Nuclear receptors (NRs) are ligand‐inducible transcription factors that mediate complex effects on development, differentiation and homeostasis. They regulate the transcription of their target genes through binding to cognate DNA sequences as homodimers or heterodimers. The molecular mechanisms underlying transcriptional activation by NRs are still poorly understood, although intermediary factors (mediators) appear to be involved in mediating the transactivation functions of NRs. TIF1 has been identified previously as a protein that interacts specifically with the ligand binding domain of several nuclear receptors, both in yeast and in vitro. The characteristics of these interactions have led us to suggest that TIF1 might be a mediator of the NR ligand‐inducible activation function AF‐2. Using a two‐hybrid screening in yeast, we have now identified two TIF1‐binding proteins, mHP1 alpha and mMOD1, that are mouse homologues of the Drosophila heterochromatinic protein 1. Using mHP1 alpha as a bait in a second two‐hybrid screening, we have isolated cDNAs encoding proteins that are also very likely to be involved in chromatin structure and function, as well as a protein structurally and functionally related to TIF1 (renamed TIF1 alpha), which was named TIF1 beta. Here we discuss how the function of members of the TIF1 family in the control of transcription could be exerted at the level of the structure of the chromatin template.

IFN-functionally resembles type I IFN, inducing antiviral protection in vitro (10,23,27) as well as in vivo (1). Activation of the IFN-receptor leads to the phosphorylation of STAT1, STAT2, and STAT3 and the formation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor (10) and to the induction of typical IFN-induced genes like the OAS and MxA genes. IFN-can reduce cell growth in vitro and possesses antitumor activity in several rodent models (11,25). However, a number of cytokines with very different biological effects activate STAT transcription factors, and pronounced functional differences between type I and type III IFNs exist. The in vivo antiviral activity of IFN-against herpes simplex virus 2 (HSV-2) has been shown to be comparable to that of IFN-␣ in a systemic model. However, a model for the clinically relevant vaginal HSV-2 infection revealed an antiviral activity of IFN-that surpassed that of IFN-␣ (1).The biological effect of the cytokine-receptor system is determined primarily by three factors: the expression profile of the cytokine itself, the expression profile of the receptor, and the set of target genes for regulation. We decided to start our investigation of the function of the IFN-system by asking which genes are regulated by IFN-. A gene array experiment covering the whole human genome revealed that all IFN--induced genes were also induced by type I IFN. Thus, no genes

contributed equally to this work Mammalian TIF1α and TIF1β (KAP-1/KRIP-1) are related transcriptional intermediary factors that possess intrinsic silencing activity. TIF1α is believed to be a euchromatic target for liganded nuclear receptors, while TIF1β may serve as a co-repressor for the large family of KRAB domain-containing zinc finger proteins. Here, we report an association of TIF1β with both heterochromatin and euchromatin in interphase nuclei. Co-immunoprecipitation of nuclear extracts shows that endogenous TIF1β, but not TIF1α, is associated with members of the heterochromatin protein 1 (HP1) family. However, in vitro, both TIF1α and TIF1β interact with and phosphorylate the HP1 proteins. This interaction involves a conserved amino acid motif, which is critical for the silencing activity of TIF1β but not TIF1α. We further show that trichostatin A, an inhibitor of histone deacetylases, can interfere with both TIF1 and HP1 silencing. The silencing activity of TIF1α appears to result chiefly from histone deacetylation, whereas that of TIF1β may be mediated via both HP1 binding and histone deacetylation.

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Kr€ uppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or beta-globin mRNAs, harboring wild-type or various 5' splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter docking of transcription initiation factors TFIID, TFIIB, and TFIIH on a gene containing a functional 5' splice site. In addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. Our data suggest a model in which a promoter-proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly.

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.

In an effort to develop a porcine model of Alzheimer's disease we used handmade cloning to produce seven transgenic Göttingen minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences to induce splicing and a human PDGF beta promoter fragment to drive transcription. Transgene insertion had occurred only at the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length transcript was verified. Consistent with PDGF beta promoter function, high levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that accumulation of the A beta peptide in the brain may develop at the age of 1-2 years.

We describe a new human isoform, GFAP⑀, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP⑀ mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP⑀ has a new C-terminal protein sequence. This provides GFAP⑀ with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP⑀ is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP⑀ should be taken into consideration when studying neurodegenerative diseases.