Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.The form of RNA polymerase II (RNAPII) 1 responsible for transcript elongation is fundamentally different from the form that enters a promoter to form a preinitiation complex (1, 2). During initiation, RNAPII is hypo-phosphorylated and associated with the functionally conserved Mediator complex, a multisubunit factor required for regulation of transcription (3, 4). The association of RNAPII with Mediator and the general transcription factors is severed during promoter clearance, triggered by TFIIH-mediated hyperphosphorylation of the carboxyl-terminal repeat domain (CTD) of the largest RNAPII subunit (5-7). During elongation, hyperphosphorylated yeast RNAPII is associated with the Elongator complex. Elongator binds directly to RNAPII, at least partly via the CTD, and the interaction is stabilized by CTD hyperphosphorylation (8).

Smads are intracellular transducers for TGF-b superfamily ligands, but little is known about the mechanism by which complexes of receptor-phosphorylated Smad2 and Smad4 regulate transcription. Using an in vitro transcription system, we have discovered that, unlike most transcription factors that are sufficient to recruit the basal transcription machinery and therefore activate transcription on both naked DNA and chromatin templates, the Smads only activate transcription from chromatin templates. We demonstrate that Smad2-mediated transcription requires the histone acetyltransferase, p300. Smad2-recruited p300 exhibits an altered substrate specificity, specifically acetylating nucleosomal histone H3 at lysines 9 and 18, and these modifications are also detected on an endogenous Smad2-dependent promoter in a ligand-induced manner. Furthermore, we show that endogenous Smad2 interacts with the SWI/SNF ATPase, Brg1, in a TGF-b-dependent manner, and demonstrate that Brg1 is recruited to Smad2-dependent promoters and is specifically required for TGF-b-induced expression of endogenous Smad2 target genes. Our data indicate that the Smads define a new class of transcription factors that absolutely require chromatin to assemble the basal transcription machinery and activate transcription.

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxynucleoside salvage pathway in mammalian cells and plays a key role in the activation of several pharmacologically important nucleoside analogs. Using a highly specific polyclonal antibody raised against a C-terminal peptide of the human dCK, we analyzed its subcellular localization by Western blots of biochemically fractionated nuclear and cytoplasmic fractions as well as by in situ immunochemistry. Native dCK was found to be located mainly in the cytoplasm in several cell types, and the enzyme was more concentrated in the perinuclear and cellular membrane area. In contrast, when dCK was overexpressed in the cells, it was mainly located in the nucleus. The results demonstrate that native dCK is a cytoplasmic enzyme. However, it has the ability to enter the nucleus under certain conditions, suggesting the existence of a cytoplasmic retention mechanism that may have an important function in the regulation of the deoxynucleoside salvage pathway.

The molecular architecture of six-subunit yeast holoElongator complex was investigated by the use of immunoprecipitation, two-hybrid interaction mapping, and in vitro studies of binary interactions between individual subunits. Surprisingly, Elp2 is dispensable for the integrity of the holo-Elongator complex, and a purified five-subunit elp2⌬ Elongator complex retains histone acetyltransferase activity in vitro. These results indicate that the WD40 repeats in Elp2 are required neither for subunit-subunit interactions within Elongator nor for Elongator interaction with histones during catalysis. Elp2 and Elp4 were largely dispensable for the association of Elongator with nascent RNA transcript in vivo. In contrast, Elongator-RNA interaction requires the Elp3 protein. Together, these data shed light on the structure-function relationship of the Elongator complex.

Cells lacking KTI12 or Elongator (ELP) genes are insensitive to the toxin zymocin and also share more general phenotypes. Moreover, data from low stringency immunoprecipitation experiments suggest that Elongator and Kti12 may interact. However, the precise relationship between these factors has not been determined. Here we use a variety of approaches to investigate the possibility that Elongator and Kti12 functionally overlap. Native Kti12 purified to virtual homogeneity under stringent conditions is a single polypeptide, but depletion of Kti12 from a yeast extract results in co-depletion of Elongator, indicating that these factors do interact. Indeed, biochemical evidence suggests that Elongator and Kti12 form a fragile complex under physiological salt conditions. Purified Kti12 does not affect Elongator histone acetyltransferase activity in vitro. However, a variety of genetic experiments comparing the effects of mutation in ELP3 and KTI12 alone and in combination with other transcription factor mutations clearly demonstrate a significant functional overlap between Elongator and Kti12 in vivo. Intriguingly, chromatin immunoprecipitation experiments show that Kti12 is associated with chromatin throughout the genome, even in non-transcribed regions and in the absence of Elongator. Conversely, RNA-immunoprecipitation experiments indicate that Kti12 only plays a minor role for Elongator association with active genes. Together, these experiments indicate a close physical and functional relationship between Elongator and the highly conserved Kti12 protein.

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Deoxyguanosine kinase (dGK) is a nuclear gene product that catalyzes the phosphorylation of purine deoxyribonucleosides and their analogues. The human enzyme is located predominantly in the mitochondria, as shown by biochemical fractionation studies and in situ localization of the overexpressed recombinant protein.Here we describe the cloning of mouse dGK cDNA and the identification of a novel amino-terminally truncated isoform that corresponds to about 14% of the total dGK mRNA population in mouse spleen. In situ fluorescence assays suggest that the new isoform cannot translocate into the mitochondria and thus may represent a cytoplasmic enzyme. Expression of mouse dGK mRNA was highly tissue-specific and differed from the tissue distribution observed in humans. Recombinant mouse dGK showed similar specific activity and substrate specificity as compared with the human enzyme. The broad specificity, restricted tissue distribution, and location of mouse dGK in multiple cellular compartments raise new considerations with respect to the role of the individual deoxynucleoside kinases in nucleotide metabolism.

Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.