A first exon termination checkpoint preferentially suppresses extragenic transcription.

Interactions between the splicing machinery and RNA polymerase II increase protein-coding gene transcription. Similarly, exons and splicing signals of enhancer-generated long noncoding RNAs (elncRNAs) augment enhancer activity. However, elncRNAs are inefficiently spliced, suggesting that, compared with protein-coding genes, they contain qualitatively different exons with a limited ability to drive splicing. We show here that the inefficiently spliced first exons of elncRNAs as well as promoter-antisense long noncoding RNAs (pa-lncRNAs) in human and mouse cells trigger a transcription termination checkpoint that requires WDR82, an RNA polymerase II-binding protein, and its RNA-binding partner of previously unknown function, ZC3H4. We propose that the first exons of elncRNAs and pa-lncRNAs are an intrinsic component of a regulatory mechanism that, on the one hand, maximizes the activity of these cis-regulatory elements by recruiting the splicing machinery and, on the other, contains elements that suppress pervasive extragenic transcription.

High constitutive activity of a broad panel of housekeeping and tissue-specific cis-regulatory elements depends on a subset of ETS proteins.

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of cis-regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the cis-regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping cis-regulatory elements with the ability to efficiently promote transcription.

A shortcut for early macrophage recruitment into tumors by activated oncogenes.

Macrophages play an important role in tumor promotion, usually acting as facilitators of cancer initiation and progression. However, it is not clear how macrophages impact early phases of tumorigenesis. Using genetically modified mouse models, Guo et al. (pp. 247-259) demonstrated that tumor-initiating cells with an activated Hippo pathway are able to recruit macrophages starting from the very early phases of cancer development, mainly through direct activation of genes encoding macrophage chemoattractants and survival factors. The recruited macrophages were of vital importance for protection of tumor-initiating cells against eradication by lymphocyte-mediated immune surveillance. Such a tight link between macrophages and a pathway controlling organ development and size may reflect the normal role of these cells in tissue morphogenesis.

Transcription of Mammalian cis-Regulatory Elements Is Restrained by Actively Enforced Early Termination.

Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs.

The histone methyltransferase Wbp7 controls macrophage function through GPI glycolipid anchor synthesis.

Histone methyltransferases catalyze site-specific deposition of methyl groups, enabling recruitment of transcriptional regulators. In mammals, trimethylation of lysine 4 in histone H3, a modification localized at the transcription start sites of active genes, is catalyzed by six enzymes (SET1a and SET1b, MLL1-MLL4) whose specific functions are largely unknown. By using a genomic approach, we found that in macrophages, MLL4 (also known as Wbp7) was required for the expression of Pigp, an essential component of the GPI-GlcNAc transferase, the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor synthesis. Impaired Pigp expression in Wbp7(-/-) macrophages abolished GPI anchor-dependent loading of proteins on the cell membrane. Consistently, loss of GPI-anchored CD14, the coreceptor for lipopolysaccharide (LPS) and other bacterial molecules, markedly attenuated LPS-triggered intracellular signals and gene expression changes. These data link a histone-modifying enzyme to a biosynthetic pathway and indicate a specialized biological role for Wbp7 in macrophage function and antimicrobial response.

Extract of oregano, coffee, thyme, clove, and walnuts inhibits NF-kappaB in monocytes and in transgenic reporter mice.

The transcription factor NF-kappaB is a promising target for chemoprevention. Several dietary plants are efficient inhibitors of NF-kappaB activation in vitro and could act synergistically on the NF-kappaB signaling pathway. In this study, we tested whether dietary plant extracts could inhibit NF-kappaB activation in a synergistic manner in vitro. Second, we investigated the potency of the same dietary plant extracts in the inhibition of NF-kappaB activation in vivo. A combined extract of clove, oregano, thyme, walnuts, and coffee synergistically inhibited lipopolysaccaride (LPS)-induced NF-kappaB activation in a monocytic cell line, compared with the sum of effects from the single extracts. Transgenic NF-kappaB luciferase reporter mice were given a single dose of the combined extract and subsequently challenged with LPS. NF-kappaB activation was monitored by in vivo imaging for 6 hours. In addition, NF-kappaB activity in organs and the expression of immune-related genes in liver were investigated. Based on the area under the curve, the extract decreased whole body LPS-induced NF-kappaB activity the first 6 hours by 35% compared with control mice. Organ-specific NF-kappaB activation was inhibited in intestine, liver, testis, and epididymis of the mice receiving the combination extract. In addition, dietary plants reduced the expression of genes related to inflammation, cell migration, and proliferation in liver. This study shows that dietary plants may be potent modulators of NF-kappaB signaling both in vitro and in vivo, and thus support further investigation of consumption of these plant foods as part of a healthy diet or as a mode of chemoprevention.

Jmjd3 contributes to the control of gene expression in LPS-activated macrophages.

Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF-kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation-sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol_II). Moreover, 70% of lipopolysaccharide (LPS)-inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine-tunes the transcriptional output of LPS-activated macrophages in an H3K27 demethylation-independent manner.

Retinoic acid inhibits in vivo interleukin-2 gene expression and T-cell activation in mice.

