Epigenetics, Chromatin Architecture and a Judo Mechanism to Attack Cancer

Epigenetics is the study of how genetic information is context-dependent: it is organized so it can be repressed, but also read, repaired and replicated. For example, transcription factors can “communicate” with each other through the chromatin-DNA interface, and work in combinations to regulate which genes are expressed. In many types of cancer, transcription factors can drive altered gene expression networks.

Transcription factors don’t bind to “flat surfaces” but rather to the hills and valleys of the epigenetic landscape. But how are these hills and valleys structured?

Chromatin is the material that packages DNA in higher organisms, and the most basic unit of chromatin is called the “nucleosome,” which behaves like a spool around which the thread (DNA) is wrapped. These nucleosome-DNA particles form the basic structures that assemble into chromosomes inside our cells. Our DNA accessibility is influenced by the occupancy, spacing and positioning of nucleosomes.

Interestingly, the “spool” [nucleosome] has protein tails (histones) that can be modified (marked), and there is growing evidence that this, in addition to DNA accessibility, can instruct epigenetic changes.

A modification to nucleosomes called acetylation is highly associated with more open chromatin states, where DNA is more accessible, and can be read more easily. Chromatin acetylation changes as different genes are activated and repressed.

Researchers who are interested in DNA’s accessibility frequently investigate how and why chromatin becomes acetylated and deacetylated.

One of the researchers investigating the relationships among chromatin structure, acetylation and transcription is Ben Stanton, PhD, principal investigator in the Center for Childhood Cancer and Blood Diseases at the Abigail Wexner Research Institute at Nationwide Children’s Hospital.

“It’s important to remember that while the genetic code is essentially linear, DNA is packaged into a three-dimensional chromatin landscape,” Dr. Stanton says. “We have been highly motivated to understand how histone marks and genome architecture are integrated, and the degree to which this interplay has network-level effects on gene expression.”

Researchers have been working to measure chromatin architecture for the last two decades: In 2002, Job Dekker, PhD, and colleagues reported a PCR-based method to define chromatin interactions (3C). In 2009, Erez Lieberman-Aiden, PhD, and colleagues reported 3D genome sequencing (Hi-C) and refined the method with new insights in 2014. In 2016, Howard Chang, MD, PhD, Will Greenleaf, PhD, and co-workers published a protein-centric 3D genome sequencing method (HiChIP).

These approaches enabled definition of chromatin interactions, the basis of architecture, but it has remained challenging to address a central question: Is the chromatin structure in a cancer cell different to a healthy cell?

To address this question, Dr. Stanton — in collaboration with Berkley Gryder, PhD, and Javed Khan, MD, both at the National Cancer Institute — developed a method of absolute comparison for chromatin interactions using “spike-in” chromatin loops from another organism that can be sequenced together.

The method is called AQuA (Absolute Quantification of Chromatin Architecture)-HiChIP. And it has already been useful for defining chromatin acetylation’s architecture in rhabdomyosarcoma, as published in Nature Genetics.

With the AQuA-HiChIP method, defined ratios of mouse and human fixed nuclei are processed within the same experiment, enabling an internal control for chromatin interactions. The paired-end sequencing tags associated with interactions across “interacting chromatin” from the human genome are normalized to interactions within the mouse genome.

To show how AQuA-HiChIP can help researchers better understand changes in chromatin architecture in context, Dr. Stanton and his colleagues investigated how histone acetylation is associated with disruption of the chromatin architecture in rhabdomyosarcoma: chromatin interactions were altered with HDAC inhibitors, and the tumor cells lost the capacity to proliferate.

Histone deacetylase (HDAC) inhibitors are a class of drugs that can increase the acetylation of chromatin on short timescales but can result in severe loss of acetylation on longer timescales. They have been shown to be useful in treating cancers in clinical and preclinical studies.

“We found that core regulatory transcription factors are essential to the tumor. They need certain patterns of DNA accessibility to keep the cancer going. Chromatin acetylation is associated with increased DNA accessibility and may fine-tune the binding sites for these transcription factors,” says Dr. Stanton.

HDAC inhibitors push the cell into “hyperacetylation mode” on short timescales, changing the chromatin architecture.

“HDAC inhibitors can be quite toxic to cancers,” says Dr. Stanton. “They operate through this ‘judo mechanism’ — using the chromatin’s momentum to toxify the cell. HDAC inhibitors push the tumor cells further and further in the acetylation direction, eventually causing a loss of the cell’s capacity to regulate its own gene expression.”

Moving forward, Dr. Stanton and his colleagues plan the continued application of AQuA-HiChIP to central questions regarding transcription control, accessibility and tissue-specific epigenetic memory.

According to Dr. Stanton, the quantitative nature of the data produced through the method allows for specific comparisons of chromatin interactivity, not limited to rank-ordering interactivity, to compare between experimental conditions.

“More than anything else, I want this technique to be applied to other systems and to be useful for the broader community. It is my hope that this spike-in approach will broadly enable insights into the architectural features in human disease,” says Dr. Stanton. “There’s a lot of very exciting basic science to be done in childhood cancer research. Increasing our fundamental understanding of the epigenetic memory of architecture can lead to new insights.”

References:

1. Dekker J, Rippe K, Dekker M, Kleckner N. Capturing chromosome conformation. Science. 2002;295:1306-1311.
2. Gryder BE, Pomella S, Sayers C, Wu XS, Song Y, Chiarella AM, Bagchi S, Chou H-C, Sinniah RS, Walton A, Wen X, Rota R, Hathaway NA, Zhao K, Chen J, Vakoc CR, Shern JF, Stanton BZ, Khan J. Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma. Nature Genetics. 2019;51:1714-1722.
3. Gryder BE, Khan J, Stanton BZ. Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP). Nature Protocols. 2020 Mar;15(3):1209-1236.
4. Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature Methods. 2016 Nov;13:919-922.
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6. Lin GL, Wilson KM, Ceribelli M, et al. Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening. Science Translational Medicine. 2019;11(519):eaaw0064.
7. Rao SS, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014; 159:1665-1680.

Image credit: Nationwide Children’s