S. J. Flint

The Double Bromodomain Proteins Brd2 and Brd3 Couple Histone Acetylation to Transcription

Posttranslational histone modifications are crucial for the modulation of chromatin structure and regulation of transcription. Bromodomains present in many chromatin-associated proteins recognize acetylated lysines in the unstructured N-terminal regions of histones. Here, we report that the double bromodomain proteins Brd2 and Brd3 associate preferentially in vivo with hyperacetylated chromatin along the entire lengths of transcribed genes. Brd2- and Brd3-associated chromatin is significantly enriched in H4K5, H4K12, and H3K14 acetylation and contains relatively little dimethylated H3K9. Both Brd2 and Brd3 allowed RNA polymerase II to transcribe through nucleosomes in a defined transcription system. Such activity depended on specific histone H4 modifications known to be recognized by the Brd proteins. We also demonstrate that Brd2 has intrinsic histone chaperone activity and is required for transcription of the cyclin D1 gene in vivo. These data identify proteins that render nucleosomes marked by acetylation permissive to the passage of elongating RNA polymerase II.

Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus.

The mechanism by which adenovirus type 2 inhibits HeLa cell protein synthesis has been investigated by a quantitative analysis of the synthesis and metabolism of cellular and viral hnRNA† sequences. By the time labeling of cellular proteins is maximally inhibited, 16 hours following adenovirus infection under the conditions we have used, all of the newly made messenger RNA sequences reaching the cytoplasm are viral-specific. Thus, the gradual shift from translation of cellular to translation of viral mRNA species that begins following entry of adenovirus-infected cells into the late phase of the productive cycle, can probably be explained simply by competition between the two types of mRNA for available ribosomes. In order to investigate the mechanism(s) by which adenovirus infection exerts this dramatic change in the biogenesis of HeLa cellular mRNA sequences, the hnRNA fraction has been isolated quantitatively from infected cells after pulselabeling or a pulse-chase. Cellular and viral transcripts were then distinguished by hybridization to viral DNA. The results of these experiments reveal that the rate of synthesis of cellular hnRNA sequences remains constant throughout the course of adenovirus type 2 infection. Moreover, poly(A) is apparently added normally to cellular hnRNA sequences synthesized during the late phase of productive adenovirus infection. This newly synthesized, poly(A)-containing hnRNA cannot be distinguished from the corresponding fraction isolated from mockinfected cells by hybridization to cDNA prepared using uninfected HeLa cell, cytoplasmic, poly(A)-containing RNA as template: it also appears to be metabolized with kinetics similar to those observed in uninfected cells.

Nucleolin Is Required for RNA Polymerase I Transcription In Vivo

ABSTRACT Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin.

Regulation of mRNA Production by the Adenoviral E1B 55-kDa and E4 Orf6 Proteins

The E1B 55-kDa and E4 Orf6 proteins of human subgroup C adenoviruses both counter host cell defenses mediated by the cellular p53 protein and regulate viral late gene expression. A complex containing the two proteins has been implicated in induction of selective export of viral late mRNAs from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of newly synthesized cellular mRNAs. The molecular mechanisms by which these viral proteins subvert cellular pathways of nuclear export are not yet clear. Here, we review recent efforts to identify molecular and biochemical functions of the E1B 55-kDa and E4 Orf6 proteins required for regulation of mRNA export, the several difficulties and discrepancies that have been encountered in studies of these viral proteins, and evidence indicating that the reorganization of the infected cell nucleus and production of viral late mRNA at specific intra-nuclear sites are important determinants of selective mRNA export in infected cells. In our view, it is not yet possible to propose a coherent molecular model for regulation of mRNA export by the E1B 55-kDa and E4 Orf6 proteins. However, it should now be possible to address specific questions about the roles of potentially relevant properties of these viral proteins.

Characterization of the adenovirus 2 virion protein, Mu

Adenovirus 2 virions contain a small, highly basic protein known as mu (mu). Partial sequence analysis of mu labeled with radioactive amino acids showed that it is derived from an 11-kDa virion precursor protein, L2-79R. Amino acid analysis, direct microsequence analysis, time-of-flight mass spectrometer analysis, and chemical synthesis demonstrated that mu is the unmodified, 19 amino acid peptide obtained from the 79-residue precursor by adenovirus-encoded proteinase-mediated cleavage after glycine31 and glycine50. Mu bound tightly to DNA and was located in the virion core. In vitro, mu could precipitate DNA fragments, suggesting that it may have a role in viral chromosome condensation.

Effects of mutations in the adenoviral E1B 55-kilodalton protein coding sequence on viral late mRNA metabolism.

