by Caitlin Smith
The power of nuclear receptors should not be underestimated. We have yet to understand fully how these fascinating molecules work—though we know they are crucial for many important developmental processes, as well as in major human diseases such as cancer. Nuclear receptors are many things at once: they are environmental detectors that sense levels of certain hormones or other chemicals near the cells in which they are expressed, binding them as ligands; they are mediators of this detection by conveying the message of ligand binding to the nucleus of the cell; and they are effectors when the ligand-bound, activated receptors bind to DNA at specific sites within the genome.
Even with their potential to effect profound change, many nuclear receptors are still not well-characterized. Some remain “orphan receptors,” meaning that their natural ligands are yet to be found. Others participate in complex signaling interactions that are the subject of intense investigation. Here is a look at some recent research and technology in the area of nuclear receptors.
Nuclear receptors and deep sequencing
Transcription is the main event for nuclear receptors and the scientists studying them, according to Neil McKenna, assistant professor in the department of molecular and cellular biology at Baylor College of Medicine. “Nuclear receptor and coregulator biologists are, fundamentally, interested in transcription, and all the events in the cell that combine to enable nuclear receptors to regulate their target genes in highly specific spatiotemporal contexts,” he says. “The most powerful new technique in the field is deep sequencing. It has the ability to rapidly and efficiently interrogate the nuclear-receptor-regulated transcriptome of any given cell in a quantitative manner—for assaying fluctuations in the levels of multiple transcripts at once—and [a] qualitative [manner], allowing researchers to distinguish between different variants of a given transcript.”
Deep sequencing can produce more gene hits and reveal rare or novel transcripts. The drawback is that the read lengths are short, but most are long enough to map to unique locations. “When combined with chromatin immunoprecipitation,” says McKenna, “[deep sequencing] can yield accurate information about where in the genome of a given cell a nuclear receptor binds DNA (its cistrome), which can then be correlated with expression array data to give clues as to where and when nuclear receptors regulate transcription, both directly and indirectly.” McKenna’s group is active in developing software for bench researchers to integrate transcriptomic datasets from expression arrays, cistromic datasets from genome-wide location analyses, and proteomic datasets from immunoprecipitation and mass spectrometry. “The challenge is to equip researchers with the software tools that will allow them to pose questions of these data and permit them to test hypotheses and assemble models in silico, leading to substantial savings by avoiding unnecessary and repetitive bench experiments, and to significant increases in the rate of knowledge advancement.”
McKenna says that managing data will become increasingly important in the coming years. “Over the next five years, the amount of data generated in the field will most likely match the research output of the last 25 years in terms of the number of data points,” he says. “Moreover, it will be highly accurate and highly contextual data. If correctly managed, the data will allow for the rapid development of translational solutions to those pathologies in which nuclear receptors and their ligands are intimately involved, both as causative agents and therapeutics. These diseases include some of the most pressing public health concerns of modern times, including cancer, diabetes, obesity, cardiovascular disease, senescent diseases and inflammatory pathologies such as asthma.”
Nuclear receptors and disease
Nuclear receptors are known to play a role in many types of cancer, as well as other diseases. For example, prostate cancer has been linked to androgen receptors, bone cancer to the nor-1 receptor, and colon cancer to PPARgamma. Many types of breast cancer are related to elevated activity levels of estrogen receptors; these can be treated with drugs (called selective estrogen receptor modulators) that compete with endogenous estrogen for binding access to receptors. The drugs bind the receptors but do not activate transcription.
Other types of immune and inflammatory diseases are also mediated by nuclear receptors. For research in this area, Bio-Rad’s Bio-Plex® Pro™ magnetic cytokine assays are designed for high-performance precision in measuring immune system modulators or inflammatory cytokines. Their magnetic bead-based assays coupled with their new Bio-Plex® Pro wash stations can increase your throughput and reproducibility as well. Mencarelli et al. 20091 used the Bio Plex® cytokine assays to measure plasma cytokine concentrations in studying activation of the farnesoid X receptor, a nuclear receptor that is also a bile sensor, highly expressed in the liver, in a model of autoimmune hepatitis. Gene ablation for the farnesoid X receptor resulted in an increase in the expression of the immunoregulatory cytokine osteopontin in the liver.
Bio-Rad’s Bio-Plex® cytokine assay kit was also used by Sonoda et al. 20072 to measure serum cytokine levels on a Bio-Plex suspension array system. Studying the activation pathway induced by the proinflammatory cytokine interferon-gamma (IFN-G), they showed that IFN-G activates the nuclear receptor ERRalpha (estrogen-related receptor alpha, NR3B1). ERRalpha, together with its coactivator PGC-1beta, work to activate mitochondrial ROS (reactive oxygen species) production, in a host defense against bacterial pathogenesis in macrophages.
Invitrogen also offers assay kits and other tools for nuclear receptor research, such as purified steroid hormone receptors, nuclear receptor antibodies, and immunoassays. Their two biochemical nuclear receptor activity assays, PolarScreen™ NR Competitive Binding Assays and LanthaScreen™ NR Coregulator Interaction Assays, feature pre-configured fluorescent solutions for high-throughput screening, hit confirmation, and lead optimization for nuclear receptor targets. In addition, their cell-based GeneBLAzer® Validated Assays can measure receptor activation or inhibition to determine compound potency and selectivity.
Nuclear receptors and epigenetics
Nuclear proteins play an important role in epigenetic processes, whereby an organism develops heritable changes that don’t involve the basic structure of the DNA, or a change in the DNA sequence—for example, heritable changes in the ability of a gene to be activated or silenced. DNA methylation is a commonly studied example of an epigenetic mechanism, often occurring at a site on the DNA where a cytosine and guanine are next to one another (known as a CpG site, with the “p” indicating a phosphate group). Areas of DNA where clusters of methylated CpG sites occur together are known as CpG islands. “Emerging evidence of epigenetic regulation of the steroid hormone synthesis and gene expression provides us a new angle to understand the hormone action pathways,” says James Wang, a scientist in stem cell product development at Novus Biologicals. “Epigenetics provides an additional layer of gene regulation through DNA methylation and histone tail modifications. Further identification of genes involved in this regulatory process and characterization of their specificities and responses to the environment can further strengthen our knowledge [of] endocrinological regulation, and facilitate new drug discovery.” Indeed, studies suggest that methylated CpG sites within a gene’s promoter region can lead to its silencing, while hypomethylation of CpG sites can lead to the overexpression of oncogenes in cancer cells3.
Novus Biologicals supplies a wide range of antibodies, as well as an extensive library of lysates, proteins, and siRNA products for reagents in nuclear receptor experiments. “For future studies, it will be important to develop highly specific reagents capable of identifying and monitoring epigenetic events both in vitro and in vivo,” says Wang. “Improved technology to study genome-wide methylation with single CpG resolution is equally important to understand the dynamic nature of the epigenome.” And this would bring us one step closer to understanding nuclear receptors.
References:
1A Mencarelli, et al. "The bile acid sensor farnesoid X receptor is a modulator of liver immunity in a rodent model of acute hepatitis." J Immunol 183: 6657-6666, 2009.
2J Sonoda, et al. "Nuclear receptor ERRá and coactivator PGC-1â are effectors of IFN-ã-induced host defense." Genes & Dev 21: 1909-1920, 2007.
3J Tost, "DNA methylation: an introduction to the biology and the disease-associated changes of a promising biomarker." Mol Biotechnol. 44: 71-81, 2010.