Robotic Liquid Handling and Automation in Epigenetics

Abstract

Automated liquid-handling robots and high-throughput screening (HTS) are widely used in the pharmaceutical industry for the screening of large compound libraries, small molecules for activity against disease-relevant target pathways, or proteins. HTS robots capable of low-volume dispensing reduce assay setup times and provide highly accurate and reproducible dispensing, minimizing variation between sample replicates and eliminating the potential for manual error. Low-volume automated nanoliter dispensers ensure accuracy of pipetting within volume ranges that are difficult to achieve manually. In addition, they have the ability to potentially expand the range of screening conditions from often limited amounts of valuable sample, as well as reduce the usage of expensive reagents. The ability to accurately dispense lower volumes provides the potential to achieve a greater amount of information than could be otherwise achieved using manual dispensing technology. With the emergence of the field of epigenetics, an increasing number of drug discovery companies are beginning to screen compound libraries against a range of epigenetic targets. This review discusses the potential for the use of low-volume liquid handling robots, for molecular biological applications such as quantitative PCR and epigenetics.

Epigenetics, the study of the regulation of gene expression, is one of the most rapidly expanding and dynamic areas in biological research. Regulation of gene expression is essential in embryology, cell growth, homeostasis, and aging. Replication, recombination, transcription, repair, chromosomal stability, gene silencing, and cell cycle progression are all regulated by epigenetic events.

Although there is no change in the nucleotide sequence of the DNA, epigenetic control of gene expression is inherited. Epigenetic changes can be influenced by both internal and environmental factors. They can exert control at numerous stages of the cell’s life and within different tissues throughout development and aging.

A breakdown in the control of gene expression can result in overexpression or nonexpression of a gene and its corresponding product, which in turn may trigger a cascade of events leading to the breakdown of homeostasis within the body. Dysregulation of epigenetic controls has been linked to a number of diseases, including cancer, inflammatory disorders, neurodegenerative autoimmune and metabolic diseases. An understanding of the events that occur in the triggering of disease states will enable the identification of potential therapeutic strategies.

DNA is closely associated with histones in a structure termed chromatin. Chemical modification of key residues within specific histone proteins by acetylation, deacetylation, methylation, phosphorylation, or ubiquitation alters the intermolecular binding properties between the histone protein and DNA, affecting transcriptional activation of the gene. ¹

A number of enzyme classes have been identified that exert changes on histone proteins, including histone deacetylases (HDAC and sirtuins), histone methyltransferases (HKMT and HRMT), histone demethylases (HDM), DNA methyltransferases (DNMT), and histone acetyltransferases (HAT). These enzymes have been demonstrated to be involved in the chemical modification of histone/chromatin structure, ^2,3 and a range of assays have been developed to help gain further understanding of how these enzymes affect gene expression.

Epigenetic control can also be mediated directly by the methylation status of DNA. Increased methylation at the CpG promoter site of a gene inhibits expression of the encoded protein. ⁴ In addition, small noncoding RNA (small regulatory or micro-RNAs) can affect gene expression. Overexpression of these RNAs can result in downstream alterations in methyltransferases, affecting chromatin structure, regulation of DNA methylation, and reactivation of several genes via promoter DNA hypomethylation. ⁵

Epigenetic research has largely been dominated by the study of DNA methylation and histone modification. Two techniques have been commonly employed in academic studies. One method, termed the bisulfite treatment technique, ⁶ determines the degree of methylation of cysteine residues in CpG promoter regions of DNA. Treatment of DNA with bisulfite results in the conversion of unmethylated cysteine residues to uracil. The degree and location of methylated cysteine residues at CpG promoter regions can then be determined by PCR techniques.

The second technique, chromatin immunoprecipitation (ChIP), studies the DNA binding sites on the genome for a specific protein by analyzing DNA binding proteins associated with DNA. ⁷ ChIP analysis involves immunoprecipitation with an antibody that is specific to the putative DNA-binding protein, resulting in the isolation of the protein–DNA complex. The purified protein–DNA complex is then heated to reverse the formaldehyde cross-linking of the protein and DNA complex, allowing the DNA to be separated from the protein. The identity and quantity of the DNA fragments isolated can then be determined by PCR.

Both ChIP analysis and the bisulfite method can be used effectively to study the effect of modulators on gene expression controlled by DNA methylation and histone protein–DNA interaction, but limitations of reagents and detection tools, such as antibodies, have until recently hindered epigenetic research. Recent efforts have focused on the development of antibody-based assays, biochemical assays, and recombinant histones, coupled with existing or new readout technologies such as fluorescent readout, enzyme-linked immunosorbent assays (ELISAs), time-resolved fluorescence resonance energy transfer (TR-FRET [HTRF]), or mass spectrometry. ⁸ These studies have resulted in many companies offering a range of reagents and technologies to further the understanding of epigenetic control as well as to screen the effect of peptides and other modulatory compounds on identified epigenetic targets.

