Protein:DNA Interactions—High-Throughput Analysis

Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.

The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Promega). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.

Literature Cited

  1. Ren, B. et al. (2007) Genome-wide mapping of in vivo protein-DNA binding proteins. Science 316, 1497–502.
  2. Garner, M.M. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nuc. Acids. Res. 9, 3047-60.
  3. Glick,Y et al. (2016) Integrated microfluidic approach for quantitative high throughput measurements of transcription factor binding affinities. Nuc. Acid Res. 44, e51.

A “Spare Tire” for Proto-oncogene Promoters

A guanine tetrad (left) and G-quadruplex (right). Image courtesy of Wikimedia Commons.

A guanine tetrad (left) and G-quadruplex (right). Image courtesy of Wikimedia Commons.

Proto-oncogenes are genes that organisms rely on for normal growth and development but, when mutated or dysregulated, can cause cells to grow uncontrollably, resulting in cancer and metastasis. In some cases, a single DNA mutation is sufficient for cancer to develop. Why then, do so many proto-oncogene promoters contain strings of guanine residues, which are extremely vulnerable to DNA damage from factors such as oxidative stress and hyperinflammation, to control transcription levels? From an evolutionary viewpoint, this is a contradiction: DNA sequences that are the most vulnerable to damage and mutation are key to regulating one of the cell’s most dangerous classes of genes. This seems to be a recipe for genomic instability and disease. Fortunately, evolution has provided a very clever solution to this potential problem.

Continue reading

How MicroRNAs Have a Big Effect on Genetic Regulation

miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

Some of us scientists who have been around for a while still think about RNA molecules falling into three categories: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). However, within the past few decades I have had to revise my outdated RNA classification scheme as scientists discover exciting new classes of RNAs that do some fairly amazing things. For example, in the early 1980s, Thomas Cech discovered ribozymes, RNAs that have catalytic functions (1), and in the early 1990s, researchers began to take interest in short noncoding RNAs that act as a genetic regulators, the first of which was discovered in C. elegans (2). RNA is no longer simply a biological middleman between DNA and protein. These ephemeral nucleic acid molecules play a much bigger role of cellular physiology and gene regulation than we had previously ascribed to RNA.

Continue reading

Tools for Understanding miRNA Regulation of mRNA Expression: The Vector

7825ma_450pxmicroRNAs  (miRNA) are abundant RNA molecules around 21 nucleotides long that regulate specific mRNA expression by directly interacting with the mRNA molecule. Our understanding of miRNA function in mRNA regulation has grown exponentially as more miRNA molecules have been described. As of 2013, more than 24,000 miRNA molecules had been described from more than 140 separate species, indicating that miRNA regulation is conserved across species. In humans, 2,500 mature miRNAs have been described, and researchers predict that 60% of human protein-coding genes may be targets of miRNA regulation. Most often miRNA regulation of an mRNA results in decreased expression, either by destabilizing the mRNA or by inducing translational repression. Very recently, some researchers have reported up regulation of mRNA through miRNA activity.

Since miRNA molecules are so abundant within cells and across species and their target sequences are found in so many protein-coding genes, understanding how miRNA regulation of mRNAs acts in concert with the many other levels of gene expression regulation becomes a complex, but fundamental, biological question.

To probe miRNA regulation of mRNA, the proper tools and experimental design are essential. Continue reading

Epigenetics and Exercise

Turning on some genes

Turning on some genes

If, like me, you sometimes need more motivation to exercise consistently—even though you know that it is good for you—you may be interested in the findings of a paper published recently in PLOS Genetics. The paper showed that consistent exercise over a 6-month period caused potentially beneficial changes in gene expression. In short, regular exercise caused expression of some “good” genes, and repression of “bad” ones, and these changes appeared to be controlled by epigenetic mechanisms.

Epigenetic changes are modifications to DNA that affect gene expression but don’t alter the underlying sequence. Perhaps the best understood example of an epigenetic change is DNA methylation—where methyl groups bind to the DNA at specific sites and alter expression, often by preventing transcription. Epigenetic changes have been shown to occur throughout all stages of development and in response to environmental factors such as diet, toxin exposure, or stress. The study of epigenetics is revealing more and more about how the information stored in our DNA is expressed in different tissues at different times and under different environmental circumstances. Continue reading

Basic Biology Matters

crop image2Every scientific paper is the story of a journey from an initial hypothesis to a final conclusion. It may take months or years and consists of many steps taken carefully one at a time. The experiments are repeated, the controls verified, the negative and positive results analyzed until the story finally makes sense. Sometimes the end of the story confirms the hypothesis, sometimes it is a surprise. A paper published last week in Cell describes a study where a team of researchers investigating one problem in basic biology (how one component of a signaling complex works), found an unexpected and potentially significant application in a different field (cancer research).

