The Puzzle Solver of the Epigenome
How Steven Henikoff is Decoding Inheritance Beyond DNA
The same childhood curiosity that developed photographs in a darkroom now develops revolutionary tools to see inside our cells.
More Than Just Genes
Imagine if the pages of a cookbook couldn't be opened. Even with the best recipes, you couldn't bake a cake. Similarly, our DNA, which contains all the instructions for life, is wrapped around proteins and packed into a structure called chromatin. Whether a particular gene can be "opened" and read determines whether a cell becomes a heart cell, a brain cell, or goes awry and becomes cancerous. This layer of control—how cells use the same DNA blueprint differently—is the realm of epigenetics.
DNA Cookbook
Contains all recipes for life, but pages must be accessible to be useful.
Epigenetic Control
Determines which genes are "open" and can be read by the cell.
For decades, scientists struggled to see this epigenetic landscape clearly. The tools were expensive, slow, and often damaged the very cellular machinery they sought to study. Then, Steven Henikoff, a scientist at the Fred Hutchinson Cancer Research Center, turned the problem on its head. With his wife Jorja, he had already changed the field of bioinformatics in the 1990s by creating the BLOSUM matrices, which are now essential for every protein sequence analysis performed today 7 . But his restless curiosity soon led him to an even bigger challenge: building a better window into the epigenome. His resulting inventions are not only illuminating fundamental biology but are also so simple that, as he demonstrated during the COVID-19 pandemic, they can be performed on a home workbench 3 .
The Maker's Mind: From Childhood Puzzles to Scientific Tools
Henikoff's career as an inventor was born from a childhood love of puzzles and photography. He was less interested in taking pictures than in the "discovery process" of the darkroom, where images emerged through chemistry 1 . This fascination with process led him from chemistry to biochemistry and eventually to Harvard for his PhD.
Childhood Curiosity
Developed a love for puzzles and photography, fascinated by the darkroom development process.
Academic Journey
Pursued chemistry and biochemistry, eventually earning a PhD from Harvard.
Peace Corps & Military
His studies were interrupted by the Vietnam War draft; joined Peace Corps where he met his wife Jorja.
Tool Development
While studying a fruit fly gene, he developed a more efficient sequencing method, beginning his career as a toolmaker.
"I came up with a method for sequencing more efficiently... That got me hooked on making tools," he says 1 . This mindset—that necessity is the mother of invention—has driven a career of creating widely used biotechnologies.
The Toolkit Revolution: CUT&RUN and CUT&Tag
For years, the standard method for mapping epigenetic marks was ChIP-seq (Chromatin Immunoprecipitation followed by sequencing). However, ChIP-seq is a brutal process. It involves cross-linking proteins to DNA with formaldehyde, shredding the chromatin with sound waves (sonication), and then using an antibody to pull down the protein of interest 5 9 . It requires millions of cells, is time-consuming, and the harsh treatment creates high background noise, requiring vast amounts of sequencing to find a true signal.
Comparison of Chromatin Profiling Methods
| Feature | ChIP-seq | CUT&RUN | CUT&Tag |
|---|---|---|---|
| Workflow | Complex: cross-linking, sonication, immunoprecipitation 5 | Simpler: no cross-linking or sonication 5 | Simplest: all steps in one tube 5 |
| Time | ~5 days 5 | ~3 days | ~2 days 5 |
| Cells Required | Millions 5 | ~500,000 5 | As few as 10,000 5 |
| Signal-to-Noise | Low (high background) 5 | High | Very High 5 |
| Sequencing Reads Needed | ~30 million 5 | Reduced | 5-8 million 5 |
Henikoff's team set out to develop a gentler, more precise method. The result was CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and its successor, CUT&Tag (Cleavage Under Targets and Tagmentation) 2 5 .
CUT&RUN
Uses MNase enzyme to snip DNA around the target 2 .
CUT&Tag
Uses Tn5 transposase to simultaneously cut DNA and attach sequencing adapters 5 .
The benefits are profound. CUT&Tag requires 90% fewer cells and 75% fewer sequencing reads than ChIP-seq, slashing costs and enabling studies on rare cell types previously impossible to analyze 5 . The process is so clean and efficient that Henikoff's lab has even automated it for high-throughput clinical applications 2 .
A Closer Look: The CUTAC Experiment
The story of one of Henikoff's experiments provides a perfect window into how science truly advances: through serendipity and sharp observation. While optimizing CUT&Tag, his team made a simple change—they lowered the salt concentration during one step of the protocol. They noticed that when they used antibodies against specific histone marks (H3K4me2 and H3K4me3), this change caused the Tn5 enzyme to behave differently 3 .
