Chromosome condensation
Chromosome condensation refers to the process by which dispersed interphase chromatin is transformed into a set of compact, rod-shaped structures during mitosis and meiosis.
The term "chromosome condensation" has long been used in biology. However, it is now increasingly recognized that mitotic chromosome condensation proceeds through mechanisms distinct from those governing "condensation" in physical chemistry or the formation of "biomolecular condensates" in cell biology. Consequently, some researchers have argued that the term "chromosome condensation" may be misleading in this context. For this reason, alternative terms such as "chromosome assembly" or "chromosome formation" are also commonly used.
Processes of chromosome condensation
From DNA to chromosomes
A diploid human cell contains 46 chromosomes: 22 pairs of autosomes and one pair of sex chromosomes. The total length of DNA within a single nucleus reaches ~2 m. These DNA molecules are initially wrapped around histones to form nucleosomes, which are further compacted into chromatin fibers, commonly referred to as 30-nm fibers. During interphase, these fibers are confined within the nucleus, which has a diameter of only ~10–20 um. During mitosis, chromatin is reorganized into a set of rod-shaped structures that can be individually distinguished under a microscope.This transformation was first described meticulously in the late 19th century by the German cytologist Walther Flemming. Originally, the term "chromosome" referred specifically to these highly condensed mitotic structures, although its meaning has since broadened.
In mitotic chromosomes of higher eukaryotes, DNA is compacted lengthwise by a factor of ~10,000. For example, human chromosome 8 contains a DNA molecule about 50 mm long, yet it is folded into a metaphase chromosome only ~5 um in length. This degree of compaction is comparable to folding a 600-meter-long thread into the size of an AA battery.
Physiological significance of chromosome condensation
As described above, although DNA in interphase is already organized into chromatin, it is dispersed throughout the nucleus and therefore not observed as individual chromosomes. Upon entry into prophase, condensation begins near the nuclear periphery, and fibrous structures gradually become visible. After nuclear envelope breakdown in prometaphase, condensation proceeds further. By metaphase, when condensation is apparently complete, the two sister chromatids of each chromosome can be clearly distinguished. This entire sequence of processes is often collectively referred to as chromosome condensation; however, due to our currently limited understanding of the higher-order structure of chromosomes, the precise definition of this term remains ambiguous.In principle, the process of chromosome condensation can be divided into three sequential but overlapping steps :
1. Individualization – Disentanglement of chromatin fibers dispersed throughout the nucleus into discrete chromosome units.
2. Shaping/Compaction – Organization of each chromosome into a compact, rod-like structure.
3. Resolution – Resolution of replicated DNA strands within each chromosome into two distinct sister chromatids.
Although conceptually distinct, these steps occur concurrently and synergistically during mitosis. For this reason, the entire process is often collectively referred to as chromosome condensation.
Importantly, chromosome condensation is not merely a reduction in chromatin length. Rather, it involves the organized folding of chromatin, initially in a random-coil–like state, into a highly structured rod-shaped form. This structural transformation is critical for ensuring the proper separation of sister chromatids during anaphase and provides the mechanical stiffness necessary for their faithful segregation. Defects in chromosome condensation can impair chromosome segregation and ultimately lead to genome instability.
Protein factors involved in chromosome condensation
Identification of essential factors
Eukaryotic chromosome condensation has long been regarded as a highly complex process involving numerous proteins. However, recent studies have shown that single chromatids can be reconstituted in vitro by mixing sperm nuclei with only six purified proteins: core histones, three histone chaperones, topoisomerase II, and condensin I. The three histone chaperones serve distinct roles in this reconstitution assay: Npm2 removes basic sperm-specific proteins from sperm chromatin; Nap1 deposits core histones H2A-H2B onto DNA to form nucleosomes; FACT dynamically remodels nucleosomes, thereby aiding the actions of topoisomerase II and condensin I. These chaperones do not remain associated with the final product of mitotic chromatids. In other words, the core reactions of mitotic chromosome condensation can be recapitulated using only three structural proteins, core histones, topoisomerase II, and condensin I, provided that their actions are aided by appropriate chaperone-mediated regulation.Independent lines of previous evidence support this simple picture of chromosome condensation. For example, it has long been known that histones account for approximately half of the total protein mass in mitotic chromosomes. Both topoisomerase II and condensin I have been identified as major structural components of mitotic chromosomes as well as of the so-called chromosome scaffold. Functional assays using Xenopus egg extracts and genetic analyses in fission yeast have demonstrated that both proteins are essential for properly assembling mitotic chromosomes.
