The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure.[Keywords: Gene expression; transcription elongation; polymerase stalling; chromatin structure] Supplemental material is available at http://www.genesdev.org.

The physiological responses of the rodent uterus to acute estrogen (E) dosing can be divided into early and late events. Examples of early responses include increased RNA transcription, hyperemia, and water imbibition 2 and 6 h following E administration respectively, whereas later responses include cycles of DNA synthesis and mitosis of epithelial cells beginning 10 and 16 h after E. The development of estrogen receptor (ER) knockout (ERKO) mice, combined with microarray technology, has allowed us to design a genomic approach to study the acute response of the rodent reproductive tract to E. To determine whether early and late biological responses are correlated with altered regulation of a single set of genes or distinct sets of genes characteristic of early and late responses, uterine RNA was obtained from ovariectomized mice that were treated with vehicle or with estradiol for 2 h (early) or 24 h (late). Samples were also prepared from identically treated mice that lacked either ERalpha (alphaERKO) or ERbeta (betaERKO) to address the relative contributions of the ERs in the uterine responses. Microarray analysis of the relative expression of 8700 mouse cDNAs indicated distinct clusters of genes that were regulated both positively and negatively by E in the early or late phases as well as clusters of genes regulated at both times. Both early and late responses by the betaERKO samples were indistinguishable from those of WT samples, whereas the alphaERKO showed little change in gene expression in response to E, indicating the predominant role for ERalpha in the genomic response. Further studies indicated that the genomic responses in samples from intermediate time points (6 h, 12 h) fall within the early or late clusters, rather than showing unique clusters regulated in the intermediary period. The use of this genomic approach has illustrated how physiological responses are reflected in genomic patterns. Furthermore, the identification of functional gene families that are regulated by E in the uterus combined with the utilization of genetically altered experimental animal models can help to uncover and define novel mechanisms of E action.

DNA double strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiologic processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell cycle checkpoints and cell survival1–5. DNA double strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we demonstrate that these physiologic DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiologic DNA double strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.

Retinoid-related orphan receptors alpha (ROR alpha) and gamma (ROR gamma) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The ROR alpha1 and ROR gamma 1 isoforms, but not ROR alpha 4, show an oscillatory pattern of expression during circadian rhythm. To obtain insight into the physiological functions of ROR receptors in liver, we analyzed the gene expression profiles of livers from WT, ROR alpha-deficient staggerer (sg) mice (ROR alpha(sg/sg)), ROR gamma(-/-), and ROR alpha(sg/sg)ROR gamma(-/-) double knockout (DKO) mice by microarray analysis. DKO mice were generated to study functional redundancy between ROR alpha and ROR gamma. These analyses demonstrated that ROR alpha and ROR gamma affect the expression of a number of genes. ROR alpha and ROR gamma are particularly important in the regulation of genes encoding several phase I and phase II metabolic enzymes, including several 3beta-hydroxysteroid dehydrogenases, cytochrome P450 enzymes, and sulfotransferases. In addition, our results indicate that ROR alpha and ROR gamma each affect the expression of a specific set of genes but also exhibit functional redundancy. Our study shows that ROR alpha and ROR gamma receptors influence the regulation of several metabolic pathways, including those involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that RORs are important in the control of metabolic homeostasis.