Understanding biological networks as dynamic, beyond static sequences and protein types, has initiated investigations into regulatory processes, transcription factors, binding patterns, and chromatin structure. These protein-DNA interactions are increasingly considered significant to what keeps us healthy, how diseases progress, and what environmental changes occur over a lifetime.
New methods for studying these interactions have opened up the door to a constantly changing world of integrated systems where much more of the human genome is transcribed and contains more regulatory information than was previously thought. By deciphering which factors work together to regulate which genes, we can analyze the role of epigenetics and regulatory sequences in metabolism, cancer, and other processes.
The forefront of genetics research
As investigations into fixed characteristics in the genome, such as mutations or SNPs, have become routine, biological research has been shifting focus to other more functional areas, specifically how proteins and DNA interact to regulate the genome. Several analytical methods have been developed to assess protein-DNA interactions, including chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), yeast one-hybrid (Y1H) assays, and DNase footprinting assays. Many of these have become key technologies utilized by large consortiums such as ENCODE or The Cancer Genome Atlas to examine the mechanisms responsible for genome regulation on a larger scale.
“DNA-protein interactions are paramount to cellular physiology,” comments Bhaskar Vadla, Ph.D., senior study manager in next-generation sequencing at GENEWIZ. “ChIP followed by next-generation sequencing (ChIP-Seq) has proven to be a very useful technique in answering biological questions related to transcriptional regulation: For instance, identification of genome-wide transcriptional factor DNA-binding sites in an unbiased manner and epigenetic transcriptional regulation via histone modifications.”
Michael Snyder, Ph.D., professor and chair of the department of genetics at Stanford University and director of the Stanford Center for Genomics and Personalized Medicine, participates in ENCODE, mapping regulatory regions of the human genome. His lab uses ChIP-Seq to study the fundamental principles of regulatory networks. In ChIP-Seq assays, a transcription factor, cofactor, or other chromatin protein of interest is enriched by immunoprecipitation from crosslinked cells, along with its associated DNA, then the DNA is isolated and sequenced. Snyder views ChIP-Seq as an important technique because of its utility for regulatory network inference and for diverse integrative analyses such as the effects of genetic variation on human traits and disease.
Research like Snyder’s is bringing epigenetics and associated methods to the forefront of genetics investigations. “Many fields focus on finding rare variants or polymorphisms in the genome or organ of interest, something encoded in the DNA sequence. However, more and more researchers are turning to epigenetics to look at differences in the way genomes are regulated rather than static mutations. Epigenetics and ChIP are at the core of that,” explains Adam Blattler, Ph.D., research scientist at Active Motif.
While ChIP-Seq is becoming a widely accepted method to identify binding sites of proteins associated with DNA…there is still quite a ways to go.
While ChIP-Seq is becoming a widely accepted method to identify binding sites of proteins associated with DNA, Epigentek CSO, Adam Li, M.D., Ph.D., notes that there is still quite a ways to go “as the complex nature of the bioinformatics is still somewhat of an art. Scientific community consensus on acceptable standards of data interpretation needs to continue to develop for ChIP-Seq to mature.”
Li emphasizes that bioinformatics analysis can still be a huge barrier of entry both financially and technically for most researchers interested in a ChIP-Seq approach. FASTQ analysis of sequencing data is still most commonly based on standard protocols such as Bowtie or MACS for peak calling, and Bioconductor packages and ChIPpeakAnno for differential binding analysis. While these programs are time tested, robust, and the open-source code makes them trustworthy, they are not very user friendly and require a good grasp of code. Improved commercial software has been coming to market but Li believes that a balance between usability and trust will be important for better adoption of these programs.
Key innovations can make a difference
As ChIP-Seq expands into labs seeking new approaches for regulatory studies, companies are striving to support research with innovative technologies. “ChIP and ChIP-Seq are applicable to so many disease types and other biology. We are seeing more advances in drug discovery as organizations target these genetic mechanisms through ChIP. By making ChIP and thus epigenetics more approachable with easier and more high-throughput methods, we are able to support this growth. This includes initial measurements of global changes in the epigenome using, for example, our histone H3 ELISAs for pre-ChIP analysis,” says Blattler.