Condensins
Among the three major structural proteins of mitotic chromosomes, condensin was the last to be discovered. However, it is now widely recognized as playing a central role in mitotic chromosome condensation. Most eukaryotes possess two types of condensin complexes, condensin I and condensin II, which partially overlap in function. In some organisms or cell types, condensin I alone is sufficient to support essential mitotic functions. Condensin exhibits ATPase activity and utilizes the energy from ATP hydrolysis to form DNA loops. Among the various models proposed for loop formation, the loop extrusion mechanism attracts much attention. However, an alternative mechanism and higher-order assembly functions have also been suggested.The spatiotemporal regulation of condensins is tightly coordinated with the progression of the cell cycle. In vertebrate cells, condensin II localizes to the nucleus or chromosomes throughout the cell cycle, whereas condensin I remains in the cytoplasm during interphase. Upon entry into prophase, chromosome condensation is initiated by condensin II. After nuclear envelope breakdown in prometaphase, condensin I gains access to chromosomes, and the two complexes work cooperatively to promote further condensation.
Condensins are subject to various post-translational modifications, among which phosphorylation has been most extensively studied. In vertebrates, Cdk1-mediated phosphorylation is essential for both the DNA supercoiling activity and chromosome assembly activity of condensin I. Experiments using Xenopus egg extracts have shown that phosphorylation of the N-terminal region of the CAP-H subunit relieves its autoinhibitory function, thereby activating the complex. In condensin II, cdk1-dependent phosphorylation of the C-terminal region of the CAP-D3 subunit is similarly involved in releasing inhibitory constraints and promoting its activity.
Topoisomerase II
is an enzyme that controls DNA topology by catalyzing the transient cleavage and re-ligation of double-stranded DNA. Through this activity, topoisomerase II resolves DNA entanglements between sister chromatids or different chromosomes, thereby aiding the action of condensins. Interestingly, recent studies suggest that within individualized chromatids, topoisomerase II may also introduce DNA entanglements, which contribute to morphological shaping and structural stabilization of mitotic chromosomes. Thus, topoisomerase II appears to play a dual role in chromosome architecture: both resolving and introducing DNA entanglements. The C-terminal domain of topoisomerase II is required for the latter function. Recently, topoisomerase II has been shown to mediate liquid-liquid phase separation in a DNA- and CTD-dependent manner. These non-enzymatic properties may also contribute to mitotic chromosome condensation.Topoisomerase II resides in the nucleus during interphase and becomes associated with chromosomes during mitosis. Its chromosomal binding is more dynamic than that of condensins. Although topoisomerase II undergoes various post-translational modifications, it remains unclear whether any of these modifications specifically regulate its activity during mitosis
Histones
Histones are major structural components of chromatin and chromosomes throughout the cell cycle. It has long been known that core histone H3 and linker histone H1 are phosphorylated specifically during mitosis, suggesting their specific contributions to chromosome condensation. However, direct evidence that these phosphorylations directly induce chromosome condensation remains lacking. In contrast, recent studies have shown that histone deacetylation contributes significantly to mitotic chromosome condensation via phase separation mechanisms.Remarkably, in Xenopus egg extracts, it is possible to assemble chromosome-like structures even under conditions that inhibit nucleosome formation, provided that condensins and topoisomerase II are present. These "nucleosome-depleted" chromosomes consist of a central axis enriched in condensin and large lateral DNA loops extending from it. This observation suggests that condensins play a central role in shaping the mitotic chromosome, while nucleosomes contribute to the compaction of DNA loops around the axis.