After playing hide-and-seq with chromatin for a while, we’re sure you’ve been begging for a new plan of attack to help you see what’s going on. Thankfully, a Northern California collaboration led by the lab of Howard Chang alongside the labs of William Greenleaf, Jan Liphardt, and Jennifer Doudna has brought forth a tactic to both visualize and sequence accessible chromatin.
Previously, the Greenleaf and Chang labs developed the Assay of Transposase-Accessible Chromatin with Sequencing (ATAC-seq), a technique that identifies regions of open chromatin. The method of ATAC(K) makes use of a hyperactive mutant Tn5 transposase that can simultaneously fragment and tag DNA with its sequencing adapter payload. The Tn5 transposase has a strong preference for open chromatin since the steric hindrance of closed chromatin makes transposition a much less likely event. Then, adapter-ligated fragments are isolated, PCR amplified, and sent off for some next-generation sequencing, which produces reads of accessible chromatin.
ATAC-see: Assay of Transposase-Accessible Chromatin with Visualization
ATAC-see is a modified version of ATAC-seq that takes the ATAC to next level and allows for in situ visualization by fluorescence. To accomplish such a task, the team engineered a bifunctional Tn5 transposase with a special payload: DNA adapters conjugated to fluorophores. Thus, the adapters allow for both the visualization and sequencing of open chromatin from the same fixed cells. Furthermore, multi-modal imaging is possible. Closed chromatin can be visualized by DAPI, while landmark proteins can be visualized by immunofluorescence, which creates a complete chromatin composite for confocal microscopy.
ATAC-see’s Insight into Nuclear Architecture
Demonstrating the insight to be gained from their technique, the team ATAC’d 5 different human cell types to study differences in nuclear architecture. They focused on neutrophils and uncovered a unique organization – the majority of signal comes from a rim structure at the nuclear periphery that is correlated with lamina-associated domains (LADs).
Next, they studied how mature neutrophils capture and kill bacteria, which is achieved by them extruding their chromatin to create neutrophil extracellular traps (NETs). This attack results in the ultimate sacrifice, a unique type of cellular death known as NETosis. The group found that when neutrophils are activated chromatin begins to disassemble into mononucleosomes. The event starts at the nuclear periphery and then spreads to the rest of the genome. Then, the mononucleosomes are further disassembled into free DNA and histones by histone citrullination.
Sorting Cells by Chromatin: ATAC-see and Flow Cytometry
Finally, ATAC-see can also be combined with fluorescence-activated cell sorting (FACS) to allow for cells to be sorted by their accessibility. This showed that accessibility was dependent on the stage of the cell cycle. Thus, ATAC-see also has the potential to uncover cellular heterogeneity.
Ultimately, ATAC-see offers up a visually appealing upgrade for ATAC-seq by allowing for a more complete molecular portrait of nuclear architecture.
Go see the whole ATAC on chromatin over at Nature Methods, October 2016