generations. Sum1p is a local repressor protein that mediates repression of meiosis-specific genes in mitotic cells while the Sir proteins are long-range repressors that stably silence genes at HML, HMR, and telomeres. The SUM1-1 mutation is a dominant neomorphic mutation that enables the mutant protein to be recruited to the HMR locus and repress genes, even in the absence of the Sir proteins. In this study we show that the mutation in Sum1-1p enabled it to spread, and the native HMR barrier blocked it from spreading. Thus, like the Sir proteins, Sum1-1p was a long-range repressor, but unlike the Sir proteins, Sum1-1p-mediated repression was more promoter specific, repressing certain genes better than others. Furthermore, repression mediated by Sum1-1p was not stably maintained or inherited and we therefore propose that Sum1-1p-mediated long-range repression is related but distinct from silencing.

Integration profiling of gene function with dense maps of transposon integration.

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.

Monoclonal antibodies to gonadotropin-releasing hormone (GnRH) inhibit binding of the hormone to its receptor.

Monoclonal antibodies (MAbs) specific to gonadotropin-releasing hormone (GnRH) were obtained using different strategies of conjugation of the peptide to carrier protein and immunization. Of several antibodies obtained, two, namely F1D3C5 and E2D2 bound GnRH in solution phase. Though the epitopes corresponding to the two overlapped, there was a one amino acid shift in the core epitope. These two antibodies were characterized with respect to inhibition of GnRH induced responses in rat pituitary cultures and alpha-T3.1 mouse gonadotrope cell line.