Development of High-Throughput Screening Assays and Compound Libraries

Identification and characterization of novel epigenetic enzymes and targets enable further understanding for the development of compounds and peptides that modulate their activity. Many of the assays and techniques used in these studies, such as PCR and ELISA, have already been successfully incorporated into the highly automated research environment of today’s pharmaceutical industry. Examples of assays include HDAC-Glo or SIRT-Glo assays (Promega, Madison, WI), which assess the relative activity of histone deacetylase or sirtuins, or the LANCE and AlphaScreen assays (PerkinElmer, Waltham, MA), which employ proximity assay technology to detect the methylation or acteylation states of peptide histone proteins. More recently, Larsen ⁹ demonstrated a simple automated solution for performing HDAC selectivity profiling using an automated pipetting station and a Lys Green assay for a wide range of HDAC enzymes.

Liquid-handling robots for pipetting reagents and samples are routinely used for the setup of a range of assays in research laboratories, including biochemical assays, antibody-based assays, molecular assays, and crystallography studies to determine enzyme structure and function. These robots offer the reproducibility and accuracy of low-volume dispensing, which is difficult to achieve using manual dispensing methods. Indeed, successful PCR results rely heavily on the accuracy and reproducibility of pipetting, requiring considerable skill and practice. Even small errors in the dispense accuracy of sample DNA or RNA can translate into huge differences after amplification. Furthermore, large sample numbers in screening assays make manual setup time-consuming, error prone, and tedious.

Automated liquid-handling instruments, such as the mosquito HTS and HV (TTP LabTech, Hertfordshire, UK) and the Freedom EVO (Tecan, Männedorf, Switzerland), use multichannel pipettes for the serial dilution and plate preparation stages of assay setup and are able to dispense into 96-, 384-, or 1536-well plates, whereas robots such as the mosquito X1 (TTP LabTech) or the Echo 555 (Labcyte, Sunnyvale, CA) can operate using only one or two channels and are suitable for nucleic acid extraction or PCR reaction preparation. The ability of these instruments to accurately, rapidly, and reproducibly pipette volumes under the 100-nL volume range minimizes the amount of reagents, saving valuable sample and ensuring low-cost assays. Employment of robotic liquid handlers enables high throughput with multiple analyses performed rapidly and accurately. Liquid handlers may be automatically coupled with accessories such as barcode readers and, following assay setup, linked via robot arms to automated equipment for assay processing and readout.

More recently, the development of second-generation or high-throughput sequencing technologies has involved the introduction of dedicated automated instruments for epigenetic and genetic research. The VERSA Mini PCR Setup workstation (Aurora Biomed, Vancouver, BC, Canada) performs the automated setup of all stages of PCR reaction setup and liquid handling. The IP-Star (Diagnode) performs the liquid-handling stages of immunoprecipitation for ChIP analysis. Instruments such as these speed up the transition from arrays to sequencing and reduce the cost and tedium of analysis, enabling effective and rapid screen setup of peptides and compound libraries for the discovery of therapeutic agents.

Although the science of epigenetics is still very much in its infancy, there is a move toward high-throughput screening for modulators of epigenetic targets by the pharmaceutical industry. Compound screening for modulators of epigenetic target enzymes and DNA activity has been limited by the lack of available compound libraries, with availability of these libraries being at the stage of research similar to that seen with the protein kinase class of drug targets 20 years ago.

Although some drugs have already been successfully introduced for the treatment of some leukemias and lymphoma, ^10,11 further studies elucidating enzyme binding sites and interactions by protein crystallography and mutational analysis need to be carried out to help design more extensive compound libraries for screening against these targets. Companies such as BioFocus (Saffron Walden, UK), which routinely uses liquid handlers and robotic technology for plate transfer and assays, offer a compound library focused on these enzyme targets.

In conclusion, the development of a wide range of epigenetic assays and the tools required to support epigenetic screening are fast emerging as our knowledge and experience with these targets increase. The study of epigenetics involves a large number of existing assays and techniques that have been incorporated successfully into the highly automated research environment of the pharmaceutical industry today. Although a field still in its infancy, robots are widely used in a number of research laboratories to identify and characterize epigenetic targets, enabling the development of compound libraries for effective screening for therapeutic agents to treat a wide range of diseases.