Keith R. Yamamoto

Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids

Airway inflammation and epithelial remodeling are two key features of asthma. IL-13 and other cytokines produced during T helper type 2 cell-driven allergic inflammation contribute to airway epithelial goblet cell metaplasia and may alter epithelial–mesenchymal signaling, leading to increased subepithelial fibrosis or hyperplasia of smooth muscle. The beneficial effects of corticosteroids in asthma could relate to their ability to directly or indirectly decrease epithelial cell activation by inflammatory cells and cytokines. To identify markers of epithelial cell dysfunction and the effects of corticosteroids on epithelial cells in asthma, we studied airway epithelial cells collected from asthmatic subjects enrolled in a randomized controlled trial of inhaled corticosteroids, from healthy subjects and from smokers (disease control). By using gene expression microarrays, we found that chloride channel, calcium-activated, family member 1 (CLCA1), periostin, and serine peptidase inhibitor, clade B (ovalbumin), member 2 (serpinB2) were up-regulated in asthma but not in smokers. Corticosteroid treatment down-regulated expression of these three genes and markedly up-regulated expression of FK506-binding protein 51 (FKBP51). Whereas high baseline expression of CLCA1, periostin, and serpinB2 was associated with a good clinical response to corticosteroids, high expression of FKBP51 was associated with a poor response. By using airway epithelial cells in culture, we found that IL-13 increased expression of CLCA1, periostin, and serpinB2, an effect that was suppressed by corticosteroids. Corticosteroids also induced expression of FKBP51. Taken together, our findings show that airway epithelial cells in asthma have a distinct activation profile and identify direct and cell-autonomous effects of corticosteroid treatment on airway epithelial cells that relate to treatment responses and can now be the focus of specific mechanistic studies.

DNA binding site sequence directs glucocorticoid receptor structure and activity.

Genes are not simply turned on or off, but instead their expression is fine-tuned to meet the needs of a cell. How genes are modulated so precisely is not well understood. The glucocorticoid receptor (GR) regulates target genes by associating with specific DNA binding sites, the sequences of which differ between genes. Traditionally, these binding sites have been viewed only as docking sites. Using structural, biochemical, and cell-based assays, we show that GR binding sequences, differing by as little as a single base pair, differentially affect GR conformation and regulatory activity. We therefore propose that DNA is a sequence-specific allosteric ligand of GR that tailors the activity of the receptor toward specific target genes.

Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region

Glucocorticoid receptor protein stimulates transcription initiation within murine mammary tumor virus (MTV) DNA sequences in vivo, and interacts selectively with MTV DNA in vitro. We mapped and compared five regions of MTV DNA that are bound specifically by purified receptor; one resides upstream of the transcription start site, and the others are distributed within transcribed sequences between 4 and 8 kb from the initiation site. Each region contains at least two strong binding sites for receptor, which itself appears to be a tetramer of 94,000 dalton hormone-binding subunits. Three of the five binding regions contain nine nuclease footprints that lack extensive homology, although a family of related octanucleotides can be discerned. Receptor interacts with the different regions with similar efficiencies, suggesting that receptor affinity for upstream and internal regions may differ by less than one order of magnitude. Moreover, each region appears to be bound independent of the others. A restriction fragment containing four footprint sequences from one of the regions has previously been shown to act in vivo as a receptor-dependent transcriptional enhancer element, implying that the binding sites detected in vitro may be biologically functional.

Allosteric effects of DNA on transcriptional regulators

Selective gene transcription is mediated in part by regulatory proteins that bind to DNA response elements. These regulatory proteins receive global information from signal-transduction events. But transcriptional regulators may also be modified in an allosteric manner by response elements themselves to generate the pattern of regulation that is appropriate to an individual gene.

A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor

The glucocorticoid receptor is a signal transducer that interacts both with the signal and with the genes it regulates. We showed previously that nuclear localization of the receptor requires hormone binding. We have now constructed recombinant receptors that relieve hormonal control of nuclear localization, and we demonstrate that the DNA binding/transcriptional regulatory functions of the receptor are also regulated directly by hormone. Surprisingly, regulation by the steroid binding domain appears to be relatively independent of protein structure. For example, regulation is maintained when the steroid binding region is repositioned from the C-terminus to the N-terminus of the receptor. Furthermore, the activity of an unrelated protein, the adenovirus E1A gene product, becomes hormone regulated upon fusion to the steroid binding domain. We speculate that the inhibitory effect of the unliganded steroid binding domain may be mediated by heat shock protein hsp90, which binds selectively to the unliganded receptor.

Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor.

Cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) phosphorylate the rat glucocorticoid receptor in vitro at distinct sites that together correspond to the major phosphorylated receptor residues observed in vivo; MAPK phosphorylates receptor residues threonine 171 and serine 246, whereas multiple CDK complexes modify serines 224 and 232. Mutations in these kinases have opposite effects on receptor transcriptional activity in vivo. Receptor-dependent transcriptional enhancement is reduced in yeast strains deficient in the catalytic (p34CDC28) or certain regulatory (cyclin) subunits of CDK complexes and is increased in a strain devoid of the mammalian MAPK homologs FUS3 and KSS1. These findings indicate that the glucocorticoid receptor is a target for multiple kinases in vivo, which either positively or negatively regulate receptor transcriptional enhancement. The control of receptor transcriptional activity via phosphorylation provides an increased array of regulatory inputs that, in addition to steroid hormones, can influence receptor function.

A prudent path forward for genomic engineering and germline gene modification

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed Genome engineering technology offers unparalleled potential for modifying human and nonhuman genomes. In humans, it holds the promise of curing genetic disease, while in other organisms it provides methods to reshape the biosphere for the benefit of the environment and human societies. However, with such enormous opportunities come unknown risks to human health and well-being. In January, a group of interested stakeholders met in Napa, California (1), to discuss the scientific, medical, legal, and ethical implications of these new prospects for genome biology. The goal was to initiate an informed discussion of the uses of genome engineering technology, and to identify those areas where action is essential to prepare for future developments. The meeting identified immediate steps to take toward ensuring that the application of genome engineering technology is performed safely and ethically.

Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans.

Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid β-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.

Regulatory crosstalk at composite response elements

Transcriptional regulatory factors from different families interact with each other when bound to DNA at composite response elements. This level of communication has two striking consequences: ubiquitous factors can effect cell specificity, and closely related factors from a given family can produce very different regulatory patterns.

Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation.