The studies and approaches described above provide insights into structural aspects of chromatin at length scales from ∼1 bp to several megabases. However, at present, there exists a “blind spot” in chromosome folding assays at the length scale between ∼200 bp and ∼1–2 kb. Specifically, 3C-based methods rely on fragmentation of the genome to assay chromatin fragments that can be crosslinked to one another. Typically, this fragmentation relies on restriction enzyme digestion, meaning that, for typical 3C and Hi-C experiments, the average fragment size is ∼4 kb. Even with four-cutter restriction enzymes, heterogeneous site locations across the genome and incomplete restriction result in a typical resolution of, at best, ∼1 kb. Thus, the length scale between 200 bp and ∼1–4 kb remains invisible with current chromosome folding assays. It is at this length scale that one would expect signal from secondary structures in chromatin, such as 30-nm fiber (Tremethick 2007), or short gene loops in budding yeast, and is thus a key area of interest for future studies in chromosome folding.
