Scientists Establish a Genome-Wide Map of HIF1α Binding Sites in the Eye Lens Using CUT&RUN

Gene expression can be regulated by a number of different mechanisms, including transcription factors, histones, and other DNA-binding proteins. Mapping out the interactions between DNA and proteins is crucial to understanding gene activation or repression and other cell processes.

Traditionally, these interactions are analyzed by performing chromatin immunoprecipitation followed by sequencing (ChIP-seq). But this method requires a large amount of starting material to produce a signal strong enough to eliminate background noise and often returns false-positive signals. ChIP-seq also requires optimized sonication to fragment the chromatin, which can be time-consuming and could result in sample loss.

CUT&RUN (Cleavage Under Target & Release Using Nuclease) is a technique that was developed to measure protein/DNA interactions in cells while addressing the limitations of ChIP-seq. This method utilizes a fusion protein called pA/MNase to digest the chromatin into fragments, thus eliminating the hassle that comes with sonication.

In a recent study, scientists from Florida Atlantic University and the National Institute of Diabetes and Digestive and Kidney Diseases looked to employ the CUT&;RUN technique to establish a functional genome-wide map of specific binding sites of a protein complex called hypoxia inducible factor (HIF1α) in the eye lens. The team expressed their interest in choosing CUT&RUN for their project over ChIP-seq analysis because it does not require DNA-protein crosslinking. Plus, the cell count needed for a successful experiment is extremely low compared to ChIP-seq.

As the eye develops after birth, the blood vessels that provide a healthy blood supply begin to wither away with time. As a result, oxygen levels in the eye steadily decrease, creating a hypoxic environment. HIF1α is a protein complex that regulates the body’s response to hypoxic environments, implying that it could play a role in lens cell development and homeostasis.

The researchers began by isolating lens cells from 10-day-old White Leghorn chicken embryos.  The harvested cells were treated with an HIF1α activator called DMOG for 4 hours with ConA-coated magnetic beads to immobilize the nuclei. Afterwards, the samples were divided into two groups: one group was treated with an HIF1α antibody, the other with an IgG antibody.

pA/MNase was added to the samples to initiate the fragmentation. This fusion protein cleaves DNA at specific regions that interact with the protein of interest—that protein being HIF1α in this case. The MNase was activated by adding a CaCL2 solution to the samples, releasing DNA fragments into the supernatant. The CUT&RUN fragments were treated with Proteinase K for 1 hour, and the DNA was then isolated for sequencing.

The CUT&RUN identified over 8000 HIF1α-DNA specific complexes in primary lens cells after HIF1α activation. They were able to validate and complete their mapping of binding sites using ATAC-seq and RNA-seq, which showed that about 1200 of these complexes were tightly clustered in chromatin accessible regions. Further analysis revealed the activation or repression of 526 genes, with 116 of those genes displaying HIF1α binding sites within 10kB of the transcription start sites.

The data gathered in this experiment helped establish the first functional map of HIF1α DNA complexes in the eye lens, while also highlighting the need for HIF1α in healthy lens development. Ultimately, this successful application of the CUT&RUN technique may help yield numerous future studies focused on understanding the roles that certain proteins could have on gene expression.

EpiGentek has developed the EpiNext CUT&RUN Fast Kit as an elaboration on the CUT&RUN technique, with the goal of rapidly enriching protein-bound DNA and mapping genome-wide protein/DNA interactions. This kit offers sonication-free fragmentation from low inputs at a cost-effective price to reliably identify true target protein-enriched regions and achieve high-resolution mapping.