The association of genes with the nuclear pore complex (NPC) and nuclear transport factors has been implicated in transcriptional regulation. We therefore examined the association of components of the nuclear transport machinery including karyopherins, nucleoporins, and the Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome. We find that most nucleoporins and karyopherins preferentially associate with a subset of highly transcribed genes and with genes that possess Rap1 binding sites whereas the RanGEF preferentially associates with transcriptionally inactive genes. Consistent with coupling of transcription to the nuclear pore, we show that transcriptional activation of the GAL genes results in their association with nuclear pore proteins, relocation to the nuclear periphery, and loss of RanGEF association. Taken together, these results indicate that the organization of the genome is coupled via transcriptional state to the nuclear transport machinery.

Objective. Prior reports document macrophage and lymphocyte infiltration with proinflammatory cytokine expression in pathologic intervertebral disc (IVD) tissues. Nevertheless, the role of the Th17 lymphocyte lineage in mediating disc disease remains uninvestigated. We undertook this study to evaluate the immunophenotype of pathologic IVD specimens, including interleukin-17 (IL-17) expression, from surgically obtained IVD tissue and from nondegenerated autopsy control tissue.Methods. Surgical IVD tissues were procured from patients with degenerative disc disease (n ‫؍‬ 25) or herniated IVDs (n ‫؍‬ 12); nondegenerated autopsy control tissue was also obtained (n ‫؍‬ 8) from the anulus fibrosus and nucleus pulposus regions. Immunohistochemistry was performed for cell surface antigens (CD68 for macrophages, CD4 for lymphocytes) and various cytokines, with differences in cellularity and target immunoreactivity scores analyzed between surgical tissue groups and between autopsy control tissue regions.Results. Immunoreactivity for IL-4, IL-6, IL-12, and interferon-␥ (IFN␥) was modest in surgical IVD tissue, although expression was higher in herniated IVD samples and virtually nonexistent in control samples. The Th17 lymphocyte product IL-17 was present in >70% of surgical tissue fields, and among control samples was detected rarely in anulus fibrosus regions and modestly in nucleus pulposus regions. Macrophages were prevalent in surgical tissues, particularly herniated IVD samples, and lymphocytes were expectedly scarce. Control tissue revealed lesser infiltration by macrophages and a near absence of lymphocytes.Conclusion. Greater IFN␥ positivity, macrophage presence, and cellularity in herniated IVDs suggests a pattern of Th1 lymphocyte activation in this pathology. Remarkable pathologic IVD tissue expression of IL-17 is a novel finding that contrasts markedly with low levels of IL-17 in autopsy control tissue. These findings suggest involvement of Th17 lymphocytes in the pathomechanism of disc degeneration.

To determine whether mononuclear cell secretory products contribute to the changes in bone turnover that characterize the development of postmenopausal osteoporosis, we evaluated the effects of oophorectomy and subsequent estrogen replacement on the spontaneous secretion of interleukin 1 (IL-1) and tumor necrosis factor a (TNF-a) and on the phytohemagglutinin A-induced secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) from peripheral blood mononuclear cells. In 15 healthy premenopausal women who underwent oophorectomy, increases in GM-CSF activity were observed as early as 1 week after surgery, whereas elevations in IL-1 and TNF-a and in hydroxyproline/creatinine and calcium/creatinine ratios, two urinary indices of bone resorption, were detectable 2 weeks after the surgical procedure. Six of the oophorectomized women received no estrogen therapy after surgery and in these subjects hydroxyproline/creatinine and calcium/creatinine ratios plateaued 6 weeks postoperatively, and all three cytokines reached the highest levels 8 weeks after oophorectomy, when the study ended. In the remaining 9 women, who were started on estrogen replacement therapy 4 weeks after oophorectomy, decreases in the indices of bone resorption paralleled decreases in the secretion of the cytokines, with lower levels detected after 2 weeks of therapy. In the women who did not receive estrogen therapy, circulating osteocalcin, a marker of bone formation, increased beyond preoperative levels 8 weeks after oophorectomy, whereas in the estrogen-treated subjects osteocalcin remained unchanged in the entire study period. In 9 female controls who underwent simple hysterectomy, cytokine release and biochemical indices of bone turnover did not change after surgery. These data indicate that changes in estrogen status in vivo are associated with the secretion of mononuclear cell immune factors in vitro and suggest that alterations in the local production of bone-acting cytokines may underlie changes in bone turnover caused by surgically induced menopause and estrogen replacement.Postmenopausal osteoporosis, a common disorder characterized by a decreased bone mass and increased fracture risk (1), stems from an accelerated loss of bone that begins after natural or surgically induced menopause and progresses rapidly for 5 or 10 years thereafter (2, 3). That estrogen deficiency plays a major role in this condition is well supported by the higher prevalence of osteoporosis in women than in men (4), by the increase in the rate of bone mineral loss detectable by bone densitometry after artificial or natural menopause (5, 6), and by the protective effect of estrogen replacement with respect to both bone mass loss and fracture incidence (7,8). Although the bone-sparing effect of estrogen appears to be related to an inhibitory effect on bone resorption (9), the mechanism of the estrogen response remains unknown.The discovery of estrogen receptors in osteoblasts (10-12) and osteoclasts (13) suggests that a direct mechanism(s) may be in...

Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.

Electron microscopy of single gene molecules and mathematical modeling shows that a promoter stochastically transitions between transcriptionally favorable and unfavorable nucleosome configurations, providing a mechanism for transcriptional bursting.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5′-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5′-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc–siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

The nuclear localization of genes is intimately tied to their transcriptional status in Saccharomyces cerevisiae, with populations of both active and silent genes interacting with components of the nuclear envelope. We investigated the relationship between the mammalian nuclear pore and the human genome by generating high-resolution, chromosome-wide binding maps of human nucleoporin 93 (Nup93) in the presence and absence of a potent histone deacetylase inhibitor (HDACI). Here, we report extensive genomic reorganization with respect to the nuclear pore following HDACI treatment, including the recruitment of promoter regions, euchromatin-rich domains, and differentially expressed genes. In addition to biochemical mapping, we visually demonstrate the physical relocalization of several genomic loci with respect to the nuclear periphery. Our studies show that inhibiting HDACs leads to significant changes in genomic organization, recruiting regions of transcriptional regulation to mammalian nuclear pore complexes.[Keywords: NPC; nucleoporin; nuclear organization; Nup93; ChIP-chip] Supplemental material is available at http://www.genesdev.org.

Although the spatial location of genes within the nucleus has been implicated in their transcriptional status, little is known about the dynamics of gene location that accompany large-scale changes in gene expression. The mating of haploid yeast Saccharomyces cerevisiae is accompanied by a large-scale change in transcription and developmental program. We examined changes in nuclear organization that accompany stimulus by the mating pheromone ␣ factor and found that most ␣-factor-induced genes become associated with components of the nuclear envelope. The myosin-like protein Mlp1, which has been implicated in mRNA export, was further shown to exhibit RNA dependence in its association with ␣-factor-induced genes. High-resolution mapping of association of chromosome III with Mlp1 revealed ␣-factor-dependent determinants of nuclear pore association, including origins of replication, specific intergenic regions, and the 3 ends of transcriptionally activated genes. Taken together, these results reveal RNA-and DNA-dependent determinants of nuclear organization as well as a detailed picture of how an entire chromosome alters its spatial conformation in response to a developmental cue.[Keywords: Nuclear organization; Mlp1; RanGEF; genome polarization; nucleoporin; NPC] Supplemental material is available at http://www.genesdev.org.