Instead of cutting just the nucleosome where the antibody was bound, the enzyme was now drawn to nearby nucleosome-depleted regions (NDRs)—the very "open" areas of chromatin that signify active regulatory elements 3 . They realized that by tethering Tn5 to histones marked by active transcription, they could use it as a beacon to map all the nearby accessible DNA. They called this new application CUTAC (Cleavage Under Targeted Accessible Chromatin) 3 .
Methodology: Mapping Accessibility Step-by-Step
The CUTAC protocol is elegantly simple, building on the CUT&Tag-direct method :
Isolate Nuclei
Cells are processed to isolate their nuclei.
Immobilize
The nuclei are bound to magnetic beads coated with Concanavalin A, which keeps them steady for all subsequent steps 5 .
Add Target Antibody
A primary antibody against H3K4me2 or RNA Polymerase II Serine-5-phosphate (a mark of initiating transcription) is added. It enters the nuclei and binds to its target .
Tether the Transposase
A secondary antibody and then the pAG-Tn5 fusion protein are added. This fusion protein binds to the primary antibody, effectively tethering the Tn5 enzyme directly to the active epigenetic mark.
Low-Salt Tagmentation
A low-salt buffer containing magnesium is added. This critical step activates Tn5 and, under low-salt conditions, encourages it to "reach" and tagment the accessible DNA in the nearby NDR 3 .
Extract and Sequence
The tagmented DNA is purified and amplified by PCR, creating a library ready for high-throughput sequencing.
Results and Analysis: A Sharper Image of Activity
When the sequencing data came back, the CUTAC maps were stunning. They were virtually indistinguishable from the best maps produced by the popular ATAC-seq method but with even higher resolution and less background noise 3 . The data provided powerful evidence that chromatin accessibility is directly coupled to transcriptional activity.
Performance Comparison of Chromatin Accessibility Methods
Key Research Reagents for CUTAC
| Reagent | Function | Example |
|---|---|---|
| Concanavalin A Beads | Magnetic beads that immobilize nuclei for easy processing 5 . | Bangs Laboratories BP531 |
| Primary Antibody | Binds the specific epigenetic target to guide the transposase. | Anti-H3K4me2 or Anti-RNA Polymerase II Ser5P |
| pAG-Tn5 Fusion Protein | Engineered protein that binds antibodies and tagments DNA. | Epicypher 15-1117 |
| Low-Salt Tagmentation Buffer | Activates Tn5 under conditions that favor tagmentation of accessible DNA 3 . | Custom buffer with MgCl₂ and diluted salts |
The scientific importance of CUTAC is twofold. First, it provides a superior technical method that is faster, cheaper, and more precise. Second, and more profoundly, it delivers a fundamental biological insight: the enzymes that modify histones during transcription are physically positioned to make the adjacent DNA accessible 3 . This directly links the histone mark to the function of the regulatory element.
A Lasting Legacy: From Fundamental Biology to Cancer Research
Henikoff's work has always been driven by a desire to understand fundamental mechanisms, such as how epigenetic information is passed on when cells divide and how chromosomes' centromeres—critical for cell division—are maintained 1 4 . His tools have now empowered the entire scientific community to ask these questions with unprecedented clarity.
Fewer cells needed with CUT&Tag vs ChIP-seq
Fewer sequencing reads needed with CUT&Tag
Minimum cells for CUT&Tag analysis
Days for CUT&Tag protocol vs 5 for ChIP-seq
His lab continues to innovate at a breathtaking pace, developing new methods like TMP-Seq to measure DNA supercoiling and MINCE-seq to see what happens to chromatin right after it is replicated 2 . This toolkit is now being applied to pressing medical problems. In a landmark 2025 study published in Science, his team used these methods to show that the behavior of RNA Polymerase II at histone genes can predict outcomes in human cancer, opening new avenues for diagnosis and therapy 8 .
Recent Innovations from Henikoff's Lab
TMP-Seq
Measures DNA supercoiling dynamics in the genome.
MINCE-seq
Examines chromatin immediately after replication.
Cancer Applications
Using epigenetic tools to predict cancer outcomes.
Steven Henikoff's story demonstrates that the most powerful scientific advances are not always just discoveries of what exists, but the creation of new ways to see. From a boy in a darkroom to a leading scientist, his journey reminds us that the drive to build better tools—to develop better film—is what ultimately illuminates the hidden worlds around us and within us.