Mitotic chromosome scaffold structure: New approaches to an old controversy

In this modern era of genome sequencing and structural genomics, the fundamental question of how chromatin folds into mitotic chromosomes remains a mystery. In higher eukaryotic cells, mitotic chromosome condensation leads to an ≈10,000- to 20,000-fold compaction, as defined by the ratio of the DNA length to metaphase chromosome length (1). Compaction is achieved through at least three levels of chromatin folding (2). The first two, the coiling of DNA around the histone octamer to form nucleosomes, and the folding of regularly spaced nucleosomes into ≈30 nm “higher order” chromatin fibers, together account for only a ≈40:1 compaction ratio. This leaves the majority of mitotic chromosome compaction, ≈500-fold, the result of poorly characterized large-scale chromatin folding above the level of 30 nm chromatin fibers. Nearly all textbooks feature the radial loop model of mitotic chromosome condensation, derived from experiments largely conducted 15–25 years ago. By a neat trick of high-salt or polyanion-driven protein extraction under conditions preserving remnants of mitotic chromosome structure, the difficult issue of higher order and large-scale chromatin folding was effectively bypassed. Striking images of extended DNA “halos” surrounding a linear protein core, roughly the shape of the native chromosome, led to a simple model in which a central protein “scaffold” constrained DNA into a set of loops, ≈50 kb in length. Yet the radial loop model has remained highly controversial. This controversy derives in large part from key assumptions that are difficult to address experimentally but implicit in the interpretation of the original experiments. Two recently published experimental approaches effectively reevaluate these questions of interpretation by testing predictions, rather than assumptions, of the radial loop model. In the first (3, 4), the in vivo dynamics of the scaffold protein topoisomerase 2 were examined by using a GFP protein. In the second, published in a recent …