The paper, published in the June 6 issue of Cell, describes a previously unknown interaction between two cellular proteins—the transcription factor HIF1A and the cyclin-dependent kinase CDK8—in the regulation of genes associated with cellular survival under low-oxygen conditions. An accompanying press release describes how the discovery of a role for CDK8 in this process may have implications for cancer research, as CDK8 may be a potential target for drugs to combat “hypoxic” tumors. Continue reading

Another Step Closer to Understanding Epigenetic Gene Regulation

Chromatin fiberBack when I was a graduate student (more than a few years ago), I remember hearing another student joke that if a member of his thesis committee asked him to explain an unexpected or unusual result, he was going to “blame” epigenetics. At that time, the study of epigenetic gene regulation was in its infancy, and scientists had much to learn about this mysterious regulatory process. Fast forward to today, and you’ll find that scientists know a lot more about basic epigenetic mechanisms, although there is still plenty to learn as scientists discover that the topic is much more complicated than initially thought, as is often the case in science. A recent EMBO Journal article is contributing to our knowledge by shedding light on the role of the TET family of DNA-modifying enzymes in epigenetics (1).

Continue reading

It’s a Jungle in There: ENCODE and the Redefinition of the Gene

September 5th saw the simultaneous publication of more than 30 papers in Nature, Genome Research and Genome Biology detailing the findings of the ENCODE project (Encyclopedia of DNA Elements)—an international collaborative research effort involving the work of more than 400 scientists in 32 groups over the last eight years. Building on the work of the human genome project, the goal of the ENCODE project was to catalog and describe all the functional elements in the human genome.

The human genome project revealed the surprising fact that only 1% of our genome encodes proteins–a mere 20,000 genes. The function of the other 99% of our DNA remained a mystery. The ENCODE project was established in 2003 to survey and catalog these unknown DNA regions in a systematic manner. Continue reading

Capture Intracellular Protein:DNA Complexes without Antibodies

2 days to results compared to 5 days with standard CHiP

2 days to results compared to 5 days with standard CHiP

Regulation of chromatin structure and gene expression is essential for normal development and cellular growth, and gene transcription involves tightly controlled  protein:DNA interactions. Currently there is a rapidly growing trend towards genome-wide identification of protein binding sites on chromatin and characterization of the transcriptome using the chromatin immunoprecipitation (ChIP) method coupled with either DNA microarray or ultrahigh-throughput sequencing analysis. While significant advances and the development of new technology in the fields of DNA microarrays and sequencing have allowed genomic-level characterization of protein binding sites, the upstream ChIP process for the capture of DNA fragments remains cumbersome and time consuming.

The HaloCHIP™ System uses a protein tag, the HaloTag® moiety, which can be fused to any protein and which mediates a covalent interaction with various interchangeable ligands. In the HaloCHIP™ System, DNA binding proteins fused to the HaloTag® moiety can be transiently or stably expressed in cells. Similar to the ChIP protocol, cells are treated with formaldehyde, generating covalent protein:DNA crosslinks, lysed, and sonicated to shear chromatin into 500–1500 bp fragments. Unique to the HaloCHIP™ System, the crosslinked complexes are then directly and covalently captured from cell lysates on HaloLink™ Resin and washed stringently to remove nonspecific proteins and DNA. Captured DNA fragments are released from the HaloLink™ Resin using heat, which reverses the formaldehyde crosslinks. The DNA can be purified using a PCR purification kit and analyzed by the researcher’s preferred method.

The entire process—from crosslinking to DNA release—can be accomplished in 24 hours using the HaloCHIP™ System, compared to five days for standard ChiP.

A paper by Hartzell et al. analyzes the CREB signaling pathway by using the HaloCHIP™ System to map the genome-wide targets of the CREB protein:

Hartzell, D. et al. (2009) A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high-throughput reporter assays. BMC Genomics 10, 497.

And two publications have used the HaloCHIP™ System to identify transcriptional regulators:

Du, X., Rosenfield, R.L, and Qin, K. (2009) KLF15 is a transcriptional regulator of human 17 beta-Hydroxysteroid Dehydrogenase Type 5 Gene. A potential link between regulation of testosterone production and fat stores in women. J. Clin. Endocrinol. Metab. 94, 2594–601.

Jag, U.R., Zavadil, J., and Stanley, F.M. (2009) Insulin acts through FOX03a to activate transcription of plasminogen activator inhibitor type 1. Molecular Endocrinology 23, 1587–602.

The Ongoing Legacy of the Human Genome Sequence

When the first draft sequence of the human genome was announced, I was a research assistant for a lab that was part of the Genome Center of Wisconsin where I created shotgun libraries of bacterial genomes for sequencing. Of course, the local news organizations were all abuzz with the news and sought opinions on what this meant for the future, including that of the lab’s PI and oddly enough, my own. While I do not recall the exact words I offered on camera, I believe they were something along the lines of this is only the first step toward the future of human genetics. Ten years later, we have not fulfilled the potential of the grandiose words used to report the first draft sequence but have gained enough knowledge of what our genome holds to only intrigue scientists even more.

Continue reading