Valeria Buggiano

The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene.

Two novel exogenous mouse mammary tumor viruses (MMTV), BALB2 and BALB14, that encode superantigens (Sags) with Vbeta2+ and Vbeta14+ specificities, respectively, were found in the BALB/cT mouse strain. BALB/cT females were crossed with AKR/J males to generate F1 females. Foster nursing of BALB/cT mice on (BALB/cT x AKR/J)F1 mothers resulted in the generation of a new mouse strain, BALB/cLA, that had acquired a new exogenous MMTV (hereafter called LA) with a Vbeta6+/Vbeta8.1+-T-cell-specific Sag. Sequence analysis of the long terminal repeats of the BALB2, BALB14, and LA viruses indicated that LA virus resulted from recombination between BALB14 and the endogenous Mtv-7 provirus. Mtv-7 is expressed only in lymphoid tissues but not the mammary glands of Mtv-7-containing mouse strains such as AKR. In contrast, LA virus was highly expressed in the mammary gland, although it had the sag-specific region from Mtv-7. The LA virus, as well as different recombinant viruses expressed in the mammary glands of (BALB/cT x AKR/J)F1 mice, acquired a specific DNA sequence from BALB14 virus that is required for the mammary-gland-specific expression of MMTV. Since the Sag encoded by LA virus strongly stimulated cognate T cells in vivo, selection for recombinant virus with the Mtv-7 sag most likely occurred because the increased T-cell proliferation resulted in greater lymphoid and mammary gland cell infection. As a result of the higher virus titer, 80% of BALB/cLA females developed mammary gland tumors, although the incidence was only 40% in BALB/cT mice.

It has previously been shown that transgenic female mice expressing TGFbeta1 under control of regulatory elements of the whey-acidic protein (WAP) gene were unable to lactate. This was due to the increased apoptosis of the cells committed to the lobular-lactogenic phenotype. Our goal was to determine whether the expression of WAP-TGFbeta1 transgene could inhibit MMTV (mouse mammary tumor virus) tumorigenic activity in the mammary gland. It is well known that the infection with this virus produces focal hyperplastic secretory nodules (HANs) and, some variants can also induce ductal pregnancy-dependent lesions (plaques). In either case, MMTV infection leads ultimately to the appearance of malignant mammary tumors. The results shown herein demonstrate that TGFbeta1 expression in the secretory mammary epithelium does not suppress mammary tumorigenesis in MMTV infected mice. Although MMTV infected WAP-TGFbeta1 transgenic females displayed a strong impairment of lobule-alveolar development, carcinogenesis induced by any of the four MMTV variants used herein proceeded unabated. WAP-TGFbeta1 tumors that showed a strong expression at the WAP promoter, appeared later and grew more slowly than their wild-type counterparts. Transgenic females also had a lower incidence of HANs and plaques. Our study suggests that the epithelial target cells for tumorigenic mutations are probably progenitor cells that are not susceptible to the apoptotic effect of TGFbeta1. Alternatively, their daughters cells that display the secretory phenotype and could be more involved in the formation of premalignant lesions continue to die due to the expression of the transgene.