Genome-wide approaches for identification of nuclear receptor target genes

Large-scale genomics analyses have grown by leaps and bounds with the rapid advances in high throughput DNA sequencing and synthesis techniques. Nuclear receptor signaling is ideally suited to genomics studies because receptors function as ligand-regulated gene switches. This review will survey the strengths and limitations of three major classes of high throughput techniques widely used in the nuclear receptor field to characterize ligand-dependent gene regulation: expression profiling studies (microarrays, SAGE and related techniques), chromatin immunoprecipitation followed by microarray (ChIP-on-chip), and genome-wide in silico hormone response element screens. We will discuss each technique, and how each has contributed to our understanding of nuclear receptor signaling

Stimulation of Sirt1-Regulated FoxO Protein Function by the Ligand-Bound Vitamin D Receptor▿

Hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signals through the nuclear vitamin D receptor (VDR). 1,25D regulates cell proliferation and differentiation and has been identified as a cancer chemopreventive agent. FoxO proteins are transcription factors that control cell proliferation and survival. They function as tumor suppressors and are associated with longevity in several organisms. Accumulating data have revealed that 1,25D and FoxO proteins regulate similarly common target genes. We show here that the ligand-bound VDR regulates the posttranslational modification and function of FoxO proteins. 1,25D treatment enhances binding of FoxO3a and FoxO4 within 4 h to promoters of FoxO target genes and blocks mitogen-induced FoxO protein nuclear export. The VDR associates directly with FoxO proteins and regulators, the sirtuin 1 (Sirt1) class III histone deacetylase (HDAC), and protein phosphatase 1. In addition, phosphatase activity and trichostatin A-resistant HDAC activity coimmunoprecipitate with the VDR. 1,25D treatment rapidly (in <4 h) induces FoxO deacetylation and dephosphorylation, consistent with activation. In contrast, ablation of VDR expression enhances FoxO3a phosphorylation, as does knockdown of Sirt1, consistent with the coupling of FoxO acetylation and phosphorylation. 1,25D regulation of common VDR/FoxO target genes is attenuated by blockade of phosphatase activity or by small interfering RNA (siRNA)-mediated knockdown of Sirt1 or FoxO protein expression. Finally, 1,25D-dependent cell cycle arrest is blocked in FoxO3a-deficient cells, indicating that FoxO proteins are key downstream mediators of the antiproliferative actions of 1,25D. These studies link 1,25D signaling through the VDR directly to Sirt1 and FoxO function and provide a molecular basis for the cancer chemopreventive actions of 1,25D

Ligand-dependent Corepressor LCoR Is an Attenuator of Progesterone-regulated Gene Expression*

Ligand-dependent corepressor LCoR interacts with the progesterone receptor (PR) and estrogen receptor ERα in the presence of hormone. LCoR contains tandem N-terminal PXDLS motifs that recruit C-terminal-binding protein (CtBP) corepressors as well as a C-terminal helix-turn-helix (HTH) domain. Here, we analyzed the function of these domains in coregulation of PR- and ERα-regulated gene expression. LCoR and CtBP1 colocalize in nuclear bodies that also contain CtBP-interacting protein CtIP and polycomb group repressor complex marker BMI1. Coexpression of CtBP1 in MCF7 or T47D breast cancer cells augmented corepression by LCoR, whereas coexpression of CtIP did not, consistent with direct interaction of LCoR with CtBP1, but not CtIP. The N-terminal region containing the PXDLS motifs is necessary and sufficient for CTBP1 recruitment and essential for full corepression. However, LCoR function was also strongly dependent on the helix-turn-helix domain, as its deletion completely abolished corepression. LCoR, CtBP, and CtIP were recruited to endogenous PR- and ERα-stimulated genes in a hormone-dependent manner. Similarly, LCoR was recruited to estrogen-repressed genes, whereas hormone treatment reduced CtBP1 binding. Small interfering RNA-mediated knockdown of LCoR or CtBP1 augmented expression of progesterone- and estrogen-stimulated reporter genes as well as endogenous progesterone-stimulated target genes. In contrast, their ablation had gene-specific effects on ERα-regulated transcription that generally led to reduced gene expression. Taken together, these results show that multiple domains contribute to LCoR function. They also reveal a role for LCoR and CtBP1 as attenuators of progesterone-regulated transcription but suggest that LCoR and CtBP1 can act to enhance transcription of some genes