Interleukin-2 (IL-2) is an essential cytokine for T-lymphocyte homeostasis. We have previously reported that all-trans retinoic acid (atRA) enhances the secretion of IL-2 from human peripheral blood T cells in vitro, followed by increased proliferation and inhibition of spontaneous cell death. In this study we used a transgenic IL-2 gene luciferase reporter model to examine the effects of atRA in vivo. In contrast to the observations in human T cells, we found an overall reduction in luciferase-reported IL-2 gene expression in mice treated with atRA. Whole-body luminescence of anti-CD3-treated and non-treated mice was reduced in mice receiving atRA. Accordingly, after 7 hr, IL-2 gene expression was on average 55% lower in the atRA-treated mice compared with the control mice. Furthermore, mice fed a vitamin A-deficient diet had a significantly higher basal level of luciferase activity compared with control mice, demonstrating that vitamin A modulates IL-2 gene expression in vivo. Importantly, the atRA-mediated inhibition of IL-2 gene expression was accompanied by decreased DNA synthesis in murine T cells, suggesting a physiological relevance of the reduced IL-2 gene expression observed in transgenic reporter mice.

Retinoic acid dampens LPS-induced NF-kappaB activity: results from human monoblasts and in vivo imaging of NF-kappaB reporter mice.

Bacterial lipopolysaccharide (LPS) is a major inducer of systemic inflammatory reactions and oxidative stress in response to microbial infections and may cause sepsis. In the present study, we demonstrate that retinoic acid inhibits LPS-induced activation in transgenic reporter mice and human monoblasts through inhibition of nuclear factor kappaB (NF-kappaB). By using noninvasive molecular imaging of NF-kappaB luciferase reporter mice, we showed that administration of retinoic acid repressed LPS-induced whole-body luminescence, demonstrating in vivo the dynamics of retinoic acid's ability to repress physiologic response to LPS. Retinoic acid also inhibited LPS-induced NF-kappaB activity in the human myeloblastic cell line U937. Retinoic-acid-receptor-selective agonists mimicked - while specific antagonists inhibited - the effects of retinoic acid, suggesting the involvement of nuclear retinoic acid receptors. Retinoic acid also repressed LPS-induced transcription of NF-kappaB target genes such as IL-6, MCP-1 and COX-2. The effect of retinoic acid was dependent on new protein synthesis, was obstructed by a deacetylase inhibitor and was partly eliminated by a signal transducer and activator of transcription-1 (STAT1)/methyltransferase inhibitor, indicating that retinoic acid induces a new protein, possibly STAT1, that is involved in inhibiting NF-kappaB. This provides more evidence for retinoic acid's anti-inflammatory potential, which may have clinical implications in terms of fighting microbial infections.

Molecular imaging of the transcription factor NF-kappaB, a primary regulator of stress response.

A wide range of environmental stress and human disorders involves inappropriate regulation of NF-kappaB, including cancers and numerous inflammatory conditions. We have developed transgenic mice that express luciferase under the control of NF-kappaB, enabling real-time non-invasive imaging of NF-kappaB activity in intact animals. We show that, in the absence of stimulation, strong, intrinsic luminescence is evident in lymph nodes in the neck region, thymus, and Peyer's patches. Treating mice with stressors, such as TNF-alpha, IL-1alpha, or lipopolysaccharide (LPS) increases the luminescence in a tissue-specific manner, with the strongest activity observable in the skin, lungs, spleen, Peyer's patches, and the wall of the small intestine. Liver, kidney, heart, muscle, and adipose tissue exhibit less intense activities. Exposure of the skin to a low dose of UV-B radiation increases luminescence in the exposed areas. In ocular experiments, LPS- and TNF-alpha injected NF-kappaB-luciferase transgenic mice exhibit a 20-40-fold increase in lens NF-kappaB activity, similar to other LPS- and TNF-alpha-responsive organs. Peak NF-kappaB activity occurs 6h after injection of TNF-alpha and 12h after injection of LPS. Peak activities occur, respectively, 3 and 6h later than that in other tissues. Mice exposed to 360J/m(2) of UV-B exhibit a 16-fold increase in NF-kappaB activity 6h after exposure, characteristically similar to TNF-alpha-exposed mice. Thus, in NF-kappaB-luciferase transgenic mice, NF-kappaB activity also occurs in lens epithelial tissue and is activated when the intact mouse is exposed to classical stressors. Furthermore, as revealed by real-time non-invasive imaging, induction of chronic inflammation resembling rheumatoid arthritis produces strong NF-kappaB activity in the affected joints. Finally, we have used the model to demonstrate NF-kappaB regulation by manipulating the Vitamin A status in mice. NF-kappaB activity is elevated in mice fed a Vitamin A deficient (VAD) diet, and suppressed by surplus doses of retinoic acid (RA). We thus demonstrate the development and use of a versatile model for monitoring NF-kappaB activation both in tissue homogenates and in intact animals after the use of classical activators, during disease progression and after dietary intervention.

Vitamin A status significantly alters nuclear factor-kappaB activity assessed by in vivo imaging.

Our study aimed to investigate, in vivo, the relationship between vitamin A status and NF-kappaB activity, a transcription factor central in regulating inflammatory and immune responses. We used a novel transgenic murine NF-kappaB-luciferase reporter model that enabled molecular imaging of NF-kappaB activity in live mice via an intensified image-capture apparatus. Whole-body luminescence, which reflects overall NF-kappaB activity, was elevated 2.2-fold in vitamin A-deficient (VAD) mice compared with control mice. Specifically, NF-kappaB activity in VAD mice was increased 1.8-fold in the lymph nodes and 1.4-fold in the thymus and, NF-kappaB induction in UVB radiation-exposed skin was also enhanced in VAD mice compared with control mice. The administration of all-trans retinoic acid to VAD mice resulted in a transient reduction in NF-kappaB activity and, conversely, a single dose of the RAR-pan-antagonist, AGN 194310, administered to control mice, led to a marked, transient induction of whole-body luminescence. Our results suggest that vitamin A status, and vitamin A itself, affects NF-kappaB activity in vivo and that the elevated NF-kappaB activity in VAD may be a mechanism underlying some of the features of VAD syndrome.