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.

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.Eukaryotic cells contain three nuclear enzymes that transcribe DNA, RNA polymerases I, II, and III, which are responsible for transcription of rRNA genes, all protein coding genes, and genes encoding various small RNAs, respectively. Although these are large, multisubunit enzymes, none alone is capable of specific initiation of transcription. Rather, initiation from their cognate promoters requires the participation of polymerase-dedicated initiation proteins such as UBF and SL1 in the case of RNA polymerase I and the six general transcription factors (GTFs) for RNA polymerase II (see references 25,56,60,67,70, and 75 for reviews). The biochemical studies that led to the identification of the RNA polymerases and these accessory proteins focused on initiation and used naked templates. However, in eukaryotes, genomic DNA is packaged with histones and non-histone-associated proteins in the form of chromatin. Consequently, RNA polymerases must negotiate chromatin during transcription in vivo.

Host-cell regulation through adenovirus type 5

The effects of the adenovirus Ad5 on basic host cell programs, such as cell-cycle regulation, were studied in a microarray analysis of human fibroblasts. About 2,000 genes were up-or down-regulated after Ad5 infection and Ad5 infection was shown to induce reversal of the quiescence program and recapitulation of the core serum response.

The human adenovirus type 5 (Ad5) E1B 55-kDa protein modulates several cellular processes, including activation of the tumor suppressor p53. Binding of the E1B protein to the activation domain of p53 inhibits p53-dependent transcription. This activity has been correlated with the transforming activity of the E1B protein, but its contribution to viral replication is not well understood. To address this issue, we used microarray hybridization methods to examine cellular gene expression in normal human fibroblasts (HFFs) infected by Ad5, the E1B 55-kDa-protein-null mutant Hr6, or a mutant carrying substitutions that impair repression of p53-dependent transcription. Comparison of the changes in cellular gene expression observed in these and our previous experiments (D. L. Miller et al., Genome Biol. 8:R58, 2007) by significance analysis of microarrays indicated excellent reproducibility. Furthermore, we again observed that Ad5 infection led to efficient reversal of the p53-dependent transcriptional program. As this same response was also induced in cells infected by the two mutants, we conclude that the E1B 55-kDa protein is not necessary to block activation of p53 in Ad5-infected cells. However, groups of cellular genes that were altered in expression specifically in the absence of the E1B protein were identified by consensus k-means clustering of the hybridization data. Statistical analysis of the enrichment of genes associated with specific functions in these clusters established that the E1B 55-kDa protein is necessary for repression of genes encoding proteins that mediate antiviral and immune defenses.The genomes of human subgroup C adenoviruses, such as adenovirus type 5 (Ad5), encode more than 20 proteins that are made prior to the onset of viral DNA synthesis in infected cells (reviewed in reference 1). The great majority of these immediate-early and early viral gene products interact with cellular proteins to optimize expression of viral genes or to block potentially deleterious host responses to infection. The 289R and 243R E1A proteins are prime examples of the former class. The larger E1A protein binds via a unique internal sequence to a specific subunit of the host cell mediator, a component of the RNA polymerase II transcriptional machinery, to stimulate transcription from viral early promoters by this enzyme in vitro and in infected cells (7,77,85). The 243R E1A protein interacts with the Rb protein and the related family members p107 and p130 to liberate transcriptional regulators of the E2F family, which are required for efficient transcription from the viral E2 early promoter (2, 13). This interaction is also crucial for the mitogenic and transforming activities of the E1A proteins. Adenoviral proteins that protect infected cells against antiviral defenses include E3 gene products, such as the 19-kDa glycoprotein that sequesters major histocompatibility complex class I molecules in the endoplasmic reticulum and several small proteins that prevent induction of apoptosis in response to external signa...