Function of Histone Deacetylase 6 as a Cofactor of Nuclear Receptor Coregulator LCoR*

Ligand-dependent corepressor LCoR was identified as a protein that interacts with the estrogen receptor α (ERα) ligand binding domain in a hormone-dependent manner. LCoR also interacts directly with histone deacetylase 3 (HDAC3) and HDAC6. Notably, HDAC6 has emerged as a marker of breast cancer prognosis. However, although HDAC3 is nuclear, HDAC6 is cytoplasmic in many cells. We found that HDAC6 is partially nuclear in estrogen-responsive MCF7 cells, colocalizes with LCoR, represses transactivation of estrogen-inducible reporter genes, and augments corepression by LCoR. In contrast, no repression was observed upon HDAC6 expression in COS7 cells, where it is exclusively cytoplasmic. LCoR binds to HDAC6 in vitro via a central domain, and repression by LCoR mutants lacking this domain was attenuated. Kinetic chromatin immunoprecipitation assays revealed hormone-dependent recruitment of LCoR to promoters of ERα-induced target genes in synchrony with ERα. HDAC6 was also recruited to these promoters, and repeat chromatin immunoprecipitation experiments confirmed the corecruitment of LCoR with ERα and with HDAC6. Remarkably, however, although we find evidence for corecruitment of LCoR and ERα on genes repressed by the receptor, LCoR and HDAC6 failed to coimmunoprecipitate, suggesting that they are part of distinct complexes on these genes. Although small interfering RNA-mediated knockdown of LCoR or HDAC6 augmented expression of an estrogen-sensitive reporter gene in MCF7 cells, unexpectedly their ablation led to reduced expression of some endogenous estrogen target genes. Taken together, these data establish that HDAC6 can function as a cofactor of LCoR but suggest that they may act in enhance expressing some target genes

International Nosocomial Infection Control Consortiu (INICC) report, data summary of 43 countries for 2007-2012. Device-associated module

We report the results of an International Nosocomial Infection Control Consortium (INICC) surveillance study from January 2007-December 2012 in 503 intensive care units (ICUs) in Latin America, Asia, Africa, and Europe. During the 6-year study using the Centers for Disease Control and Prevention's (CDC) U.S. National Healthcare Safety Network (NHSN) definitions for device-associated health care–associated infection (DA-HAI), we collected prospective data from 605,310 patients hospitalized in the INICC's ICUs for an aggregate of 3,338,396 days. Although device utilization in the INICC's ICUs was similar to that reported from ICUs in the U.S. in the CDC's NHSN, rates of device-associated nosocomial infection were higher in the ICUs of the INICC hospitals: the pooled rate of central line–associated bloodstream infection in the INICC's ICUs, 4.9 per 1,000 central line days, is nearly 5-fold higher than the 0.9 per 1,000 central line days reported from comparable U.S. ICUs. The overall rate of ventilator-associated pneumonia was also higher (16.8 vs 1.1 per 1,000 ventilator days) as was the rate of catheter-associated urinary tract infection (5.5 vs 1.3 per 1,000 catheter days). Frequencies of resistance of Pseudomonas isolates to amikacin (42.8% vs 10%) and imipenem (42.4% vs 26.1%) and Klebsiella pneumoniae isolates to ceftazidime (71.2% vs 28.8%) and imipenem (19.6% vs 12.8%) were also higher in the INICC's ICUs compared with the ICUs of the CDC's NHSN