Active Motif has partnered with Swift BioSciences to couple their ChIP technology with Swift’s library prep kit, aimed specifically to solve the issue of limited starting material and improve assay sensitivity. When working with fewer cells, starting material amount is precious. Using a Low Cell ChIP-Seq kit, along with unique molecular identifiers to increase the number of distinct alignments, researchers can extract more information from and do more with each sample.
“ChIP-Seq typically requires millions of cells,” adds Vadla. “However, recent advances in the field enable development of protocols that use a very limited number of cells, on the order of thousands.” As innovations enable lesser sample amounts to be used as input, these methods could aid in the assessment of the epigenetic landscape from rare cell populations, such as those in stem cell differentiation or tumor development. GENEWIZ assists clients with ChIP-Seq projects by offering a range of workflows including library preparation of immunoprecipitated DNA, sequencing, and bioinformatics analysis, applying these new technologies to get the most out of their data.
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Another innovation employs Tn5 transposase conjugated to an antibody that can direct the transposase to the sites of a specific protein of interest, allowing IP of the regions of interest and tagmentation for sequencing simultaneously. TAM-ChIP allows ChIP and next-generation sequencing library prep to occur in one step, speeding preparation and expanding use of limited material. This shortcut provides higher resolution data in the form of narrower peaks, as Tn5 can cut right against the protein of interest. Active Motif’s TAM-ChIP technology offers a highly robust procedure validated to profile both histone and transcription factor DNA binding sites.
Image: In ChIP-IT High Sensitivity, intact cells are fixed with a specially formulated formaldehyde buffer, which cross-links and preserves protein/DNA interactions. DNA is then sheared into fragments using sonication and incubated with an antibody directed against the DNA-binding protein of interest. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase K, and the DNA is recovered and purified. ChIP-enriched DNA can be used for either gene-specific or whole-genome analysis. Image courtesy of Active Motif.
In addition to expanding their ChIP-grade antibody collection to ensure proper immunoprecipitation and accurate results, Epigentek also launched a circulating modified histone multiplex ELISA that can help screen for histone modifications cost-effectively prior to any potential ChIP-Seq work. Histone post-translational modifications can tell a lot about how a genome is regulated, especially when examining several together. They are also currently looking into their own development of microplate-based transposase-assisted ChIP-Seq for high sensitivity for as low as 1,000 cells.
ChIP-Seq for the future
The future for ChIP-Seq is bright, with a widely expanding base of assays and applications as well as growing interest in what the technology can accomplish. Combining approaches can deliver more from samples and yield deeper insights into questions asked. As Li suggests, ChIP-Seq data could be integrated into other functional genomics assays, such as prediction of regulatory sequence variants by integrative analysis with ChIP-Seq combined with the study of functional genomic status in a single cell.
“Most of the ChIP-Seq data that is available thus far is derived from populations of cells, which tend to be heterogeneous,” Vadla adds. “The single-cell Chip-Seq workflow could be an invaluable tool in understanding chromatin status at the individual cell level. This approach coupled with orthogonal single-cell transcriptomics data may provide an unprecedented survey of transcriptional regulation and help identify novel cell populations.”
With this widening reach comes progressions into other areas such as off-target effects, an important assessment when investigating new drug candidates. When considering a specific histone methyltransferase and its associated methyl lysine profiles, Blattler offers, a company can also examine how other acetyl groups are changing the genome, including other promoters or enhancers that can be used to fine-tune the targeted system with epigenetic inhibitors.
Developing higher throughput methods for ChIP-Seq in the study of epigenomes can have a large impact, standardizing protocols for drug development and screening or contributing to data generation in labs or in large consortiums like ENCODE. As Snyder puts it, “ChIP-Seq has helped us understand fundamental aspects of human development and disease and functions of the human genome, enabling us to better understand and appreciate its complexity.”