ABSTRACT The human subgroup C adenoviral E1B 55-kDa protein cooperates with the viral E4 Orf6 protein to induce selective export of viral, late mRNAs from the nucleus to the cytoplasm. Previous studies have suggested that such preferential transport of viral mRNA and the concomitant inhibition of export of cellular mRNAs are the result of viral colonization of specialized microenvironments within the nucleus. However, neither the molecular basis of this phenomenon nor the mechanism by which the E1B 55-kDa protein acts has been elucidated. We therefore examined viral late mRNA metabolism in HeLa cells infected with a series of mutant viruses that carry insertions at various positions in the E1B protein coding sequence (P. R. Yew, C. C. Kao, and A. J. Berk, Virology 179:795-805, 1990). All the mutations examined impaired cytoplasmic accumulation of viral L2 mRNAs and reduced L2 mRNA export efficiency. However, in most cases these defects could be ascribed to reduced E1B 55-kDa protein concentration or the unexpected failure of the altered E1B proteins to enter the nucleus efficiently. The latter property, the pleiotropic defects associated with all the mutations that impaired nuclear entry of the E1B protein, and consideration of its primary sequence suggest that these insertions result in misfolding of the protein. Insertion of four amino acids at residue 143 also inhibited viral mRNA export but resulted in increased rather than decreased accumulation of the E1B 55-kDa protein in the nucleus. This mutation specifically impaired the previously described association of the E1B protein with intranuclear structures that correspond to sites of adenoviral DNA replication and transcription (D. Ornelles and T. Shenk, J. Virol. 65:424-439, 1991) and the colocalization of the E1B and E4 Orf6 proteins. As this insertion has been shown to inhibit the interaction of the E1B with the E4 Orf6 protein in infected cell extracts (S. Rubenwolf, H. Schütt, M. Nevels, H. Wolf, and T. Dobner, J. Virol. 71:1115-1123, 1997), these phenotypes provide direct support for the hypothesis that selective viral mRNA export is determined by the functional organization of the infected cell nucleus.

Identification of the adenovirus early proteins and their genomic map positions

Six different group C adenovirus transformed hamster cell lines were employed to produce tumors in hamsters. The sera from these animals were then used to immunoprecipitate [35S]methionine-labeled adenovirus induced tumor antigens from virus infected and transformed cells. Collectively, these sera detect 14 virus induced tumor antigens. Based upon the regions of the adenoviral genome present and transcribed in each of the transformed cell lines, an estimated position of the genomic map location responsible for the induction of each of the tumor antigens or viral early proteins was determined. These sera were also employed to follow the synthesis of the adenovirus proteins during productive infection and in transformed cells. Based upon this analysis the tumor antigens can be divided into two groups, early and delayed early proteins, depending upon the time after infection that a protein was synthesized and detected by immunoprecipitation. A comparison of the Ad2 and Ad5 early proteins produced in virus infected and transformed cells indicated that several proteins have different apparent molecular weights that are serotype specific but independent of the species of host cell employed.

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

The proteins associated with parental, adenoviral DNA in productively‐infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P‐labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross‐linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co‐migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross‐linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross‐linkable to viral DNA in cells infected by wild‐type virus and a non‐infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre‐early and early viral genes.

Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo

A new approach to the identification of DNA binding proteins has been developed and used to study the DNA-protein interactions within the nucleoprotein core of subgroup C adenoviruses. Virions labelled in vivo with [32P]orthophosphate were exposed to ultraviolet light and the DNA digested by chemical or enzymatic methods. Labelled phosphoamino acids of the virion phosphoproteins were selectively hydrolysed by alkali, permitting proteins crosslinked to DNA to be identified by virtue of their covalently attached, 32P-labelled nucleotides. In parallel experiments, [3H]arginine-labelled virions were crosslinked by exposure to ultraviolet light and analysed by more conventional methods. The results indicate that proteins VII and V lie in close contact with viral DNA within the core. The compact arrangement of the nucleoprotein core appears to be capable of trapping protein VII molecules that are not covalently attached to DNA after exposure to ultraviolet light, suggesting that viral DNA might be wrapped around clusters of protein VII molecules. The domains of protein VII that lie in contact with DNA were identified by partial proteolytic mapping of the sites of covalent-attachment of the 32P-labelled oligonucleotides. The implications of these data for the nature of the interactions that mediate the packaging of viral DNA within the nucleoprotein core of adenovirions are discussed.

The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription

ABSTRACT The adenovirus late IVa2 protein is required for maximally efficient transcription from the viral major late (ML) promoter, and hence, the synthesis of the majority of viral late proteins. This protein is a sequence-specific DNA-binding protein that also promotes the assembly of progeny virus particles. Previous studies have established that a IVa2 protein dimer (DEF-B) binds specifically to an intragenic ML promoter sequence necessary for late phase-specific stimulation of ML transcription. However, activation of transcription from the ML promoter correlates with binding of at least one additional infected-cell-specific protein, termed DEF-A, to the promoter. Using an assay for the DNA-binding activity of DEF-A, we identified the unknown protein by using conventional purification methods, purification of FLAG-tagged IVa2-protein-containing complexes, and transient synthesis of viral late proteins. The results of these experiments established that the viral L4 33-kDa protein is the only component of DEF-A: the IVa2 and L4 33-kDa proteins are necessary and sufficient for formation of all previously described complexes in the intragenic control region of the ML promoter. Furthermore, the L4 33-kDa protein binds to the promoter with the specificity characteristic of DEF-A and stimulates transcription from the ML promoter in transient-expression assays.