Chromatin conformation capture (3C) assays have been used for ~20 years to elucidate complex folding of genomic sequence in three-dimensional space.
All 3C-based assays follow the same core workflow:
Chromatin fixation → Digestion → Ligation of proximal cut ends → Removal of cross-links
What type of data is generated?
The result is chimeric DNA that is reflective of DNA fragments that were close in physical space.
Auxillary molecular biology processes can be over laid on the core workflow to:
- Detect specific chimeric constructs by qPCR (3C)
- Amplify all interactions that occur with known loci (4C)
- Convert all chimeric DNA into an NGS sequencing library (Hi-C)
- Target many loci through hybrid capture-NGS (Capture-C or Chi-C)
- Conduct chromatin immunoprecipitation on a protein of interest (HiChIP or PLAC-seq)
All of these approaches capture the 3D topology of the genome through the lens of the chimeric DNA molecule.
That’s great! So what’s the problem?
Historically, these data types, regardless of the chimeric DNA of focus, have one thing in common – the chromatin fragmentation is performed using restriction enzymes (REs) that recognize and cut at specific motifs. RE adoption was largely a product of convenience as they:
- Provided an endpoint assay.
- Acted as a primary-sequence touchstone to computationally assess the data.
While convenient, due to their sequence-dependence, the use of REs does have three primary downsides:
- Generation of highly variable chromatin fragment sizes.
- Uneven read distribution with data stacked at restriction sites.
- Limited contact matrix resolution preventing detection of finer chromatin features.
- Literature reviews reveal contact matrix limits are ~1 kb.
1 kb matrices require an extraordinary amount sequencing – 1.2-1.6 billion paired-reads (yes, that’s billion with a B).
How can you overcome these downsides?
Micro-C, a Hi-C approach using micrococcal nuclease (MNase) in place of REs, offers a solution.
For those of you not familiar, MNase is an endo/exonuclease that cuts at nucleosome-free (linker DNA) and then digests DNA back to the nucleosome. The resulting fragments are short and consistently sized – roughly the length of DNA wrapped around the nucleosome (i.e. 146 bp). Following proximity-ligation and cross-link reversal, the resulting chimeric DNA reveals the positioning of nucleosomes in physical space. Moreover, the short and consistent fragment sizes do not require sheering thereby offering two key advantages:
- Reduced hands-on time.
- Eliminates capture of self-ligation events in the final library increasing signal-to-noise and making better use of the sequence space.
Thus, replacing REs with MNase results in the enrichment of nucleosome-protected sequence and depletion of linker DNA (Figure 1).