We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

The identification of regulatory regions is one of the most important and challenging problems toward the functional annotation of the human genome. In higher eukaryotes, transcription-factor (TF) binding sites are often organized in clusters called cis-regulatory modules (CRM). While the prediction of individual TF-binding sites is a notoriously difficult problem, CRM prediction has proven to be somewhat more reliable. Starting from a set of predicted binding sites for more than 200 TF families documented in Transfac, we describe an algorithm relying on the principle that CRMs generally contain several phylogenetically conserved binding sites for a few different TFs. The method allows the prediction of more than 118,000 CRMs within the human genome. A subset of these is shown to be bound in vivo by TFs using ChIP-chip. Their analysis reveals, among other things, that CRM density varies widely across the genome, with CRM-rich regions often being located near genes encoding transcription factors involved in development. Predicted CRMs show a surprising enrichment near the 3′ end of genes and in regions far from genes. We document the tendency for certain TFs to bind modules located in specific regions with respect to their target genes and identify TFs likely to be involved in tissue-specific regulation. The set of predicted CRMs, which is made available as a public database called PReMod (http://genomequebec.mcgill.ca/PReMod), will help analyze regulatory mechanisms in specific biological systems.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

The orphan nuclear receptor ERR␤ is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR␤ results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage. Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR␤ and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DEStreated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast. These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development. Received December 12, 2000; revised version accepted February 7, 2001. Nuclear receptors constitute a large family of transcription factors that mediate responses to small lipophilic hormones and play essential roles in embryonic development and maintenance of homeostasis in adult animals (Mangelsdorf et al. 1995). Orphan nuclear receptors are members of the nuclear receptor family that lack identified ligands (Giguère 1999). Recent studies have identified several natural and synthetic ligands for a number of orphan nuclear receptors, which led to the discovery of new hormone-response systems implicated in the control of cell fate, organogenesis, and basic metabolic functions Kliewer et al. 1999). While the estrogen-receptor-related receptor (ERR) ␣ (NR3B1) and ERR␤ (NR3B2) were the first orphan nuclear receptors identified more than a decade ago (Giguère et al. 1988), the identification of natural and synthetic ERR ligands has remained elusive. Despite being closely related to the estrogen receptors, ERRs are not activated by natural estrogens (Giguère et al. 1988). In contrast, members of the ERR family, now known to contain a third member referred to as ERR␥ (NR3B3) (Eudy et al. 1998), display various levels of constitutive activity and can interact with coactivators in the absence of ligand Vanacker et al. 1999;Xie et al. 1999).Molecular and genetic studies have shown that ERR␤ plays an important role in early placentation. ERR␤ is expressed in undifferentiated trophoblast stem cell lines (Tanaka et al. 1998) and in a subset of cells in extraembryonic ectoderm destined to make up the chorion (Pettersson et al. 1996;Luo et al. 1997). Genetic ablation of ERR␤ in the mouse results in abnormal chorion formation followed by failure of diploid trophoblast self-renewal and an increase in trophoblast giant cells (Luo et al. 1997). Thus, ERR␤ is essential for normal placental formation, and pharmacological modulation of its activity could influence reproductive function.Diethylstilbestrol (DES) is a potent synthetic estrogen that was used clinically for the prevention of spontaneous abortions from the 1940s to 1971 (Smith 1948). The mechanism of action and factual effectiveness of DES ...

The Vpr gene product of human immunodeficiency virus type 1 is a virion-associated protein that is important for efficient viral replication in nondividing cells such as macrophages. At the cellular level, Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Incorporation of Vpr into viral particles requires a determinant within the p6 domain of the Gag precursor polyprotein Pr55 gag. In the present study, we have used site-directed mutagenesis to identify a domain(s) of Vpr involved in virion incorporation and nuclear localization. Truncations of the carboxyl (C)-terminal domain, rich in basic residues, resulted in a less stable Vpr protein and in the impairment of both virion incorporation and nuclear localization. However, introduction of individual substitution mutations in this region did not impair Vpr nuclear localization and virion incorporation, suggesting that this region is necessary for the stability and/or optimal protein conformation relevant to these Vpr functions. In contrast, the substitution mutations within the amino (N)-terminal region of Vpr that is predicted to adopt an alpha-helical structure (extending from amino acids 16 to 34) impaired both virion incorporation and nuclear localization, suggesting that this structure may play a pivotal role in modulating both of these biological properties. These results are in agreement with a recent study showing that the introduction of proline residues in this predicted alpha-helical region abolished Vpr virion incorporation, presumably by disrupting this secondary structure (S.