Name: Benjamin Garcia, assistant professor of molecular biology

Invention: Reverse ChIP for Exploring Epigenetic Oncology and for Anti-Cancer Drug Screening

What It Is: A method for extracting specific target genes along with regulatory proteins that bind to the gene and then analyzing their identity and epigenetic modifications.

How It Works: The genome contains vast amounts of genetic information relevant to human health. In the past few decades, researchers have realized that the proteins that surround the DNA can control whether the DNA is active (expressed) or silent.  Some of these proteins, known as histones, bind to DNA and help it to coil into the kinked structure known as chromatin, which in turn forms chromosomes.

Modifications to histone proteins can change whether individual genes are read and translated, and thus alter biological processes ranging from embryo development to tumor growth. These modifications come in the form of additions or removals of chemical groups that are attached to the histones and can control whether a particular chromosomal region is open to reading by the cell’s DNA transcription machinery. In this way, modifications to histones make up a secondary code, known as the epigenomic code or histone code. “These are mechanisms that control DNA expression and yet exist outside the genes themselves,” said Garcia.

These epigenetic modifications occur through the addition or removal of chemical units, such as methyl and acetyl groups, to the proteins. Certain enzymes have already been identified that add or remove these units from histones, and companies are developing drugs that inhibit these enzymes for use in potential cancer therapies.

Reverse ChIP consists of gene-specific chromatin preparation followed by analysis of DNA-bound proteins by mass spectroscopy (see Fig. 1).  After cell lysis (Fig.1a) and restriction digest (Fig.1b), the targeted loci are captured by primers that bind to a specific genomic region.  An enzymatic step incorporates biotin labels only to chromosomal fragments that contain the targeted sequence and streptavidin-coated magnetic particles isolate the targeted chromatin (Fig.1c).  Proteins associated with the isolated regions are then released and analyzed by high-end mass spectrometry either on peptides generated from an enzymatic digest or from intact protein (Fig.1d). Provided by epitrac LLC.

Garcia, Gary LeRoy (a postdoctoral scientist in the Garcia group) and their collaborator Johannes Dapprich of Generation Biotech, a biotechnology company in Lawrenceville, NJ, have created a tool called reverse chromatin immunoprecipitation, or “Reverse ChIP”.  Reverse ChIP isolates a target gene in a sequence-specific way and explores the modifications of the histones or other proteins that are bound to that gene using a technique known as mass spectrometry.  This is an improvement over existing techniques which can only detect aggregated histone changes over all the genes. “We are developing a method for researchers who want to look at only the histones on a certain gene of interest, so for example, involved in cancer,” said Garcia.

Reverse ChIP consists of two steps. The first is to efficiently capture the gene and the proteins bound to it, and the second step is to analyze the proteins that were isolated along with the gene by mass spectrometry.

To capture a gene and its histones, Reverse ChIP uses a strand of DNA, called a primer, that binds to the gene of interest.  After binding, the primer is elongated by an enzyme in a sequence-specific way.  This process creates a ‘handle’ that allows the researchers to capture the targeted gene on magnetic nanoparticles along with its histones and other DNA-binding proteins intact. These proteins are removed from the gene and then broken into smaller segments, called peptides, for analysis.

In the second step, exploring the modifications that have been made to these proteins, the researchers use a method called high resolution mass spectrometry, which can detect minute changes to the proteins, such as the addition or removal of chemical groups.

Reverse ChIP is an improvement over other methods of studying histone modifications because it is rapid and can be carried out in an automated and medium-throughput format.  It is also possible to use capture primers that are designed to be specific to individual DNA base changes, called single-nucleotide polymorphisms (SNPs).  SNPs are widely used as markers in genome-wide association studies.  The ability to directly link the histone code to specific SNPs can provide valuable information for genetic research and pharmaceutical drug development.

Other techniques, such as the use of site-specific antibodies, suffer drawbacks such as cross reactivity with similar regions on the same or different proteins and are tedious and hard to validate.

Applications:

Reverse ChIP has many potential uses in cancer research and drug development. For example, researchers can use Reverse ChIP to compare histone modification patterns in healthy versus diseased cells. By understanding the histone modifications involved in cancer, and pinpointing the enzymes involved in these modifications, it may be possible to create drugs that reset the modification patterns and return the patient to a healthy state.

The Reverse ChIP technique represents a merging of proteomics and genomics techniques.  The ability to potentially identify any DNA binding protein – such as transcription factors, histones and other proteins - that bind to a given candidate gene can be highly valuable in order to understand the regulatory pathways that may act on a particular gene of interest. This systems biology approach could lead to more effective drug treatments for human diseases that are caused by epigenetic mechanisms.

A number of enzymes have been identified that modify histones on genes implicated in breast cancer, myeloma, and leukemia. Many pharmaceutical companies have active programs in epigenetic oncology and are interested in screening small molecule libraries for activity against enzymes that acetylate, deacetylate and otherwise modify histones associated with cancer genes.

Inspiration: In addition to cancer, several other diseases such as metabolic, autoimmune and neurologic disorders are suspected of having epigenetic causes. 

Collaborators: The isolation of histones using magnetic beads was done in collaboration with Johannes Dapprich of epitrac, a Lawrenceville, NJ, start-up company using a transgene system developed by Gary LeRoy, a postdoctoral scientist in Garcia’s group.  Garcia collaborates with Constellation Pharmaceuticals in Cambridge, MA, to look at enzymes that act on histones bound to HOX genes, which are suspected in the development of cancer. Certain enzymes have been identified that modify these histones by adding and removing methyl groups. Constellation and other companies have developed libraries of small molecules that can inhibit these enzymes and thus change the modification patterns on HOX-related histones.

Commercialization Status

The group is in discussion with several biotech and pharmaceutical companies with applications in oncology for potential strategic partnering and licensing.  Funding for the application of the technology to the isolation of neuroblastoma-related histones is anticipated for 2012 through the NIH’s small business innovation research (SBIR) program.  The method is protected by US and international patent applications.